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SEBASTIANO CALANDRA BUONAURA

Professore emerito
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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Pubblicazioni

2023 - Identification and Molecular Characterization of a Novel Large-Scale Variant (Exons 4_18 Loss) in the LDLR Gene as a Cause of Familial Hypercholesterolaemia in an Italian Family [Articolo su rivista]
Concolino, P.; De Paolis, E.; Moffa, S.; Onori, M. E.; Soldovieri, L.; Ricciardi Tenore, C.; De Bonis, M.; Rabacchi, C.; Santonocito, C.; Rinelli, M.; Calandra, S.; Giaccari, A.; Urbani, A.; Minucci, A.
abstract

Abstract: Next-generation sequencing (NGS) is nowadays commonly used for clinical purposes, and represents an efficient approach for the molecular diagnosis of familial hypercholesterolemia (FH). Although the dominant form of the disease is mostly due to the low-density lipoprotein receptor (LDLR) small-scale pathogenic variants, the copy number variations (CNVs) represent the underlying molecular defects in approximately 10% of FH cases. Here, we reported a novel large deletion in the LDLR gene involving exons 4–18, identified by the bioinformatic analysis of NGS data in an Italian family. A long PCR strategy was employed for the breakpoint region analysis where an insertion of six nucleotides (TTCACT) was found. Two Alu sequences, identified within intron 3 and exon 18, could underlie the identified rearrangement by a nonallelic homologous recombination (NAHR) mechanism. NGS proved to be an effective tool suitable for the identification of CNVs, together with small-scale alterations in the FH-related genes. For this purpose, the use and implementation of this cost-effective, efficient molecular approach meets the clinical need for personalized diagnosis in FH cases.

2023 - Lipoprotein(a) Genotype Influences the Clinical Diagnosis of Familial Hypercholesterolemia [Articolo su rivista]
Olmastroni, Elena; Gazzotti, Marta; Averna, Maurizio; Arca, Marcello; Tarugi, Patrizia; Calandra, Sebastiano; Bertolini, Stefano; Catapano, Alberico L; Casula, Manuela; Marcello, Arca; Laura, D'Erasmo; Maurizio, Averna; Angelo Baldassare Cefalù, ; Andrea, Bartuli; Paola Sabrina Buonuomo, ; Andrea, Benso; Guglielmo, Beccuti; Giacomo, Biasucci; Maria Elena Capra, ; Gianni, Biolo; Pierandrea, Vinci; Luca, Bonanni; Claudio, Borghi; Sergio, D'Addato; Antonio Carlo Bossi, ; Giancarla, Meregalli; Adriana, Branchi; Paolo, Calabrò; Carubbi, Francesca; Nascimbeni, Fabio; Francesco, Cipollone; Marco, Bucci; Nadia, Citroni; Maria Del Ben, ; Francesco, Baratta; Massimo, Federici; Martina, Montagna; Claudio, Ferri; Serena, Notargiacomo; Anna Maria Fiorenza, ; Emanuela, Colombo; Giuliana, Fortunato; Maria Donata Di Taranto, ; Andrea, Giaccari; Simona, Moffa; Francesco, Giorgino; Sergio Di Molfetta, ; Ornella, Guardamagna; Luisa De Sanctis, ; Arcangelo, Iannuzzi; Raimondo, Cavallaro; Gabriella, Iannuzzo; Marco, Gentile; Iughetti, Lorenzo; Bruzzi, Patrizia; Salvatore, Lia; Alessandro, Lupi; Giuseppe, Mandraffino; Arianna, Toscano; Rossella, Marcucci; Martina, Berteotti; Lorenzo, Maroni; Fabiana, Locatelli; Tiziana, Montalcini; Giuliana, Mombelli; Sandro, Muntoni; Davide, Baldera; Gianfranco, Parati; Angelina, Passaro; Valerio, Pecchioli; Cristina, Pederiva; Giuseppe, Banderali; Antonio, Pipolo; Debora, D'Elia; Matteo, Pirro; Vanessa, Bianconi; Livia, Pisciotta; Elena, Formisano; Francesco, Purrello; Roberto, Scicali; Elena, Repetti; Elena, Cantino; Elisabetta, Rinaldi; Elena, Sani; Riccardo, Sarzani; Francesco, Spannella; Francesco, Sbrana; Beatrice Dal Pino, ; Patrizia, Suppressa; Cocco, VERONICA MARGHERITA; Chiara, Trenti; Emanuele Alberto Negri, ; Josè Pablo Werba, ; Alessandra, Romandini; Sabina, Zambon; Alberto, Zambon; Maria Grazia Zenti, ; Giulia, Fainelli; Fabio, Pellegatta; Liliana, Grigore; Katia, Bonomo; Eleonora, Capatti; Ada, Cutolo; Fabio, Fimiani; Simonetta, Genovesi; Sandro, Inchiostro; Chiara, Pavanello; Roberta, Pujia; Alon, Schaffer; Stefano, Bertolini; CALANDRA BUONAURA, Sebastiano; Alberico Luigi Catapano, ; Manuela, Casula; Elena, Olmastroni; Tarugi, Patrizia Maria; Marta, Gazzotti; Federica, Galimberti
abstract

BackgroundEvidence suggests that LPA risk genotypes are a possible contributor to the clinical diagnosis of familial hypercholesterolemia (FH). This study aimed at determining the prevalence of LPA risk variants in adult individuals with FH enrolled in the Italian LIPIGEN (Lipid Transport Disorders Italian Genetic Network) study, with (FH/M+) or without (FH/M-) a causative genetic variant. Methods and ResultsAn lp(a) [lipoprotein(a)] genetic score was calculated by summing the number risk-increasing alleles inherited at rs3798220 and rs10455872 variants. Overall, in the 4.6% of 1695 patients with clinically diagnosed FH, the phenotype was not explained by a monogenic or polygenic cause but by genotype associated with high lp(a) levels. Among 765 subjects with FH/M- and 930 subjects with FH/M+, 133 (17.4%) and 95 (10.2%) were characterized by 1 copy of either rs10455872 or rs3798220 or 2 copies of either rs10455872 or rs3798220 (lp(a) score >= 1). Subjects with FH/M- also had lower mean levels of pretreatment low-density lipoprotein cholesterol than individuals with FH/M+ (t test for difference in means between FH/M- and FH/M+ groups <0.0001); however, subjects with FH/M- and lp(a) score >= 1 had higher mean (SD) pretreatment low-density lipoprotein cholesterol levels (223.47 [50.40] mg/dL) compared with subjects with FH/M- and lp(a) score=0 (219.38 [54.54] mg/dL for), although not statistically significant. The adjustment of low-density lipoprotein cholesterol levels based on lp(a) concentration reduced from 68% to 42% the proportion of subjects with low-density lipoprotein cholesterol level >= 190 mg/dL (or from 68% to 50%, considering a more conservative formula). ConclusionsOur study supports the importance of measuring lp(a) to perform the diagnosis of FH appropriately and to exclude that the observed phenotype is driven by elevated levels of lp(a) before performing the genetic test for FH.

2023 - Refinement of the diagnostic approach for the identification of children and adolescents affected by familial hypercholesterolemia: Evidence from the LIPIGEN study [Articolo su rivista]
Casula, M.; Gazzotti, M.; Capra, M. E.; Olmastroni, E.; Galimberti, F.; Catapano, A. L.; Pederiva, C.; Anesi, A.; Arca, M.; Auricchio, R.; Averna, M.; Baldera, D.; Banderali, G.; Beccuti, G.; Benso, A.; Berteotti, M.; Bertolini, S.; Bianconi, V.; Biasucci, G.; Biolo, G.; Bonanni, L.; Borghi, C.; Bossi, A. C.; Branchi, A.; Bruzzi, P.; Bucci, M.; Buonuomo, P. S.; Calabro, P.; Calandra, S.; Carubbi, F.; Cavallaro, R.; Cefalu, A. B.; Cesaro, A.; Cipollone, F.; Citroni, N.; Colombo, E.; Coppola, C.; D'Addato, S.; Dal Pino, B.; Dalla Nora, E.; De Corrado, G.; Del Ben, M.; Di Molfetta, S.; Di Taranto, M. D.; Fainelli, G.; Federici, M.; Ferri, C.; Fiorenza, A. M.; Formisano, E.; Fortunato, G.; Giaccari, A.; Giorgino, F.; Grigore, L.; Guardamagna, O.; Iannuzzi, A.; Iannuzzo, G.; Iughetti, L.; Lia, S.; Longo, S.; Lupi, A.; Mandraffino, G.; Marcucci, R.; Maroni, L.; Massini, G.; Mazza, E.; Melchioda, E.; Meregalli, G.; Minicocci, I.; Moffa, S.; Mombelli, G.; Muntoni, S.; Nascimbeni, F.; Negri, E. A.; Notargiacomo, S.; Panfili, F. M.; Parati, G.; Passaro, A.; Pavanello, C.; Pecchioli, V.; Pecchioli, L.; Pellegatta, F.; Perla, F. M.; Pipolo, A.; Piro, S.; Pirro, M.; Pisciotta, L.; Pujia, R.; Putotto, C.; Repetti, E.; Rinaldi, E.; Romandini, A.; Sani, E.; Sarnari, S.; Sarzani, R.; Sbrana, F.; Scicali, R.; Scuruchi, M.; Suppressa, P.; Tarugi, P.; Trenti, C.; Vinci, P.; Werba, J. P.; Zambon, S.; Zambon, A.; Zenti, M. G.
abstract

Background and aims: We aimed to describe the limitations of familiar hypercholesterolemia (FH) diagnosis in childhood based on the presence of the typical features of FH, such as physical sings of cholesterol accumulation and personal or family history of premature cardiovascular disease or hypercholesterolemia, comparing their prevalence in the adult and paediatric FH population, and to illustrate how additional information can lead to a more effective diagnosis of FH at a younger age. Methods: From the Italian LIPIGEN cohort, we selected 1188 (≥18 years) and 708 (<18 years) genetically-confirmed heterozygous FH, with no missing personal FH features. The prevalence of personal and familial FH features was compared between the two groups. For a sub-group of the paediatric cohort (N = 374), data about premature coronary heart disease (CHD) in second-degree family members were also included in the evaluation. Results: The lower prevalence of typical FH features in children/adolescents vs adults was confirmed: the prevalence of tendon xanthoma was 2.1% vs 13.1%, and arcus cornealis was present in 1.6% vs 11.2% of the cohorts, respectively. No children presented clinical history of premature CHD or cerebral/peripheral vascular disease compared to 8.8% and 5.6% of adults, respectively. The prevalence of premature CHD in first-degree relatives was significantly higher in adults compared to children/adolescents (38.9% vs 19.7%). In the sub-cohort analysis, a premature CHD event in parents was reported in 63 out of 374 subjects (16.8%), but the percentage increased to 54.0% extending the evaluation also to second-degree relatives. Conclusions: In children, the typical FH features are clearly less informative than in adults. A more thorough data collection, adding information about second-degree relatives, could improve the diagnosis of FH at younger age.

2022 - Worldwide experience of homozygous familial hypercholesterolaemia: retrospective cohort study [Articolo su rivista]
Tromp, Tycho R; Hartgers, Merel L; Hovingh, G Kees; Vallejo-Vaz, Antonio J; Ray, Kausik K; Soran, Handrean; Freiberger, Tomas; Bertolini, Stefano; Harada-Shiba, Mariko; Blom, Dirk J; Raal, Frederick J; Cuchel, Marina; Tromp, Tycho R.; Hartgers, Merel L.; Hovingh, G. Kees; Vallejo-Vaz, Antonio J.; Ray, Kausik K.; Soran, Handrean; Freiberger, Tomas; Bertolini, Stefano A.; Harada-Shiba, Mariko; Pang, Jing; Watts, Gerald F.; Greber-Platzer, Susanne; Mäser, Martin; Stulnig, Thomas M.; Ebenbichler, Christoph F.; Bin Thani, Khalid; Cassiman, David; Descamps, Olivier S.; Rymen, Daisy; Witters, Peter; Santos, Raul D.; Brunham, Liam R.; Francis, Gordon A.; Genest, Jacques; Hegele, Robert A.; Kennedy, Brooke A.; Ruel, Isabelle; Sherman, Mark H.; Jiang, Long; Wang, Luya; Reiner, Željko; Blaha, Vladimir; Ceska, Richard; Dvorakova, Jana; Dlouhy, Lubomir; Horak, Pavel; Soska, Vladimir; Tichy, Lukas; Urbanek, Robin; Vaverkova, Helena; Vrablik, Michal; Zemek, Stanislav; Zlatohlavek, Lukas; Emil, Sameh; Naguib, Tarek; Reda, Ashraf; Béliard, Sophie; Bruckert, Eric; Gallo, Antonio; Elisaf, Moses S.; Kolovou, Genovefa; Cohen, Hofit; Durst, Ronen; Dann, Eldad J.; Elis, Avishay; Hussein, Osama; Leitersdorf, Eran; Schurr, Daniel; Setia, Nitika; Verma, Ishwar C.; Alareedh, Mohammed D.; Al-Khnifsawi, Mutaz; Abdalsahib Al-Zamili, Ali F.; Rhadi, Sabah H.; Shaghee, Foaad K.; Arca, Marcello; Averna, Maurizio; Bartuli, Andrea; Bucci, Marco; Buonuomo, Paola S.; Calabrò, Paolo; Calandra, Sebastiano; Casula, Manuela; Catapano, Alberico L.; Cefalù, Angelo B.; Cicero, Arrigo F. G.; D'Addato, Sergio; D'Erasmo, Laura; Di Costanzo, Alessia; Fasano, Tommaso; Gazzotti, Marta; Giammanco, Antonina; Iannuzzo, Gabriella; Ibba, Anastasia; Negri, Emanuele A.; Pasta, Andrea; Pavanello, Chiara; Pisciotta, Livia; Rabacchi, Claudio; Ripoli, Carlo; Sampietro, Tiziana; Sbrana, Francesco; Sileo, Fulvio; Suppressa, Patrizia; Tarugi, Patrizia; Trenti, Chiara; Zenti, Maria G.; Hori, Mika; Ayesh, Mahmoud H.; Azar, Sami T.; Bitar, Fadi F.; Fahed, Akl C.; Moubarak, Elie M.; Nemer, Georges; Nawawi, Hapizah M.; Madriz, Ramón; Mehta, Roopa; Cupido, Arjen J.; Defesche, Joep C.; Reijman, M. Doortje; Roeters-van Lennep, Jeanine E.; Stroes, Erik S. G.; Wiegman, Albert; Zuurbier, Linda; Al-Waili, Khalid; Sadiq, Fouzia; Chlebus, Krzysztof; Bourbon, Mafalda; Gaspar, Isabel M.; Lalic, Katarina S.; Ezhov, Marat V.; Susekov, Andrey V.; Groselj, Urh; Charng, Min-Ji; Khovidhunkit, Weerapan; Aktan, Melih; Altunkeser, Bulent B.; Demircioglu, Sinan; Kose, Melis; Gokce, Cumali; Ilhan, Osman; Kayikcioglu, Meral; Kaynar, Leyla G.; Kuku, Irfan; Kurtoglu, Erdal; Okutan, Harika; Ozcebe, Osman I.; Pekkolay, Zafer; Sag, Saim; Salcioglu, Osman Z.; Temizhan, Ahmet; Yenercag, Mustafa; Yilmaz, Mehmet; Yilmaz Yasar, Hamiyet; Mitchenko, Olena; Lyons, Alexander R. M.; Stevens, Christophe A. T.; Brothers, Julie A.; Hudgins, Lisa C.; Nguyen, Christina; Alieva, Rano; Shek, Aleksandr; Do, Doan-Loi; Kim, Ngoc-Thanh; Le, Hong-An; Le, Thanh-Tung; Nguyen, Mai-Ngoc T.; Truong, Thanh-Huong; Blom, Dirk J.; Raal, Frederick J.; Cuchel, Marina
abstract

Background Homozygous familial hypercholesterolaemia (HoFH) is a rare inherited disorder resulting in extremely elevated low-density lipoprotein cholesterol levels and premature atherosclerotic cardiovascular disease (ASCVD). Current guidance about its management and prognosis stems from small studies, mostly from high-income countries. The objective of this study was to assess the clinical and genetic characteristics, as well as the impact, of current practice on health outcomes of HoFH patients globally. Methods The HoFH International Clinical Collaborators registry collected data on patients with a clinical, or genetic, or both, diagnosis of HoFH using a retrospective cohort study design. This trial is registered with ClinicalTrials.gov, NCT04815005. Findings Overall, 751 patients from 38 countries were included, with 565 (75%) reporting biallelic pathogenic variants. The median age of diagnosis was 12·0 years (IQR 5·5–27·0) years. Of the 751 patients, 389 (52%) were female and 362 (48%) were male. Race was reported for 527 patients; 338 (64%) patients were White, 121 (23%) were Asian, and 68 (13%) were Black or mixed race. The major manifestations of ASCVD or aortic stenosis were already present in 65 (9%) of patients at diagnosis of HoFH. Globally, pretreatment LDL cholesterol levels were 14·7 mmol/L (IQR 11·6–18·4). Among patients with detailed therapeutic information, 491 (92%) of 534 received statins, 342 (64%) of 534 received ezetimibe, and 243 (39%) of 621 received lipoprotein apheresis. On-treatment LDL cholesterol levels were lower in high-income countries (3·93 mmol/L, IQR 2·6–5·8) versus non-high-income countries (9·3 mmol/L, 6·7–12·7), with greater use of three or more lipid-lowering therapies (LLT; high-income 66% vs non-high-income 24%) and consequently more patients attaining guideline-recommended LDL cholesterol goals (high-income 21% vs non-high-income 3%). A first major adverse cardiovascular event occurred a decade earlier in non-high-income countries, at a median age of 24·5 years (IQR 17·0–34·5) versus 37·0 years (29·0–49·0) in high-income countries (adjusted hazard ratio 1·64, 95% CI 1·13–2·38). Interpretation Worldwide, patients with HoFH are diagnosed too late, undertreated, and at high premature ASCVD risk. Greater use of multi-LLT regimens is associated with lower LDL cholesterol levels and better outcomes. Significant global disparities exist in treatment regimens, control of LDL cholesterol levels, and cardiovascular event-free survival, which demands a critical re-evaluation of global health policy to reduce inequalities and improve outcomes for all patients with HoFH. Funding Amsterdam University Medical Centers, Location Academic Medical Center; Perelman School of Medicine at the University of Pennsylvania; and European Atherosclerosis Society

2020 - Homozygous familial hypercholesterolemia in Italy: Clinical and molecular features. [Articolo su rivista]
Bertolini, S.; Calandra, S.; Arca, M.; Averna, M.; Catapano, A. L.; Tarugi, P.; Bartuli, A.; Bucci, M.; Buonuomo, P. S.; Calabrò, P.; Casula, M.; Cefalù, A. B.; Cicero, A.; D'Addato, S.; D'Erasmo, L.; Fasano, T.; Iannuzzo, G.; Ibba, A.; Negri, E. A.; Pasta, A.; Pavanello, C.; Pisciotta, L.; Rabacchi, C.; Ripoli, C.; Sampietro, T.; Sbrana, F.; Sileo, F.; Suppressa, P.; Trenti, C.; Zenti, M. G.
abstract

2019 - Angiopoietin-like protein 3 (ANGPTL3) deficiency and familial combined hypolipidemia [Articolo su rivista]
Tarugi, P.; Bertolini, S.; Calandra, S.
abstract

Three members of the angiopoietin-like (ANGPTL) protein family-ANGPTL3, ANGPTL4 and ANGPTL8- are important regulators of plasma lipoproteins. They inhibit the enzyme lipoprotein lipase, which plays a key role in the intravascular lipolysis of triglycerides present in some lipoprotein classes. This review focuses on the role of ANGPTL3 as emerged from the study of genetic variants of Angptl3 gene in mice and humans. Both loss of function genetic variants and inactivation of Angptl3 gene in mice are associated with a marked reduction of plasma levels of triglyceride and cholesterol and an increased activity of lipoprotein lipase and endothelial lipase. In humans with ANGPTL3 deficiency, caused by homozygous loss of function (LOF) variants of Angptl3 gene, the levels of all plasma lipoproteins are greatly reduced. This plasma lipid disorder referred to as familial combined hypolipidemia (FHBL2) does not appear to be associated with distinct pathological manifestations. Heterozygous carriers of LOF variants have reduced plasma levels of total cholesterol and triglycerides and are at lower risk of developing atherosclerotic cardiovascular disease, as compared to non-carriers. These observations have paved the way to the development of strategies to reduce the plasma level of atherogenic lipoproteins in man by the inactivation of ANGPTL3, using either a specific monoclonal antibody or anti-sense oligonucleotides.

2019 - In vitro functional characterization of splicing variants of the APOB gene found in familial hypobetalipoproteinemia [Articolo su rivista]
Rabacchi, C.; Simone, M. L.; Pisciotta, L.; Di Leo, E.; Bocchi, D.; Pietrangelo, A.; D'Addato, S.; Bertolini, S.; Calandra, S.; Tarugi, P.
abstract

Background: Familial hypobetalipoproteinemia type 1 (FHBL-1) is a codominant disorder characterized by greatly reduced plasma levels of total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B. Rare exonic pathogenic variants of APOB gene (nonsense variants, minute deletions/insertions and nonsynonymous variants) have been frequently reported in subjects with FHBL-1. Also, rare intronic variants of APOB located at intron/exon junctions and assumed to affect splicing have been reported. However, the pathogenicity of most of these intronic variants remains to be established. Objective: The objective of this study was the in vitro functional characterization of six splicing variants of APOB gene identified in seven putative FHBL-1 heterozygotes. Methods: ApoB minigenes harboring each variant were expressed in COS-1 cells and their transcripts were sequenced. Results: Four novel variants (c.237+1G>A, c.818+5G>A, c.3000-1G>T, and c.3842+1G>A), predicted in silico to obliterate splice site activity, were found to generate abnormal transcripts. The abnormal transcripts were generated by the activation of cryptic splice sites or exon skipping. All these transcripts harbored a premature termination codon and were predicted to encode truncated apoBs devoid of function. The predicted translation products were: i) p.(Lys41Serfs*2) and p.(Val80Ilefs*10) for c.237+1G>A; ii) p.(Asn274*) for c.818+5G>A; iii) p.(Leu1001Alafs*10) for c.3000-1G>T, and iv) p.(Ser1281Argfs*2) for c.3842+1G>A. Two previously annotated rare variants (c.905-15C>G and c.1618-4G>A) with uncertain effect in silico were found to generate only wild-type transcripts. Conclusions: These in vitro minigene expression studies support the assignment of pathogenicity to four novel splice site variants of APOB gene found in FHBL-1.

2019 - Novel mutations of SAR1B gene in four children with chylomicron retention disease [Articolo su rivista]
Simone, M. L.; Rabacchi, C.; Kuloglu, Z.; Kansu, A.; Ensari, A.; Demir, A. M.; Hizal, G.; Di Leo, E.; Bertolini, S.; Calandra, S.; Tarugi, P.
abstract

Background: Intestinal lipid malabsorption, resulting from an impaired formation or secretion of chylomicrons and associated with severe hypobetalipoproteinemia (HBL), may be due to biallelic mutations in APOB (homozygous FHBL type-1), MTTP (abetalipoproteinemia), or SAR1B (chylomicron retention disease). Objective: We investigated four children, each born from consanguineous parents, presenting with steatorrhea, malnutrition, accumulation of lipids in enterocytes, and severe hypocholesterolemia with an apparent recessive transmission. Methods: We sequenced a panel of genes whose variants may be associated with HBL. Results: Case 1, a 9-month-old male, was found to be homozygous for a SAR1B variant (c.49 C>T), predicted to encode a truncated Sar1b protein devoid of function (p.Gln17*). Case 2, a 4-year-old male, was found to be homozygous for a SAR1B missense variant [c.409 G>C, p.(Asp137His)], which affects a highly conserved residue close to the Sar1b guanosine recognition site. Case 3, a 6-year-old male, was found to be homozygous for an ∼6 kb deletion of the SAR1B gene, which eliminates exon 2; this deletion causes the loss of the ATG translation initiation codon in the SAR1B mRNA. The same homozygous mutation was found in an 11-month-old child (case 4) who was related to case 3. Conclusions: We report 4 children with intestinal lipid malabsorption were found to have chylomicron retention disease due to 3 novel variants in the SAR1B gene.

2018 - Evaluation of the performance of Dutch Lipid Clinic Network score in an Italian FH population: The LIPIGEN study [Articolo su rivista]
Casula, Manuela; Olmastroni, Elena; Pirillo, Angela; Catapano, Alberico Luigi; Arca, Marcello; Averna, Maurizio; Bertolini, Stefano; Calandra, Sebastiano; Tarugi, Patrizia; Pellegatta, Fabio; Angelico, Francesco; Bartuli, Andrea; Biasucci, Giacomo; Biolo, Gianni; Bonanni, Luca; Bonomo, Katia; Borghi, Claudio; Bossi, Antonio Carlo; Branchi, Adriana; Carubbi, Francesca; Cipollone, Francesco; Citroni, Nadia; Federici, Massimo; Ferri, Claudio; Fiorenza, Anna Maria; Giaccari, Andrea; Giorgino, Francesco; Guardamagna, Ornella; Iannuzzi, Arcangelo; Iughetti, Lorenzo; Lupattelli, Graziana; Lupi, Alessandro; Mandraffino, Giuseppe; Marcucci, Rossella; Maroni, Lorenzo; Miccoli, Roberto; Mombelli, Giuliana; Muntoni, Sandro; Pecchioli, Valerio; Pederiva, Cristina; Pipolo, Antonio; Pisciotta, Livia; Pujia, Arturo; Purrello, Francesco; Repetti, Elena; Rubba, Paolo; Sabbà, Carlo; Sampietro, Tiziana; Sarzani, Riccardo; Tagliabue, Milena Paola; Trenti, Chiara; Vigna, Giovanni Battista; Werba, Josè Pablo; Zambon, Sabina; Zenti, Maria Grazia; Minicocci, Ilenia; Noto, Davide; Fortunato, Giuliana; Banderali, Giuseppe; Benso, Andrea; Bigolin, Paola; Bonora, Enzo; Bruzzi, Patrizia; Bucci, Marco; Buonuomo, Paola Sabrina; Capra, Maria Elena; Cardolini, Iris; Cefalù, Baldassarre; Cervelli, Nazzareno; Chiariello, Giuseppe; Cocci, Guido; Colombo, Emanuela; Cremonini, Anna Laura; D'addato, Sergio; D'erasmo, Laura; Dal Pino, Beatrice; De Sanctis, Luisa; De Vita, Emanuele; Del Ben, Maria; Di Costanzo, Alessia; Di Taranto, Maria Donata; Fasano, Tommaso; Gentile, Luigi; Gentile, Marco; Ghirardello, Omar; Grigore, Liliana; Lussu, Milena; Meregalli, Giancarla; Moffa, Simona; Montalcini, Tiziana; Morgia, Valeria; Nascimbeni, Fabio; Pasta, Andrea; Pavanello, Chiara; Saitta, Antonino; Scicali, Roberto; Siepi, Donatella; Spagnolli, Walter; Spina, Rossella; Sticchi, Elena; Suppressa, Patrizia; Vigo, Lorenzo; Vinci, Pierandrea; Manzato, Enzo; Tragni, Elena; Zampoleri, Veronica
abstract

Background and aims: Familial hypercholesterolemia (FH) is an inherited disorder characterized by high levels of blood cholesterol from birth and premature coronary heart disease. Thus, the identification of FH patients is crucial to prevent or delay the onset of cardiovascular events, and the availability of a tool helping with the diagnosis in the setting of general medicine is essential to improve FH patient identification. Methods: This study evaluated the performance of the Dutch Lipid Clinic Network (DLCN) score in FH patients enrolled in the LIPIGEN study, an Italian integrated network aimed at improving the identification of patients with genetic dyslipidaemias, including FH. Results: The DLCN score was applied on a sample of 1377 adults (mean age 42.9 ± 14.2 years) with genetic diagnosis of FH, resulting in 28.5% of the sample classified as probable FH and 37.9% as classified definite FH. Among these subjects, 43.4% had at least one missing data out of 8, and about 10.0% had 4 missing data or more. When analyzed based on the type of missing data, a higher percentage of subjects with at least 1 missing data in the clinical history or physical examination was classified as possible FH (DLCN score 3–5). We also found that using real or estimated pre-treatment LDL-C levels may significantly modify the DLCN score. Conclusions: Although the DLCN score is a useful tool for physicians in the diagnosis of FH, it may be limited by the complexity to retrieve all the essential information, suggesting a crucial role of the clinical judgement in the identification of FH subjects.

2018 - Overview of the current status of familial hypercholesterolaemia care in over 60 countries - The EAS Familial Hypercholesterolaemia Studies Collaboration (FHSC) [Articolo su rivista]
Vallejo-Vaz, A. J.; Marco, M. D.; Stevens, C. A. T.; Akram, A.; Freiberger, T.; Hovingh, G. K.; Kastelein, J. J. P.; Mata, P.; Raal, F. J.; Santos, R. D.; Soran, H.; Watts, G. F.; Abifadel, M.; Aguilar-Salinas, C. A.; Al-Khnifsawi, M.; Alkindi, F. A.; Alnouri, F.; Alonso, R.; Al-Rasadi, K.; Al-Sarraf, A.; Ashavaid, T. F.; Binder, C. J.; Bogsrud, M. P.; Bourbon, M.; Bruckert, E.; Chlebus, K.; Corral, P.; Descamps, O.; Durst, R.; Ezhov, M.; Fras, Z.; Genest, J.; Groselj, U.; Harada-Shiba, M.; Kayikcioglu, M.; Lalic, K.; Lam, C. S. P.; Latkovskis, G.; Laufs, U.; Liberopoulos, E.; Lin, J.; Maher, V.; Majano, N.; Marais, A. D.; Marz, W.; Mirrakhimov, E.; Miserez, A. R.; Mitchenko, O.; Nawawi, H. M.; Nordestgaard, B. G.; Paragh, G.; Petrulioniene, Z.; Pojskic, B.; Postadzhiyan, A.; Reda, A.; Reiner, Z.; Sadoh, W. E.; Sahebkar, A.; Shehab, A.; Shek, A. B.; Stoll, M.; Su, T. -C.; Subramaniam, T.; Susekov, A. V.; Symeonides, P.; Tilney, M.; Tomlinson, B.; Truong, T. -H.; Tselepis, A. D.; Tybjaerg-Hansen, A.; Vazquez-Cardenas, A.; Viigimaa, M.; Vohnout, B.; Widen, E.; Yamashita, S.; Banach, M.; Gaita, D.; Jiang, L.; Nilsson, L.; Santos, L. E.; Schunkert, H.; Tokgozoglu, L.; Car, J.; Catapano, A. L.; Ray, K. K.; Schreier, L.; Pang, J.; Dieplinger, H.; Hanauer-Mader, G.; Desutter, J.; Langlois, M.; Mertens, A.; Rietzschel, E.; Wallemacq, C.; Isakovic, D.; Dzankovic, A. M.; Obralija, J.; Pojskic, L.; Sisic, I.; Stimjanin, E.; Torlak, V. A.; Jannes, C. E.; Krieger, J. E.; Pereira, A. C.; Ruel, I.; Asenjo, S.; Cuevas, A.; Pecin, I.; Miltiadous, G.; Panayiotou, A. G.; Vrablik, M.; Benn, M.; Heinsar, S.; Beliard, S.; Gouni-Berthold, I.; Hengstenberg, W.; Julius, U.; Kassner, U.; Klose, G.; Konig, C.; Konig, W.; Otte, B.; Parhofer, K.; Schatz, U.; Schmidt, N.; Steinhagen-Thiessen, E.; Vogt, A.; Antza, C.; Athyros, V.; Bilianou, E.; Boufidou, A.; Chrousos, G.; Elisaf, M.; Garoufi, A.; Katsiki, N.; Kolovou, G.; Kotsis, V.; Rallidis, L.; Rizos, C.; Skalidis, E.; Skoumas, I.; Tziomalos, K.; Shawney, J. P. S.; Abbaszadegan, M. R.; Aminzadeh, M.; Hosseini, S.; Mobini, M.; Vakili, R.; Zaeri, H.; Agar, R.; Boran, G.; Colwell, N.; Crowley, V.; Durkin, M.; Griffin, D.; Kelly, M.; Rakovac-Tisdall, A.; Bitzur, R.; Cohen, H.; Eliav, O.; Ellis, A.; Gavish, D.; Harats, D.; Henkin, Y.; Knobler, H.; Leavit, L.; Leitersdorf, E.; Schurr, D.; Shpitzen, S.; Szalat, A.; Arca, M.; Averna, M.; Bertolini, S.; Calandra, S.; Tarugi, P.; Erglis, A.; Gilis, D.; Nesterovics, G.; Saripo, V.; Upena-Roze, A.; Elbitar, S.; Jambart, S.; Khoury, P. E.; Gargalskaite, U.; Kutkiene, S.; Al-Khateeb, A.; An, C. Y.; Ismail, Z.; Kasim, S.; Ibrahim, K. S.; Radzi, A. B. M.; Kasim, N. A.; Nor, N. S. M.; Ramli, A. S.; Razak, S. A.; Muid, S.; Rosman, A.; Sanusi, A. R.; Razman, A. Z.; Nazli, S. A.; Kek, T. L.; Azzopardi, C.; Aguilar Salinas, C. A.; Galan, G.; Rubinstein, A.; Magana-Torres, M. T.; Martagon, A.; Mehta, R.; Wittekoek, M. E.; Isara, A. R.; Obaseki, D. E.; Ohenhen, O. A.; Holven, K. B.; Gruchala, M.; Baranowska, M.; Borowiec-Wolny, J.; Gilis-Malinowska, N.; Michalska-Grzonkowska, A.; Pajkowski, M.; Parczewska, A.; Romanowska-Kocejko, M.; Strozyk, A.; Zarczynska-Buchowiecka, M.; Kleinschmidt, M.; Alves, A. C.; Medeiros, A. M.; Ershova, A.; Korneva, V.; Kuznetsova, T.; Malyshev, P.; Meshkov, A.; Rozhkova, T.; Popovic, L.; Lukac, S. S.; Stosic, L.; Rasulic, I.; Lalic, N. M.; Chua, T. S. J.; Ting, S. P. L.; Raslova, K.; Battelino, T.; Cevc, M.; Jug, B.; Kovac, J.; Podkrajsek, K. T.; Sustar, U.; Trontelj, K. J.; Marais, D.; Isla, L. P.; Martin, F. J.; Charng, M. -J.; Chen, P. -L.; Kayikcioglu, M.; Dell'Oca, N.; Fernandez, G.; Ressia, A.; Reyes, X.; Zelarayan, M.; Alieva, R. B.; Hoshimov, S. U.; Nizamov, U. I.; Kurbanov, R. D.; Lima-Martinez, M. M.; Nguyen, M. -N. T.; Do, D. -L.; Kim, N. -T.; Le, T. -T.; Le, H. -A.
abstract

Background and aims: Management of familial hypercholesterolaemia (FH) may vary across different settings due to factors related to population characteristics, practice, resources and/or policies. We conducted a survey among the worldwide network of EAS FHSC Lead Investigators to provide an overview of FH status in different countries. Methods: Lead Investigators from countries formally involved in the EAS FHSC by mid-May 2018 were invited to provide a brief report on FH status in their countries, including available information, programmes, initiatives, and management. Results: 63 countries provided reports. Data on FH prevalence are lacking in most countries. Where available, data tend to align with recent estimates, suggesting a higher frequency than that traditionally considered. Low rates of FH detection are reported across all regions. National registries and education programmes to improve FH awareness/knowledge are a recognised priority, but funding is often lacking. In most countries, diagnosis primarily relies on the Dutch Lipid Clinics Network criteria. Although available in many countries, genetic testing is not widely implemented (frequent cost issues). There are only a few national official government programmes for FH. Under-treatment is an issue. FH therapy is not universally reimbursed. PCSK9-inhibitors are available in ∼2/3 countries. Lipoprotein-apheresis is offered in ∼60% countries, although access is limited. Conclusions: FH is a recognised public health concern. Management varies widely across countries, with overall suboptimal identification and under-treatment. Efforts and initiatives to improve FH knowledge and management are underway, including development of national registries, but support, particularly from health authorities, and better funding are greatly needed.

2017 - CASO CLINICO: QUANDO LA RISPOSTA ALLA DIETA IPOLIPIDEMIZZANTE DETERMINA LA DIAGNOSI [Abstract in Atti di Convegno]
Bruzzi, Patrizia; Predieri, Barbara; Madeo, Simona Filomena; Cattini, Umberto; Rabacchi, Claudio; Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; Iughetti, Lorenzo
abstract

PRESENTAZIONE DEL CASO, STORIA CLINICA E SINTOMATOLOGIA G.M., maschio di 7.8 anni, è stato condotto presso il centro di Dislipidemie in età evolutiva per il riscontro occasionale di ipercolesterolemia [colesterolo totale (CT) 524 mg/dl, LDL-colesterolo (LDL-C) 412 mg/dl, HDL-colesterolo (HDL-C) 52 mg/dl, trigliceridi (TG) 55 mg/dl, Apolipoproteina A (ApoA) 104 mg/dl, ApoB100 253 mg/dl]. Obiettivamente: altezza -2.02 SDS e BMI -1.40 SDS; non riscontro clinico di xantomi e/o xantelasmi, arco corneale o splenomegalia. Anamnesi patologica remota: muta. Anamnesi familiare: positiva per moderata ipercolesterolemia paterna (CT 242 mg/dl), obesità ed ipertensione. IPOTESI DIAGNOSTICHE E INDAGINI DI I E II LIVELLO Previa esclusione di una forma di ipercolesterolemia secondaria (glicemia, funzionalità tiroidea ed epato-renale nella norma), nel sospetto di ipercolesterolemia familiare omozigote abbiamo eseguito l’analisi genetica dei geni LDL-R ed ARH, risultata negativa per mutazioni. L’ecocardiografi a non documentava alterazioni. DIAGNOSI ED EVENTUALE TERAPIA In considerazione dell’iniziale assetto lipidico, abbiamo posto immediata indicazione di seguire una dieta ipolipidemizzante come da National Cholesterol Education Program ATP III - STEP II (apporto di grassi totali pari al 30% delle kcal quotidiane, grassi saturi < 7% e colesterolo < 200 mg/die). La rivalutazione dell’assetto lipidico, eseguita a distanza di 6 mesi dall’inizio della dietoterapia esclusiva, ha documentato un importante e rapido miglioramento dei valori di CT (203 mg/dl; -39%) e LDL-C (141 mg/dl; -34%). Per questo motivo abbiamo posto il sospetto diagnostico di sitosterolemia. La sitosterolemia rappresenta un disordine raro (circa 80-100 casi descritti nel mondo) a trasmissione autosomica recessiva caratterizzato da iperassorbimento intestinale e ridotta escrezione biliare di steroli vegetali. Nel nostro paziente abbiamo, quindi, dosato le concentrazioni di steroli vegetali plasmatici (mg/L) che sono risultate elevate (betasitosterolo 228, campesterolo 77.9 e desmosterolo 2.13) ed eseguito l’analisi genetica del gene ABCG8 che ha confermato il sospetto diagnostico, identifi cando la presenza di 2 mutazioni non-senso (esone 3: c.320C>G, p.Ser107*; esone 7: c.1083 G>A, p.Trp361*) come da eterozigosi composta. In considerazione della diagnosi di sitosterolemia confermata geneticamente, abbiamo posto indicazione a seguire un approccio dietetico con restrizione di cibi ad alto contenuto di steroli (olii vegetali, margarina, frutta secca, avocado, cioccolato…) associato ad un’appropriata supplementazione vitaminica.

2017 - Familial hypercholesterolemia: The Italian Atherosclerosis Society Network (LIPIGEN) [Articolo su rivista]
Averna, Maurizio; Cefalã¹, Angelo B.; Casula, Manuela; Noto, Davide; Arca, Marcello; Bertolini, Stefano; Calandra, Sebastiano; Catapano, Alberico L.; Tarugi, Patrizia; Arca, Marcello; Averna, Maurizio; Bertolini, Stefano; Calandra, Sebastiano; Catapano, Alberico Luigi; Tarugi, Patrizia; Pellegatta, Fabio; Angelico, Francesco; Arca, Marcello; Averna, Maurizio; Bartuli, Andrea; Biasucci, Giacomo; Biolo, Gianni; Bonanni, Luca; Bonomo, Katia; Borghi, Claudio; Bossi, Antonio Carlo; Branchi, Adriana; Carubbi, Francesca; Cipollone, Francesco; Citroni, Nadia; Federici, Massimo; Ferri, Claudio; Fiorenza, Anna Maria; Giaccari, Andrea; Giorgino, Francesco; Guardamagna, Ornella; Iannuzzi, Arcangelo; Iughetti, Lorenzo; Lupattelli, Graziana; Mandraffino, Giuseppe; Marcucci, Rossella; Mombelli, Giuliana; Muntoni, Sandro; Pecchioli, Valerio; Pederiva, Cristina; Pipolo, Antonio; Pisciotta, Livia; Pujia, Arturo; Purrello, Francesco; Repetti, Elena; Rubba, Paolo; Sabbã , Carlo; Sampietro, Tiziana; Sarzani, Riccardo; Tagliabue, Milena Paola; Trenti, Chiara; Vigna, Giovanni Battista; Werba, Josà Pablo; Zambon, Sabina; Zenti, Maria Grazia; Montali, Anna; Noto, Davide; Bertolini, Stefano; Calandra, Sebastiano; Fortunato, Giuliana; Grigore, Liliana; Del Ben, Maria; Maranghi, Marianna; Cefalã¹, Angelo B.; Barbagallo, Carlo M.; Buonuomo, Paola Sabrina; Capra, Maria Elena; Vinci, Pierandrea; D'Addato, Sergio; Galbiati, Stella; Nascimbeni, Fabio; Bucci, Marco; Spagnoli, Walter; Cardolini, Iris; Cervelli, Nazzareno; Emanuela, Colombo; Vinsin, A. Sun; Laviola, Luigi; Bello, Francesca; Chiariello, Giuseppe; Predieri, Barbara; Siepi, Donatella; Saitta, Antonino; Giusti, Betti; Pavanello, Chiara; Lussu, Milena; Prati, Lucia; Banderali, Giuseppe; Balleari, Giulia; Montalcini, Tiziana; Scicali, Roberto; Gentile, Luigi; Gentile, Marco; Suppressa, Patrizia; Sbrana, Francesco; Cocci, Guido; Benso, Andrea; Negri, Emanuele Alberto; Ghirardello, Omar; Lorenzo, Vigo; Zambon, Alberto; Enzo, Bonora; Minicocci, Ilenia; Spina, Rossella; Orlando, Camilla; Tarugi, Patrizia; Di Taranto, Maria Donata; Catapano, Alberico Luigi; Casula, Manuela; Chiodo, Lorenzo; Garlaschelli, Katia; Manzato, Enzo; Tragni, Elena
abstract

Background and aims Primary dyslipidemias are a heterogeneous group of disorders characterized by abnormal levels of circulating lipoproteins. Among them, familial hypercholesterolemia is the most common lipid disorder that predisposes for premature cardiovascular disease. We set up an Italian nationwide network aimed at facilitating the clinical and genetic diagnosis of genetic dyslipidemias named LIPIGEN (LIpid TransPort Disorders Italian GEnetic Network). Methods Observational, multicenter, retrospective and prospective study involving about 40 Italian clinical centers. Genetic testing of the appropriate candidate genes at one of six molecular diagnostic laboratories serving as nationwide DNA diagnostic centers. Results and conclusions From 2012 to October 2016, available biochemical and clinical information of 3480 subjects with familial hypercholesterolemia identified according to the Dutch Lipid Clinic Network (DLCN) score were included in the database and genetic analysis was performed in 97.8% of subjects, with a mutation detection rate of 92.0% in patients with DLCN score ≥6. The establishment of the LIPIGEN network will have important effects on clinical management and it will improve the overall identification and treatment of primary dyslipidemias in Italy.

2017 - Impact of rare variants in autosomal dominant hypercholesterolemia causing genes. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Bertolini, Stefano
abstract

PURPOSE OF REVIEW: The systematic analysis of the major candidate genes in autosomal dominant hypercholesterolemia (ADH) and the use of next-generation sequencing (NGS) technology have made possible the discovery of several rare gene variants whose pathogenic effect in most cases remains poorly defined. RECENT FINDINGS: One major advance in the field has been the adoption of a set of international guidelines for the assignment of pathogenicity to low-density lipoprotein receptor (LDLR) gene variants based on the use of softwares, complemented with data available from literature and public databases. The clinical impact of several novel rare variants in LDLR, APOB, PCSK9, APOE genes have been reported in large studies describing patients with ADH found to be homozygotes/compound heterozygotes, double heterozygotes, or simple heterozygotes. In-vitro functional studies have been conducted to clarify the effect of some rare ApoB variants on LDL binding to LDLR and the impact of a rare ApoE variant on the uptake of VLDL and LDL by hepatocytes. SUMMARY: The update of the ADH gene variants database and the classification of variants in categories of pathogenicity is a major advance in the understanding the pathophysiology of ADH and in the management of this disorder. The studies of molecularly characterized patients with ADH have emphasized the impact of a specific variant and the variable clinical expression of different genotypes. The functional studies of some variants have increased our understanding of the molecular bases of some forms of ADH.

2017 - Incidental finding of severe hypertriglyceridemia in children. Role of multiple rare variants in genes affecting plasma triglyceride. [Articolo su rivista]
Buonuomo, Ps; Rabacchi, C; Macchiaiolo, M; Trenti, C; Fasano, T; Tarugi, P; Bartuli, A; Bertolini, S; Calandra, S.
abstract

BACKGROUND: The incidental finding of severe hypertriglyceridemia (HyperTG) in a child may suggest the diagnosis of familial chylomicronemia syndrome (FCS), a recessive disorder of the intravascular hydrolysis of triglyceride (TG)-rich lipoproteins. FCS may be due to pathogenic variants in lipoprotein lipase (LPL), as well as in other proteins, such as apolipoprotein C-II and apolipoprotein A-V (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface) and LMF1 (a factor required for intracellular formation of active LPL). OBJECTIVE: Molecular characterization of 5 subjects in whom HyperTG was an incidental finding during infancy/childhood. METHODS: We performed the parallel sequencing of 20 plasma TG-related genes. RESULTS: Three children with severe HyperTG were found to be compound heterozygous for rare pathogenic LPL variants (2 nonsense, 3 missense, and 1 splicing variant). Another child was found to be homozygous for a nonsense variant of APOA5, which was also found in homozygous state in his father with longstanding HyperTG. The fifth patient with a less severe HyperTG was found to be heterozygous for a frameshift variant in LIPC resulting in a truncated Hepatic Lipase. In addition, 1 of the patients with LPL deficiency and the patient with APOA-V deficiency were also heterozygous carriers of a pathogenic variant in LIPC and LPL gene, respectively, whereas the patient with LIPC variant was also a carrier of a rare APOB missense variant. CONCLUSIONS: Targeted parallel sequencing of TG-related genes is recommended to define the molecular defect in children presenting with an incidental finding of HyperTG.

2017 - Spectrum of mutations in Italian patients with familial hypercholesterolemia: New results from the LIPIGEN study [Articolo su rivista]
Pirillo, Angela; Garlaschelli, Katia; Arca, Marcello; Averna, Maurizio; Bertolini, Stefano; Calandra, Sebastiano; Tarugi, Patrizia; Catapano, Alberico L.; Arca, Marcello; Averna, Maurizio; Bertolini, Stefano; Calandra, Sebastiano; Catapano, Alberico Luigi; Tarugi, Patrizia; Pellegatta, Fabio; Angelico, Francesco; Arca, Marcello; Averna, Maurizio; Bartuli, Andrea; Biasucci, Giacomo; Biolo, Gianni; Bonanni, Luca; Bonomo, Katia; Borghi, Claudio; Bossi, Antonio Carlo; Branchi, Adriana; Carubbi, Francesca; Cipollone, Francesco; Citroni, Nadia; Federici, Massimo; Ferri, Claudio; Fiorenza, Anna Maria; Giaccari, Andrea; Giorgino, Francesco; Guardamagna, Ornella; Iannuzzi, Arcangelo; Iughetti, Lorenzo; Lupattelli, Graziana; Mandraffino, Giuseppe; Marcucci, Rossella; Mombelli, Giuliana; Muntoni, Sandro; Pecchioli, Valerio; Pederiva, Cristina; Pipolo, Antonio; Pisciotta, Livia; Pujia, Arturo; Purrello, Francesco; Repetti, Elena; Rubba, Paolo; SabbÃ&nbsp;, Carlo; Sampietro, Tiziana; Sarzani, Riccardo; Tagliabue, Milena Paola; Trenti, Chiara; Vigna, Giovanni Battista; Werba, Josà Pablo; Zambon, Sabina; Zenti, Maria Grazia; Montali, Anna; Noto, Davide; Bertolini, Stefano; Calandra, Sebastiano; Fortunato, Giuliana; Grigore, Liliana; Del Ben, Maria; Maranghi, Marianna; Cefalù, A. Baldassarre; Buonuomo, Paola Sabrina; Capra, Maria Elena; Vinci, Pierandrea; D'Addato, Sergio; Galbiati, Stella; Nascimbeni, Fabio; Bucci, Marco; Spagnoli, Walter; Cardolini, Iris; Cervelli, Nazzareno; Emanuela, Colombo; Sun, Vinsin A.; Laviola, Luigi; Bello, Francesca; Chiariello, Giuseppe; Predieri, Barbara; Siepi, Donatella; Saitta, Antonino; Giusti, Betti; Pavanello, Chiara; Lussu, Milena; Prati, Lucia; Banderali, Giuseppe; Balleari, Giulia; Montalcini, Tiziana; Scicali, Roberto; Gentile, Luigi; Gentile, Marco; Suppressa, Patrizia; Sbrana, Francesco; Cocci, Guido; Benso, Andrea; Negri, Emanuele Alberto; Ghirardello, Omar; Lorenzo, Vigo; Zambon, Alberto; Enzo, Bonora; Minicocci, Ilenia; Spina, Rossella; Orlando, Camilla; Tarugi, Patrizia; Di Taranto, Maria Donata; Catapano, Alberico Luigi; Casula, Manuela; Chiodo, Lorenzo; Garlaschelli, Katia; Manzato, Enzo; Tragni, Elena
abstract

Background Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by elevated plasma levels of LDL-cholesterol that confers an increased risk of premature atherosclerotic cardiovascular disease. Early identification and treatment of FH patients can improve prognosis and reduce the burden of cardiovascular mortality. Aim of this study was to perform the mutational analysis of FH patients identified through a collaboration of 20 Lipid Clinics in Italy (LIPIGEN Study). Methods We recruited 1592 individuals with a clinical diagnosis of definite or probable FH according to the Dutch Lipid Clinic Network criteria. We performed a parallel sequencing of the major candidate genes for monogenic hypercholesterolemia (LDLR, APOB, PCSK9, APOE, LDLRAP1, STAP1). Results A total of 213 variants were detected in 1076 subjects. About 90% of them had a pathogenic or likely pathogenic variants. More than 94% of patients carried pathogenic variants in LDLR gene, 27 of which were novel. Pathogenic variants in APOB and PCSK9 were exceedingly rare. We found 4 true homozygotes and 5 putative compound heterozygotes for pathogenic variants in LDLR gene, as well as 5 double heterozygotes for LDLR/APOB pathogenic variants. Two patients were homozygous for pathogenic variants in LDLRAP1 gene resulting in autosomal recessive hypercholesterolemia. One patient was found to be heterozygous for the ApoE variant p.(Leu167del), known to confer an FH phenotype. Conclusions This study shows the molecular characteristics of the FH patients identified in Italy over the last two years. Full phenotypic characterization of these patients and cascade screening of family members is now in progress.

2017 - The study of familial hypercholesterolemia in Italy: A narrative review. [Articolo su rivista]
Bertolini, S.; Pisciotta, L.; Fasano, T.; Rabacchi, C.; Calandra, S.
abstract

2016 - Clinical and genetic features of 3 patients with familial chylomicronemia due to mutations in GPIHBP1 gene [Articolo su rivista]
Rabacchi, Claudio; D'Addato, Sergio; Palmisano, Silvia; Lucchi, Tiziano; Bertolini, Stefano; Calandra Buonaura, Sebastiano; Tarugi, Patrizia Maria
abstract

BACKGROUND: Familial chylomicronemia is a recessive disorder that may be due to mutations in lipoprotein lipase (LPL) and in other proteins such as apolipoprotein C-II and apolipoprotein A-V (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface), and LMF1 (a factor required for intracellular formation of active LPL). METHODS: We sequenced the familial chylomicronemia candidate genes in 2 adult females presenting long-standing hypertriglyceridemia and a history of acute pancreatitis. RESULTS: Both probands had plasma triglyceride .10 mmol/L but no mutations in the LPL gene. The sequence of the other candidate genes showed that one patient was homozygous for a novel missense mutation p.(Cys83Arg), and the other was homozygous for a previously reported nonsense mutation p.(Cys 89*), respectively, in GPIHBP1. Family screening showed that the hypertriglyceridemic brother of the p.(Cys83Arg) homozygote was also homozygous for this mutation. He had no history of pancreatitis. The p.(Cys83Arg) heterozygous carriers had normal triglyceride levels. The substitution of a cysteine residue in the Ly6 domain of GPIHBP1 is predicted to abolish one of the disulfide bridges required to maintain the structure of GPIHBP1. The p.(Cys89*) mutation results in a truncated protein devoid of function. CONCLUSIONS: Both mutant GPIHBP1 proteins are expected to be incapable of transferring LPL from the subendothelial space to the endothelial surface.

2016 - Erratum: Corrigendum to “Molecular diagnosis of hypobetalipoproteinemia: An ENID review” (Atherosclerosis (2007) 192(2) (19–27)(S0021915007003280)(10.1016/j.atherosclerosis.2007.05.003)) [Articolo su rivista]
Tarugi, P.; Averna, M.; Di Leo, E.; Cefalu, A. B.; Noto, D.; Magnolo, L.; Cattin, L.; Bertolini, S.; Calandra, S.
abstract

The authors regret an error in the previously reported result concerning the nomenclature of a mutation of APOB gene detected in hypocholesterolemic blood donors. In the text on page e24 in the paragraph 6. ApoB truncations in hypocholeserolemic subjects from the general population we wrote “a carrier of a 6 nucleotide deletion in exon 3 (c.158_163del6) which eliminates two amino acids (threonine at position 26 and tyrosine at position 27 of the mature protein) and introduces an aspartic acid residue with no disruption of the reading frame (Supplementary Table&nbsp;2 available on line)”. The correct sentence is “a carrier of a six nucleotide deletion in exon 3 (c.158_163del6) which eliminates two amino acids (threonine at position 26 and tyrosine at position 27 of the mature protein) with no disruption of the reading frame (Supplementary Table&nbsp;2 available on line)”. In the appendix A, Supplementary data, we reported in the first row of Table&nbsp;2 entitled “Supplementary Table&nbsp;2. ApoB amino acid changes in hypocholesterolemic blood donors” under the heading “effect on ApoB” the nomenclature Thr26_27delinsAsn. On re-analysis we found that the nomenclature was erroneously reported. The correct designation of the mutation is the following: p.(Thr53_Tyr54del) (Thr26_Tyr27del in the mature protein).

2015 - A 3-day-old neonate with severe hypertriglyceridemia from novel mutations of the GPIHBP1 gene [Articolo su rivista]
Buonuomo, P. S.; Bartuli, A.; Rabacchi, C.; Bertolini, S.; Calandra, S.
abstract

Background Familial chylomicronemia is a genetic defect of the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. Intravascular lipolysis involves the TG-hydrolase lipoprotein lipase (LPL) as well as other factors such as apolipoprotein CII and apolipoprotein AV (activators of LPL), GPIHBP1 (the molecular platform required for LPL activity on endothelial surface), and LMF1 (a factor required for intracellular formation of active LPL). Methods We sequenced the familial chylomicronemia candidate genes in a neonate with chylomicronemia. Results A 3-day-old newborn was found to have chylomicronemia (plasma TG 18.8 mmol/L, 1.667 mg/dL). The discontinuation of breastfeeding for 24 hours reduced plasma TG to 2.3 mmol/L (201 mg/dL), whereas its resumption induced a sharp TG increase (7.9 mmol/L, 690 mg/dL). The child was switched to a low-fat diet, which was effective in maintaining TG level below 3.5 mmol/L (294 mg/dL) during the first months of life. The child was found to be a compound heterozygous for 2 novel mutations in GPIHBP1 gene. The first mutation was a 9-bp deletion and 4-bp insertion in exon 2, causing a frameshift that abolished the canonical termination codon TGA. The predicted translation product of the mutant messenger RNA is a peptide that contains 51 amino acids of the N-terminal end of the wild-type protein followed by 252 novel amino acids. The second mutation was a nucleotide change (c.319T>C), causing an amino acid substitution p.(Ser107Pro) predicted in silico to be damaging. Conclusions GPIHBP1 mutations should be considered in neonates with chylomicronemia negative for mutations in LPL gene.

2015 - Spectrum of mutations of the LPL gene identified in Italy in patients with severe hypertriglyceridemia [Articolo su rivista]
Rabacchi, Claudio; Pisciotta, Livia; Cefalù, Angelo B.; Noto, Davide; Fresa, Raffaele; Tarugi, Patrizia Maria; Averna, Maurizio; Bertolini, Stefano; CALANDRA BUONAURA, Sebastiano
abstract

Monogenic hypertriglyceridemia (HTG) may result from mutations in some genes which impair the intravascular lipolysis of triglyceride (TG)-rich lipoproteins mediated by the enzyme Lipoprotein lipase (LPL). Mutations in the LPL gene are the most frequent cause of monogenic HTG (familial chylomicronemia) with recessive transmission.

2014 - A man with low cholesterol and weakness of the lower limbs. [Articolo su rivista]
Lucchi, T.; Calandra, S.; Rabacchi, C.; Conti, G.; Ardolino, G.; Assolari, L.; Arosio, B.; Vergani, C.
abstract

2014 - Effects of miglustat treatment in a patient affected by an atypical form of Tangier disease. [Articolo su rivista]
Sechi, A.; Dardis, A.; Zampieri, S.; Rabacchi, C.; Zanoni, P.; Calandra, S.; De Maglio, G.; Pizzolitto, S.; Maruotti, V.; Di Muzio, A.; Platt, F.; Bembi, B.
abstract

2014 - Severe hypertriglyceridemia in a newborn with monogenic lipoprotein lipase deficiency: an unconventional therapeutic approach with exchange transfusion. [Articolo su rivista]
Pugni, L.; Riva, E.; Pietrasanta, C.; Rabacchi, C.; Bertolini, S.; Pederiva, C.; Mosca, F.; Calandra, S.
abstract

Severe hypertriglyceridemia (sHTG) (plasma triglyceride level &gt; 10 mmol/L) due to lipoprotein lipase (LPL) deficiency is a known risk factor for acute pancreatitis. A 23-day-old male with sHTG was admitted to the Neonatal Intensive Care Unit for plasmapheresis being at high risk for acute pancreatitis. Given the potential hazard of an extracorporeal technique in a very young infant, we decided to perform an exchange transfusion (ET), a procedure widely used by neonatologists and less invasive than plasmapheresis. ET led to a dramatic reduction in plasma triglyceride level, from 93.2 to 3.8 mmol/L at the end of the procedure, without adverse events. The subsequent administration of a special formula low in fat and high in medium-chain triglycerides was effective in keeping fasting plasma triglyceride level below 5.6 mmol/L during the first 5 months of life. The sequence of LPL gene revealed that the patient was apparently homozygous for a novel nucleotide deletion (c.840delG) in exon 6 leading to a premature termination codon (p.N281Mfs*23). However, family studies revealed that while the patient's mother was heterozygous for this mutation, the father was heterozygous for a novel deletion eliminating the whole LPL gene. The patient therefore turned out to be a compound heterozygous for two LPL gene mutations predicted to abolish LPL activity. This is the first case of sHTG treated with ET in a neonate reported in the literature. ET appears to be a safe procedure, alternative to plasmapheresis, to prevent acute pancreatitis in young infants with sHTG due to LPL deficiency.

2013 - Clinical characteristics and plasma lipids in subjects with familial combined hypolipidemia: a pooled analysis. [Articolo su rivista]
Minicocci, I; Santini, S; Cantisani, V; Stitziel, N; Kathiresan, S; Arroyo, Ja; Martí, G; Pisciotta, L; Noto, D; Cefalù, Ab; Maranghi, M; Labbadia, G; Pigna, G; Pannozzo, F; Ceci, F; Ciociola, E; Bertolini, S; CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Averna, M; Arca, M.
abstract

Angiopoietin-like 3 (ANGPTL3) regulates lipoprotein metabolism by modulating extracellular lipases. Loss-of function mutations in ANGPTL3 gene cause familial combined hypolipidemia (FHBL2). The mode of inheritance and hepatic and vascular consequences of FHBL2 have not been fully elucidated. To get further insights on these aspects, we reevaluated the clinical and the biochemical characteristics of all reported cases of FHBL2. One hundred fifteen FHBL2 individuals carrying 13 different mutations in the ANGPTL3 gene (14 homozygotes, 8 compound heterozygotes, and 93 heterozygotes) and 402 controls were considered. Carriers of two mutant alleles had undetectable plasma levels of ANGPTL3 protein, whereas heterozygotes showed a reduction ranging from 34% to 88%, according to genotype. Compared with controls, homozygotes as well as heterozygotes showed a significant reduction of all plasma lipoproteins, while no difference in lipoprotein(a) [Lp(a)] levels was detected between groups. The prevalence of fatty liver was not different in FHBL2 subjects compared with controls. Notably, diabetes mellitus and cardiovascular disease were absent among homozygotes. FHBL2 trait is inherited in a codominant manner, and the lipid-lowering effect of two ANGPTL3 mutant alleles was more than four times larger than that of one mutant allele. No changes in Lp(a) were detected in FHBL2. Furthermore, our analysis confirmed that FHBL2 is not associated with adverse clinical sequelae. The possibility that FHBL2 confers lower risk of diabetes and cardiovascular disease warrants more detailed investigation.

2013 - Familial combined hypolipidemia due to mutations in the ANGPTL3 gene [Articolo su rivista]
Calandra, S.; Tarugi, P.; Averna, M.; Bertolini, S.
abstract

The role of ANGPTL3 in lipoprotein metabolism emerged from studies in a mutant mouse strain characterized by severe hypotriglyceridemia and carrying a loss-of-function (LOF) mutation of the ANGPTL3 gene. ANGPTL3 was found to inhibit lipoprotein lipase and endothelial lipase. Genome-wide association studies in humans demonstrated the association of ANGPTL3 variants with plasma triglyceride levels and LOF mutations of ANGPTL3 were found in hypotriglyceridemic subjects in population studies. Recently, individuals originally classified as affected by familial hypobetalipoproteinemia were found to be homozygotes/compound heterozygotes for rare LOF mutations of ANGPTL3. They show a striking reduction of all lipoprotein classes (VLDL, LDL and HDL), a condition defined as familial combined hypolipidemia. This disorder, transmitted as a recessive trait, does not seem to be associated with specific clinical manifestations, such as premature atherosclerosis or fatty liver disease. © 2013 Future Medicine Ltd.

2013 - Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia. [Articolo su rivista]
Magnolo, Antonia Lucia; Najah, M; Fancello, Tatiana; Di Leo, E; Pinotti, Elisa; Brini, I; Gueddiche, Nm; CALANDRA BUONAURA, Sebastiano; Slimene, Nm; Tarugi, Patrizia Maria
abstract

Monogenic hypobetalipoproteinemias include three disorders: abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) with recessive transmission and familial hypobetalipoproteinemia (FHBL) with dominant transmission. We investigated three unrelated Tunisian children born from consanguineous marriages, presenting hypobetalipoproteinemia associated with chronic diarrhea and retarded growth. Proband HBL-108 had a moderate hypobetalipoproteinemia, apparently transmitted as dominant trait, suggesting the diagnosis of FHBL. However, she had no mutations in FHBL candidate genes (APOB, PCSK9 and ANGPTL3). The analysis of MTTP gene was also negative, whereas SAR1B gene resequencing showed that the patient was homozygous for a novel mutation (c.184G>A), resulting in an amino acid substitution (p.Glu62Lys), located in a conserved region of Sar1b protein. In the HBL-103 and HBL-148 probands, the severity of hypobetalipoproteinemia and its recessive transmission suggested the diagnosis of ABL. The MTTP gene resequencing showed that probands HBL-103 and HBL-148 were homozygous for a nucleotide substitution in the donor splice site of intron 9 (c.1236+2T>G) and intron 16 (c.2342+1G>A) respectively. Both mutations were predicted in silico to abolish the function of the splice site. In vitro functional assay with splicing mutation reporter MTTP minigenes showed that the intron 9 mutation caused the skipping of exon 9, while the intron 16 mutation caused a partial retention of this intron in the mature mRNA. The predicted translation products of these mRNAs are non-functional truncated proteins. The diagnosis of ABL and CMRD should be considered in children born from consanguineous parents, presenting chronic diarrhea associated with hypobetalipoproteinemia.

2013 - Spectrum of mutations and phenotypic expression in patients with autosomal dominant hypercholesterolemia identified in Italy [Articolo su rivista]
Bertolini, S.; Pisciotta, L.; Rabacchi, C.; Cefalù, A. B.; Noto, D.; Fasano, T.; Signori, A.; Fresa, R.; Averna, M.; Calandra, S.
abstract

2012 - Characterization of Three Kindreds with Familial Combined Hypolipidemia Due to Loss of Function Mutations of ANGPTL3. [Articolo su rivista]
Pisciotta, L; Favari, E; Magnolo, Antonia Lucia; Simonelli, S; Adorni, Mp; Sallo, R; Fancello, Tatiana; Zavaroni, I; Ardigò, D; Bernini, F; Calabresi, L; Franceschini, G; Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

BACKGROUND: -Angiopoietin-like protein 3 (ANGPTL3) affects lipid metabolism by inhibiting the activity of lipoprotein and endothelial lipases. Angptl3 knock out mice have a marked hypolipidemia and heterozygous carriers of ANGPLT3 loss of function (LOF) mutations were found among individuals in the lowest quartile of plasma triglyceride (TG) in population studies. Recently four related individuals with primary hypolipidemia were found to be compound heterozygotes for ANGPTL3 LOF mutations.METHODS AND RESULTS: -We resequenced ANGPTL3 in four members of three kindred originally identified for very low LDL-C and HDL-C levels (0.97±0.16 and 0.56±0.20 mmol/L), in whom no mutations of known candidate genes for monogenic hypobeta- and hypoalpha-lipoproteinemia had been detected. These subjects were found to be homozygous or compound heterozygous for ANGPTL3 LOF mutations (p.G400VfsX5, p.I19LfsX22/p.N147X), associated with the absence of ANGPTL3 in plasma. They had reduced plasma levels of TG containing lipoproteins and of LpA-I and preβ-HDL particles. In addition, their apoB-depleted sera had a reduced capacity to promote cell cholesterol efflux through the various pathways (ABCA1-, SR-BI- and ABCG1-mediated efflux). However, these subjects had no clinical evidence of accelerated atherosclerosis. Heterozygous carriers of the ANGPTL3 mutations had low plasma ANGPTL3, moderately reduced LDL-C (2.52±0.38 mmol/L) but normal plasma HDL-C.CONCLUSIONS: -Complete ANGPTL3 deficiency caused by LOF mutations of ANGPTL3 is associated with a recessive hypolipidemia characterized by a reduction of apolipoprotein B and apolipoprotein A-I containing lipoproteins, changes in HDL subclasses and reduced cholesterol efflux potential of serum. Partial ANGPTL3 deficiency is associated only with a moderate reduction of LDL.

2012 - Leucine 10 allelic variant in signal peptide of PCSK9 increases the LDL cholesterol-lowering effect of statins in patients with familial hypercholesterolaemia. [Articolo su rivista]
Pisciotta, L; Sallo, R; Rabacchi, Claudio; Wunsch, Alessia; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

BACKGROUND AND AIMS: In the normal population, carriers of an additional leucine residue in a stretch of nine leucines in the signal peptide of PCSK9 (L10) have lower total (TC) and low-density lipoprotein cholesterol (LDL-C) than homozygotes for the wild-type allele (L9/L9). A similar effect was detected in familial hypercholesterolaemia (FH) patients with the p.C681X mutation of LDL-receptor (LDLR). We investigated the effect of L10 variant on basal lipid profile and response to statins in molecularly characterised FH patients.METHODS AND RESULTS: Plasma lipids were determined in 322 FH patients screened for the L9/L10/L11 polymorphism and in a subgroup of 54 patients carrying the same LDLR mutation (p.Q474HfsX63). Plasma lipids were also determined in 42 FH patients carrying the L10 variant and in a parallel group of 42 FH patients, L9/L9 homozygotes, matched for gender, age, type of LDLR gene mutation, as well as for type, dose and duration of statin treatment. In FH patients, no difference in the basal plasma TC and LDL-C levels was observed between carriers of L10 variant (L9/L10+L10/L10) and L9/L9 homozygotes. The same was true in FH patients carrying the p.Q474HfsX63 LDLR mutation. In the subgroups of statin-treated patients, the reduction of TC and LDL-C was greater in carriers of L10 (-34.0% and -42.5%, respectively) than in L9/L9 homozygotes (-27.5% and -34.3%, respectively) (P&lt;0.001).CONCLUSION: The variant L10 of the leucine repeats in PCSK9 signal peptide is to be considered as a factor capable of modulating the lipid-lowering effects of statins in FH.

2012 - Lysosomal lipase deficiency: Molecular characterization of eleven patients with Wolman or cholesteryl ester storage disease [Articolo su rivista]
Fasano, Tommaso; Pisciotta, L; Bocchi, Letizia; Guardamagna, O; Assandro, P; Rabacchi, Claudio; Zanoni, Paolo; Filocamo, M; Bertolini, S; CALANDRA BUONAURA, Sebastiano
abstract

Wolman Disease (WD) and cholesteryl ester storage disease (CESD) represent two distinct phenotypes of the same recessive disorder caused by the complete or partial deficiency of lysosomal acidic lipase (LAL), respectively. LAL, encoded by the LIPA gene, hydrolyzes cholesteryl esters derived from cell internalization of plasma lipoproteins. WD is a rapidly progressive and lethal disease characterized by intestinal malabsorption, hepatic and adrenal failure. CESD is characterized by hepatic fibrosis, hyperlipidemia and accelerated atherosclerosis. Aim of the study was the identification of LIPA mutations in three WD and eight CESD patients.The WD patients, all deceased before the first year of age, were homozygous for two novel mutations (c.299+1G>A and c.419G>A) or a mutation (c.796G>T) previously reported as compound heterozygosity in a CESD patient. The two mutations (c.419G>A and c.796G>T) resulting in truncated proteins (p.W140* and p.G266*) and the splicing mutation (c.229+1G>A) were associated with undetectable levels of LIPA mRNA in fibroblasts.All eight CESD patients carried the common mutation c.894G>A known to result not only in a major non-functional transcript with the skipping of exon 8 (p.S275_Q298del), but also in a minor normally spliced transcript producing 5–10% residual LAL activity. The c.894G>A mutation was found in homozygosity in four patients and, as compound heterozygosity, in association with a known (p.H295Y and p.G342R) or a novel (p.W140*) mutation in four other CESD patients. Segregation analysis performed in all patients harboring c.895G>A showed its occurrence on the same haplotype suggesting a common founder ancestor. The other WD and CESD mutations were associated with different haplotypes.

2012 - Novel mutations of ABCA1 transporter in patients with Tangier disease and familial HDL deficiency. [Articolo su rivista]
Fasano, T.; Zanoni, P.; Rabacchi, C.; Pisciotta, L.; Favari, E.; Adorni, M. P.; Deegan, P. B.; Park, A.; Hlaing, T.; Feher, M. D.; Jones, B.; Uzak, A. S.; Kardas, F.; Dardis, A.; Sechi, A.; Bembi, B.; Minuz, P.; Bertolini, S.; Bernini, F.; Calandra, S.
abstract

2011 - Altered mRNA splicing in lipoprotein disorders. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Bertolini, S.
abstract

PURPOSE OF REVIEW: To review recent publications concerning the functional assessment on pre-mRNA splicing of genomic variants found in some monogenic dyslipidemias. Examples are derived from familial hypercholesterolemia,familial HDL deficiency/Tangier disease and familial hypobetalipoproteinemia.RECENT FINDINGS: About 5-10% of genomic variants found in familial hypercholesterolemia,FHD/Tangier disease and familial hypobetalipoproteinemia are located in the introns of the candidate genes and are classified as splicing mutations. Although variants affecting highly conserved GT/AG dinucleotides at the splice sites are likely to be pathogenic,it is difficult to predict the effects of variants located deep in the introns. Algorithms were developed to predict the effect of these variants and to provide the rationale for functional studies. Combined in-silico and wet bench analysis revealed that some intronic variants classified as pathogenic have no effect,whereas others generated abnormal transcripts. Nucleotide substitutions at the 5' and the 3' of exons might change the splice site consensus sequence,causing splicing defects. Rare silent mutations were identified which create new splice sites within exons,with the consequent production of abnormal transcripts.SUMMARY: Intronic variants,even if located deep in introns,as well as exonic variants could affect splicing with the formation of abnormal transcripts encoding structurally abnormal proteins

2011 - Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Speedy, He; Dean, Af; Bertolini, S; Shoulders, C. C.
abstract

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.

2011 - Rearrangements of the ABCC6 gene in Italian patients with PXE [Abstract in Rivista]
Guerra, Deanna; Costa, Sonia; J., Roggiani; Rabacchi, Claudio; CALANDRA BUONAURA, Sebastiano; I., PASQUALI RONCHETTI; Quaglino, Daniela
abstract

The objective of this study was to search for the deletion of exon 24 and exons 24-27 that had been previously reported in Italian patients affected by pseudoxanthoma elasticum, anb autosomal recessive disorder characterized by calcification and fragmentation of elastic fibers as a conseguence of mutations in the ABCC6 gene. To detect deletions in the ABCC6 gene the development and use of long range PCR procedure using appropriately designed primers are described.

2011 - Two novel rare variants of APOA5 gene found in subjects with severe hypertriglyceridemia. [Articolo su rivista]
Pisciotta, L; Fresa, R; Bellocchio, A; Guido, V; PRIORE OLIVA, Claudio; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

BACKGROUND: Common variants of APOA5 gene affect plasma triglyceride (TG) in the population and a number of rare variants APOA5 have been reported in individuals with hypertriglyceridemia (HTG).METHODS: APOA5 was analysed in 98 HTG individuals (plasma TG >9 mmol/L) in whom no mutations in LPL and APOC2 had been found.RESULTS: Two patients were found to be heterozygous for two novel APOA5 variants. The first variant (p.L253P) was identified in an obese male who consumed a diet rich in fat and simple sugars. He was also a carrier in trans of the common TG-raising p.S19W SNP (5*3 haplotype). The second variant (c.295-297 del GAG, p.E99 del) was found in a lean male with no life style or metabolic factors known to affect plasma TG. He was a carrier in trans of the TG-raising 5*2 haplotype and was homozygous for the rare c.1337T allele of a SNP of GCKR gene. No mutations in other genes affecting plasma TG (LMF1 and GPIHBP1) were found in these patients. These APOA5 variants, resulted to be deleterious in silico, were not found in 350 control subjects.CONCLUSIONS: These novel APOA5 variants predispose to HTG in combination with other genetic or nutritional factors.

2010 - A novel mutation in the sterol 27-hydroxylase gene of a woman with autosomal recessive cerebrotendinous xanthomatosis. [Articolo su rivista]
Schneider, H; Lingesleben, A; Vogel, Hp; Garuti, R; CALANDRA BUONAURA, Sebastiano
abstract

Mutations of the gene encoding the mitochondrial enzyme sterol 27-hydroxylase (CYP27A1 gene) cause defects in the cholesterol pathway to bile acids that lead to the storage of cholestanol and cholesterol in tendons, lenses and the central nervous system. This disorder is the cause of a clinical syndrome known as cerebrotendinous xanthomatosis (CTX). Since 1991 several mutations of the CYP27A1 gene have been reported. We diagnosed the clinical features of CTX in a caucasian woman. Serum levels of cholestanol and 7α-hydroxycholesterol were elevated and the concentration of 27-hydroxycholesterol was reduced. Bile alcohols in the urine and faeces were increased. The analysis of the CYP27A1 gene showed that the patient was a compound heterozygote carrying two mutations both located in exon 8. One mutation is a novel four nucleotide deletion (c.1330-1333delTTCC) that results in a frameshift and the occurrence of a premature stop codon leading to the formation of a truncated protein of 448 amino acids. The other mutation, previously reported, is a C - &gt; T transition (c. c.1381C &gt; T) that converts the glutamine codon at position 461 into a termination codon (p.Q461X). These truncated proteins are expected to have no biological function being devoid of the cysteine residue at position 476 of the normal enzyme that is crucial for heme binding and enzyme activity.

2010 - Identification and characterization of novel loss of function mutations in ATP-binding cassette transporter A1 in patients with low plasma high-density lipoprotein cholesterol. [Articolo su rivista]
Candini, Chiara; Schimmel, Aw; Peter, J; Bochem, Ae; Holleboom, Ag; Vergeer, M; Dullaart, Rp; Dallinga Thie, Gm; Hovingh, Gk; Khoo, Kl; Fasano, Tommaso; Bocchi, Letizia; CALANDRA BUONAURA, Sebastiano; Kuivenhoven, Ja; Motazacker, M. M.
abstract

OBJECTIVES: The current literature provides little information on the frequency of mutations in the ATP-binding cassette transporter A1 (ABCA1) in patients with low high-density lipoprotein cholesterol (HDL) levels that are referred to the clinic. In 78 patients with low plasma levels of HDL cholesterol that were referred to our clinic, we routinely screened for ABCA1 gene mutations and studied the functionality of newly identified ABCA1 missense mutations.METHODS: The coding regions and exon-intron boundaries of the ABCA1 gene were sequenced in 78 subjects with HDL cholesterol levels below the 10th percentile for age and gender. Novel mutations were studied by assessing cholesterol efflux capacity (using apolipoprotein A-I as acceptor) after transient expression of ABCA1 variants in BHK cells.RESULTS: Sixteen out of 78 patients (21%) were found to carry 19 different ABCA1 gene variants (1 frameshift, 2 splice-site, 4 nonsense and 12 missense variation) of which 14 variations were novel. Of three patients with homozygous mutations and three patients having compound heterozygous mutations only one patient presented with the clinical characteristics of Tangier Disease (TD) in the presence of nearly complete HDL deficiency. Seven out of eight newly identified ABCA1 missense mutations were found to exhibit a statistically significant loss of cholesterol efflux capacity.CONCLUSION: This study shows that one out of five patients who are referred to our hospital because of low HDL cholesterol levels have a functional ABCA1 gene mutation. It is furthermore demonstrated that in vitro studies are needed to assess functionality of ABCA1 missense mutations

2010 - Multiple abnormally spliced ABCA1 mRNAs caused by a novel splice site mutation of ABCA1 gene in a patient with Tangier disease. [Articolo su rivista]
Bocchi, Letizia; Pisciotta, L; Fasano, Tommaso; Candini, Chiara; Puntoni, Mr; Sampietro, T; Bertolini, S; CALANDRA BUONAURA, Sebastiano
abstract

BACKGROUND: Mutations in ABCA1 gene are the cause of Tangier disease (TD) and familial high density lipoprotein (HDL) deficiency. Splice site mutations of this gene were reported infrequently.METHODS: ABCA1 gene was sequenced in a TD patient and in subjects with low HDL. The effect of intronic variants on ABCA1 pre-mRNA splicing was studied in COS-1 cells expressing a mutant minigene or in patients' cells.RESULTS: A novel mutation in intron 20 (c.2961 -2 A>C) was found in the TD patient. To assess its effect, a mutant ABCA1 minigene, containing intron 18-intron 23 region, was expressed in COS-1 cells. The mutant minigene generated three transcripts: i) in the first (459bp) 61 nucleotides of intron 20 were retained; ii) in the second (384bp) exon 20 joined to exon 21 devoid of the first 14 nucleotides; and iii) in the third (255bp) the entire exon 21 was skipped. The first two transcripts were also observed in patient's peripheral blood mononuclear cells. These mRNAs encode truncated proteins. A variant in intron 8 (c.814 -14 ins A), identified in subjects with low HDL, had no effect on ABCA1 pre-mRNA splicing.CONCLUSIONS: Functional analysis is required to establish the effect of intronic mutations on ABCA1 pre-mRNA splicing.

2010 - Preemptive liver transplantation in a child with familial hypercholesterolemia [Articolo su rivista]
Maiorana, A; Nobili, V; CALANDRA BUONAURA, Sebastiano; Francalanci, P; Bernabei, S; El Hachem, M; Monti, L; Gennari, F; Torre, G; de Ville de Goyet, J; Bartuli, A.
abstract

Familial hypercholesterolemia is an autosomal codominant disorder associated with markedly elevated plasma concentration of LDL-cholesterol and increased cardiovascular risk. Homozygous patients have rapid development of atherosclerosis with death from cardiovascular disease even in childhood. Life-long recurrent apheresis to reduce plasma LDL-cholesterol is considered the gold standard for treatment. Liver transplantation can be curative for this condition, but is usually only considered after the development of cardiovascular disease. We report a 5.5-yr-old child initially misdiagnosed with heterozygous familial hypercholesterolemia and treated by low-fat diet only. In view of persistent hypercholesterolemia and development of xanthomatosis, new molecular studies indicated the presence of two different mutations in the LDL receptor gene, with one being a deletion of two exons not identifiable with standard sequencing analysis. Recurrent plasma apheresis in combination with statins lowered, but did not normalize plasma LDL-cholesterol levels. It caused progressive reduction of the size of xanthomas and prevented the development of vascular complications. After two yr, liver transplantation normalized LDL-cholesterol levels and completely resolved the skin lesions. Preemptive liver transplantation is a definitive cure of familial homozygous hypercholesterolemia and might be more effective if performed before development of vascular complications.

2010 - Pseudoxanthoma elasticum and familial hypercholesterolemia: A deleterious combination of cardiovascular risk factors [Articolo su rivista]
Pisciotta, L; Tarugi, Patrizia Maria; Borrini, C; Bellocchio, A; Fresa, R; Guerra, Deanna; Quaglino, Daniela; Ronchetti, Ivonne; Calandra Buonaura, Sebastiano; Bertolini, S.
abstract

Pseudoxanthoma Elasticum (PXE), an autosomal recessive disease due to mutations in ABCC6 gene, is characterised by fragmentation of elastic fibres with involvement of the cardiovascular system. We investigated a 60-year-old female with angina pectoris found to have PXE, associated with elevated plasma LDL-C suspected to be due to autosomal-co-dominant hypercholesterolemia. METHODS: ABCC6, LDLR, PCSK9 and exon 26 of APOB genes were re-sequenced. Cardiovascular involvement was assessed by coronary angiography, single-photon emission computed tomography (SPECT) and ultrasound examination. RESULTS AND CONCLUSIONS: The patient was a compound heterozygous for two ABCC6 mutations (p.S317R and p.R1141X) and heterozygous for a novel LDLR mutation (p.R574H). She had severe coronary stenosis and calcification of the arteries of the lower limbs. Treatment with ezetimibe/simvastatin 10/60mg/day, maintained over a 4.5-year period, reduced of LDL-C and the myocardial ischemic area. In PXE patients LDL-lowering treatment might contribute to delay macrovascular complications.

2009 - A novel deletion of BRCA1 gene that eliminates the ATG initiation codon without affecting the promoter region. [Articolo su rivista]
Marino, Marco; Rabacchi, Claudio; Simone, Maria Luisa; Medici, Veronica; Cortesi, L; CALANDRA BUONAURA, Sebastiano
abstract

BackgroundPoint mutations in the highly penetrant cancer susceptibility gene BRCA1 are responsible for the majority of hereditary breast and/or ovarian cancer. We describe a novel large rearrangement of the BRCA1 gene identified in an Italian woman affected by an early onset bilateral breast cancer and a family history of hereditary breast cancer. The proband and her parents were negative for the presence of point mutations in BRCA1 and BRCA2 genes.MethodsMultiplex ligation-dependent probe amplification (MLPA) was used to detect rearrangements in the BRCA1 gene. The breakpoint of the rearrangement identified in the proband was defined by restriction mapping and PCR amplification. BRCA1 mRNA encoded by the mutant allele was isolated from peripheral blood.ResultsThe proband was heterozygous for a 9.1 kb deletion spanning from intron 1 to intron 3 (g.1238_10350del) that eliminates exons 2 and 3 in the mature mRNA. In mutant mRNA exon 1a joins directly to exon 5 with no disruption of the reading frame.ConclusionsThis deletion that eliminates the ATG initiation site in exon 2 and the sequence located in exons 2 and 3 encoding part of the RING finger domain of BRCA1 protein, is expected to abolish the function of this protein.

2009 - A novel homozygous mutation in CETP gene as a cause of CETP deficiency in a Caucasian kindred. [Articolo su rivista]
Calabresi, L; Nilsson, P; Pinotti, Elisa; Gomaraschi, M; Favari, E; Adorni, Mp; Bernini, F; Sirtori, Cr; CALANDRA BUONAURA, Sebastiano; Franceschini, G; Tarugi, Patrizia Maria
abstract

ObjectiveTo analyze the cholesteryl ester transfer protein (CETP) gene and the plasma HDL phenotype in a Caucasian subject with extremely elevated plasma high density lipoprotein-cholesterol (HDL-C).Methods and resultsThe proband, a 63-year-old male of Swedish ancestry with elevated HDL-C (208 mg/dl) and apoA-I (and 272 mg/dl), was found to be homozygous for a point mutation in exon 2 of CETP gene (c.109 C > T) resulting in a premature termination codon (R37X). Plasma CETP mass and activity were undetectable. Plasma HDL were characterized by predominance of large HDL with enhanced preβ-HDL content. The proband's sons, heterozygotes for the mutation, had reduced plasma CETP activity and moderately elevated HDL-C. Serum of CETP deficient subjects showed a normal or enhanced cholesterol efflux capacity via ABCG1/SR-BI; cholesterol efflux via ABCA1 and macrophage cholesterol removal were lower than normal. The proband was healthy and had no atherosclerotic plaques in carotid or femoral arteries.ConclusionComplete CETP deficiency caused by mutations in CETP gene is exceedingly rare in Caucasians; the description of this single case indicates that CETP deficiency does not predispose to atherosclerosis in the absence of major cardiovascular risk factors.

2009 - An apparent inconsistency in parent to offspring transmission ofpoint mutations of LDLR gene in familial hypercholesterolemia [Articolo su rivista]
Rabacchi, Claudio; Wunsch, A; Ghisellini, Margherita; Marino, Marco; Pisciotta, L; Bertolini, S; Calandra Buonaura, Sebastiano
abstract

BackgroundFamilial Hypercholesterolemia (FH), the most common form of autosomal co-dominant hypercholesterolemia, is due to mutations in the LDLR gene, mostly minute or point mutations in the coding sequence.MethodsAnalysis of LDLR gene was performed by direct resequencing and multiplex ligation-dependent probe amplification (MLPA).ResultsLDLR gene resequencing showed that proband I.G., with the clinical diagnosis of homozygous FH, was homozygous for a mutation in exon 12 (c.1775 G&gt;A, G571E) known to be pathogenic, and heterozygous for a mutation in intron 14 (c.2140 +5G&gt;A). Proband's daughter with heterozygous FH carried only the intron 14 mutation. To explain this inconsistency we assumed that the proband was a carrier of a gene deletion. MLPA showed that the proband and her daughter were heterozygous for a deletion of exons 11 and 12. This explains the apparent homozygosity of the c.1175 G&gt;A mutation in the proband. Ex 11–12 deletion was linked to the c.2140 +5G&gt;A mutation. Other FH patients, heterozygotes for c.2140 +5G&gt;A, were found to carry the Ex 11–12 deletion found in the proband or other pathogenic mutations.ConclusionsInconsistencies in the parent to offspring transmission of point mutations in LDLR gene may be due to a large deletion not detected by resequencing.

2009 - Cholesteryl Ester Storage Disease (CESD) due to novel mutations in the LIPA gene. [Articolo su rivista]
Pisciotta, L; Fresa, R; Bellocchio, A; Pino, E; Guido, V; Cantafora, A; Di Rocco, M; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

Cholesteryl Ester Storage Disease (CESD) is a rare recessive disorder due to mutations in LIPA gene encoding the lysosomal acidic lipase (LAL). CESD patients have liver disease associated with mixed hyperlipidemia and low plasma levels of high-density lipoproteins (HDL). The aim of this study was the molecular characterization of three patients with CESD. LAL activity was measured in blood leukocytes.In two patients (twin sisters) the clinical diagnosis of CESD was made at 9 years of age, following the fortuitous discovery of elevated serum liver enzymes in apparently healthy children. They had mixed hyperlipidemia, hepatosplenomegaly, reduced LAL activity (5% of control) and heteroalleic mutations in LIPA gene coding sequence: (i) the common c.894 G>A mutation and (ii) a novel nonsense mutation c.652 C>T (p.R218X). The other patient was an 80 year-old female who for several years had been treated with simvastatin because of severe hyperlipidemia associated with low plasma HDL. In this patient the sequence of major candidate genes for monogenic hypercholesterolemia and hypoalphalipoproteinemia was negative. She was found to be a compound heterozygote for two LIPA gene mutations resulting in 5% LAL activity: (i) c.894 G>A and (ii) a novel complex insertion/deletion leading to a premature termination codon at position 82.These findings suggest that, in view of the variable severity of its phenotypic expression, CESD may sometimes be difficult to diagnose, but it should be considered in patients with severe type IIb hyperlipidemia associated with low HDL, mildly elevated serum liver enzymes and hepatomegaly.

2009 - Functional analysis of two novel splice site mutations of APOB gene in familial hypobetalipoproteinemia. [Articolo su rivista]
Di Leo, E; Magnolo, L; Pinotti, Elisa; Martini, S; Cortella, I; Vitturi, N; Rabacchi, Claudio; Wunsch, A; Pucci, F; Bertolini, S; Calandra Buonaura, Sebastiano; Tarugi, Patrizia Maria
abstract

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low density lipoprotein cholesterol (LDL-C) and its protein constituent apolipoprotein B (apoB), which may be due to mutations in APOB gene, mostly located in the coding region of this gene. We report two novel APOB gene mutations involving the acceptor splice site of intron 11 (c.1471-1G&gt;A) and of intron 23 (c.3697-1G&gt;C), respectively, which were identified in two patients with heterozygous FHBL associated with severe fatty liver disease. The effects of these mutations on APOB pre-mRNA splicing were assessed in COS-1 cells expressing the mutant APOB minigenes.The c.1471-1G&gt;A APOB minigene generated two abnormal mRNAs. In one mRNA the entire intron 11 was retained; in the other mRNA exon 11 joined to exon 12, in which the first nucleotide was deleted due to the activation of a novel acceptor splice site. The predicted products of these mRNAs are truncated proteins of 546 and 474 amino acids, designated apoB-12.03 and apoB-10.45, respectively. The c.3697-1G&gt;C APOB minigene generated a single abnormal mRNA in which exon 23 joined to exon 25, with the complete skipping of exon 24. This abnormal mRNA is predicted to encode a truncated protein of 1220 amino acids, designated apoB-26.89.These splice site mutations cause the formation of short truncated apoBs, which are not secreted into the plasma as lipoprotein constituents. This secretion defect is the major cause of severe fatty liver observed in carriers of these mutations.

2009 - Functional lecithin: Cholesterol acyltransferase Is not required for efficient atheroprotection in humans [Articolo su rivista]
Calabresi, L; Baldassarre, D; Castelnuovo, S; Conca, P; Bocchi, Letizia; Candini, Chiara; Frigerio, B; Amato, M; Sirtori, Cr; Alessandrini, P; Arca, M; Boscutti, G; Cattin, L; Gesualdo, L; Sampietro, T; Vaudo, G; Veglia, F; CALANDRA BUONAURA, Sebastiano; Franceschini, G.
abstract

Background— Mutations in the LCAT gene cause lecithin:cholesterol acyltransferase (LCAT) deficiency, a very rare metabolic disorder with 2 hypoalphalipoproteinemia syndromes: classic familial LCAT deficiency (Online Mendelian Inheritance in Man No. 245900), characterized by complete lack of enzyme activity, and fish-eye disease (Online Mendelian Inheritance in Man No. 136120), with a partially defective enzyme. Theoretically, hypoalphalipoproteinemia cases with LCAT deficiency should be at increased cardiovascular risk because of high-density lipoprotein deficiency and defective reverse cholesterol transport. Methods and Results— The extent of preclinical atherosclerosis was assessed in 40 carriers of LCAT gene mutations from 13 Italian families and 80 healthy controls by measuring carotid intima-media thickness (IMT). The average and maximum IMT values in the carriers were 0.07 and 0.21 mm smaller than in controls (P=0.0003 and P=0.0027), respectively. Moreover, the inheritance of a mutated LCAT genotype had a remarkable gene-dose–dependent effect in reducing carotid IMT (P=0.0003 for average IMT; P=0.001 for maximum IMT). Finally, no significant difference in carotid IMT was found between carriers of LCAT gene mutations that cause total or partial LCAT deficiency (ie, familial LCAT deficiency or fish-eye disease). Conclusions— Genetically determined low LCAT activity in Italian families is not associated with enhanced preclinical atherosclerosis despite low high-density lipoprotein cholesterol levels. This finding challenges the notion that LCAT is required for effective atheroprotection and suggests that elevating LCAT expression or activity is not a promising therapeutic strategy to reduce cardiovascular risk.

2009 - Molecular analysis of NPC1 and NPC2 gene in 34 Niemann-Pick C Italian Patients: Identification and structural modeling of novel mutations [Articolo su rivista]
Fancello, T; Dardis, A; Rosano, C; Tarugi, Patrizia Maria; Tappino, B; Zampieri, S; Pinotti, Elisa; Corsolini, F; Fecarotta, S; D'Amico, A; DI ROCCO, M; Uziel, G; CALANDRA BUONAURA, Sebastiano; Bembi, B; Filocamo, M.
abstract

Niemann–Pick C, the autosomal recessive neuro-visceral disease resulting from a failure of cholesterol trafficking within the endosomal–lysosomal pathway, is due to mutations in NPC1 or NPC2 genes. We characterized 34 unrelated patients including 32 patients with mutations in NPC1 gene and two patients in NPC2 gene. Overall, 33 distinct genotypes were encountered. Among the 21 unpublished NPC1 alleles, 15 were due to point mutations resulting in 13 codon replacements (p.C100S, p.P237L, p.R389L, p.L472H, p.Y634C, p.S636F, p.V780G, p.Q921P, p.Y1019C, p.R1077Q, p.L1102F, p.A1187V, and p.L1191F) and in two premature stop codons (p.R934X and p.Q447X); a new mutant carried two in cis mutations, p.[L648H;M1142T] and four other NPC1 alleles were small deletions/insertions leading both to frame shifts and premature protein truncations (p.C31WfsX26, p.F284LfsX26, p.E1188fsX54, and p.T1205NfsX53). Finally, the new intronic c.464-2A>C change at the 3′ acceptor splice site of intron 4 affected NPC1 messenger RNA processing. We also found a new NPC2 mutant caused by a change of the first codon (p.M1L). The novel missense mutations were further investigated by two bioinformatics approaches. Panther proein classification system computationally predicted the detrimental effect of all new missense mutations occurring at evolutionary conserved positions. The other bioinformatics approach was based on prediction of structural alterations induced by missense mutations on the NPC1 atomic models. The in silico analysis predicted protein malfunctioning and/or local folding alteration for most missense mutations. Moreover, the effects of the missense mutations (p.Y634C, p.S636F, p.L648H, and p.V780G) affecting the sterol-sensing domain (SSD) were evaluated by docking simulation between the atomic coordinates of SSD model and cholesterol.

2009 - Severe HDL deficiency due to novel defects in the ABCA1 transporter [Articolo su rivista]
Pisciotta, L; Bocchi, Letizia; Candini, Chiara; Sallo, R; Zanotti, I; Fasano, Tommaso; Chakrapani, A; Bates, T; Bonardi, R; Cantafora, A; Ball, S; Watts, G; Bernini, F; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

Objectives. The objective was the identification and functional characterization of mutations in the ABCA1 gene in four patients with severe HDL deficiency.Subjects. Patients were referred to the clinic because of almost complete HDL deficiency.Methods. The ABCA1 gene was sequenced directly. The analysis of the ABCA1 protein, ABCA1 mRNA and ABCA1-mediated cholesterol efflux was performed in cultured fibroblasts. Intracellular localization of ABCA1 mutants was investigated in transfected HEK293 cells.Results. Two patients were homozygous for mutations in the coding region of the ABCA1 gene, resulting in an amino acid substitution (p.A1046D) and a truncated protein (p.I74YFsX76). The third patient was homozygous for a splice site mutation in intron 35 (c.4773 + 1g>a), resulting in an in-frame deletion of 25 amino acids (del p.D1567_K1591) in ABCA1. These patients had clinical manifestations of accumulation of cholesterol in the reticulo-endothelial system. The fourth patient, with preclinical atherosclerosis, was a compound heterozygote for two missense mutations (p.R587W/p.W1699C). ABCA1-mediated cholesterol efflux was abolished in fibroblasts from patients with p.A1046D and del p.D1567_K1591 mutants and in fibroblasts homozygous for p.R587W. A reduced ABCA1 protein content was observed in these cells, suggesting an increased intracellular degradation. The mutant p.W1699C was largely retained in the endoplasmic reticulum, when expressed in HEK293 cells.Conclusions. The homozygotes for mutations which abolish ABCA1 function showed overt signs of involvement of the reticulo-endothelial system. This was not the case in the compound heterozygote for missense mutations, suggesting that this patient retains some residual ABCA1 function that reduces cholesterol accumulation in the reticulo-endothelial system

2009 - The type of LDLR gene mutationpredicts cardiovascular risk in children with familial hypercholesterolemia. [Articolo su rivista]
Guardamagna, O; Restagno, G; Rolfo, E; Pederiva, C; Martini, S; Abello, F; Baracco, V; Pisciotta, L; Pino, E; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

ObjectiveTo ascertain whether the molecular characterization of a defect in the low-density lipoprotein (LDL) receptor gene (LDLR) in children with heterozygous familial hypercholesterolemia (heFH) identifies subjects at greater risk of developing premature coronary artery disease (pCAD) later in life.Study designWe investigated 264 children with heFH from 201 families, along with 148 affected parents and 100 unaffected siblings. The lipid profile was assessed before any treatment was provided, and genotype analysis was performed to characterize LDLR defects. In a subgroup of children with heFH and controls, we measured aorta and carotid intima-media thickness (aIMT and cIMT). The prevalence of pCAD in parents and/or grandparents with heFH was recorded.ResultsThe children with heFH with a family history of pCAD had higher LDL cholesterol and apolipoprotein B levels and greater aIMT and cIMT than those with negative family history. Compared with carriers of LDLR-defective mutations, carriers of LDLR-negative mutations had a more severe phenotype, in terms of plasma lipid levels and IMT, and a higher prevalence of pCAD in first-degree relatives (36% vs 6.7%; P < .001).ConclusionsThe study of heFH in children, in which other risk factors for CAD play a minor role, allows early identification of those at increased risk for developing pCAD, who merit more stringent clinical control and early pharmacologic treatment.

2008 - A novel mutation of the apolipoprotein A-I gene in a family with familial combined hyperlipidemia [Articolo su rivista]
Pisciotta, L; Fasano, Tommaso; Calabresi, L; Bellocchio, A; Fresa, R; Borrini, C; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

We report a large family in which four members showed a plasma lipid profile consistent with the clinical diagnosis of familial combined hyperlipidemia (FCHL). One of these patients was found to have markedly reduced HDL cholesterol (HDL-C) (0.72 mmol/l) and Apo A-I (72 mg/dl) levels, a condition suggestive of the presence of a mutation in one of the HDL-related genes. The analysis of APOA1 gene revealed that this patient was heterozygous for a cytosine insertion in exon 3 (c.49–50 ins C), resulting in a frame-shift and premature stop codon at position 26 of pro-Apo A-I (Q17PFsX10). This novel mutation, which prevents the synthesis of Apo A-I, was also found in four family members, including three siblings and the daughter of the proband. Carriers of Apo A-I mutation had significantly lower HDL-C and Apo A-I than non-carriers family members (0.77 ± 0.15 mmol/l vs. 1.15 ± 0.20 mmol/l, P < 0.005; 71.4 ± 9.1 mg/dl vs. 134.0 ± 14.7 mg/dl, P < 0.005, respectively). Two of the APOA1 mutation carriers, who were also heavy smokers, had fibrous plaques in the carotid arteries causing mild stenosis (20%). The intimal-media thickness in the two other adult carriers was within the normal range. The other non-carriers family members with FCHL had either overt vascular disease or carotid atherosclerosis at ultrasound examination. This observation suggests that the low HDL-C/low Apo A-I phenotype may result from a genetic defect directly affecting HDL metabolism, even in the context of a dyslipidemia which, like FCHL, is associated with low plasma HDL-C.

2008 - Genetics and molecular biology: proprotein convertase subtilisin/kexin type 9 and LDL receptor--an intriguing story. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria
abstract

Genetic studies in humans and mice have demonstrated that proprotein convertase subtilisin/kexin type 9 (PCSK9) protein is involved in the post-translational regulation of LDL-receptor (LDL-R) protein levels in hepatocytes [1,2]. Several lines of evidence indicate that mature PCSK9 is secreted by hepatocytes and interacts with LDL-R on the cell surface. Secreted PCSK9 (or recombinant PCSK9), when added to cultured hepatocytes, readily reduces LDL-R protein levels in a time-dependent and concentration-dependent manner. PCSK9 is endocytosed and exhibits endosomal/lysosomal localization. Endocytosis of PCSK9 is mediated by LDL-R, because it is not observed in LDL-R-deficient hepatocytes or in hepatocytes deficient in autosomal recessive hypercholesterolaemia (ARH) protein [3,4•] – an adaptor protein that is required for LDL-R internalization. When infused into mice, human recombinant PCSK9 was able to reduce hepatic LDL-R protein levels and elevate plasma LDL [3]. Thus, the idea has progressively emerged that, at least in hepatocytes, secreted PCSK9 interacts directly with the extracellular domain of the LDL-R and undergoes LDL-R/ARH-mediated endocytosis, leading to intracellular degradation of the LDL-R [3,4•]. A key question underlying the interaction between PCSK9 and LDL-R concerns the site in the extracellular domain of LDL-R that binds to PCSK9. A recent study [5••] showed that PCSK9 interacts with the first epidermal growth factor repeat (EGF-A) in the EGF-precursor homology domain of LDL-R. This interaction is greatly increased with reduction in pH from 7 to 5.2, suggesting that PCSK9 binds to LDL-R at the cell surface at neutral pH and remains strongly associated with it in the acidic environment of the endosomes, where the binding affinity to LDL-R is increased by more than 100-fold. In endosomes PCSK9 would lock LDL-R in a specific conformation that favours degradation of the receptor instead of its recycling back to the plasma membrane [5••,6•]. This finding raises the question of whether naturally occurring mutations in the EGF-A domain of LDL-R increase or, vice versa, decrease the affinity of the receptor for PCSK9. In the first case, the increased PCSK9-dependent LDL-R degradation would result in hypercholesterolaemia, whereas in the second case the reduced LDL-R degradation would result in hypocholesterolaemia. The site of PCSK9 involved in binding to LDL-R remains to be defined. It is probable that functional and structural studies of PCSK9 mutants will assist with mapping this site [7••,8]. Two other questions have been addressed, namely whether the binding of LDL to LDL-R affects the interaction between PCSK9 and LDL-R, and whether PCSK9 is directly responsible for the proteolytic degradation of LDL-R. According to Fisher et al. [6•], binding of LDL diminishes PCSK9 binding to LDL-R in vitro, and partially inhibits the effect of secreted PCSK9 on LDL-R degradation within the cell. Deletion of the LDL-binding domain of the LDL-R does not affect binding of PCSK9, however, thus indicating that PCSK9 can bind to LDL-R independently of lipoproteins [5••]. With regard to the second question, two studies [9••,10] showed that catalytic activity is not required for secreted PCSK9 to induce LDL-R degradation. Catalytic inactive wild-type or mutant PCSK9 (harbouring the gain-of-function mutation D374Y) exhibited no loss of binding capacity to LDL-R, suggesting that the role played by PCSK9 is that of a molecular chaperone directing LDL-R to lysosomal degradation [9••]. Catalytic activity is, however, required for the intracellular autocatalytic cleavage of the prodomain and the secretion of PCSK9 from the cells. There are, however, gain-of-function mutations that prevent autocatalytic cleavage or secretion of PCSK9, but that nevertheless induce LDL-R degradation, suggesting that PCSK9 may also function intracellularly [11]. The story of PCSK9 continues and is full of surprises.

2008 - Hypertriglyceridaemia and low plasma HDL in a patient with apolipoprotein A-V deficiency due to a novel mutation in the APOA5 gene [Articolo su rivista]
PRIORE OLIVA, Claudio; Carubbi, Francesca; Schaap, F. G; Bertolini, S; CALANDRA BUONAURA, Sebastiano
abstract

APOA5 encodes a novel apolipoprotein (apo A-V) which appears to be a modulator of plasma triglyceride (TG). In apoA5 knock out mice plasma TG level increases almost fourfold, whereas in human APOA5 transgenic mice it decreases by 70%. Some SNPs in the APOA5 gene have been associated with variations in plasma TG in humans. In addition, hypertriglyceridaemic (HTG) patients have been identified who carried rare nonsense mutations in the APOA5 gene (Q139X and Q148X), predicted to result in apo A-V deficiency. In this study we report a 17-year-old male with high TG and low high density lipoprotein cholesterol (HDL-C), who at the age of two had been found to have severe HTG and eruptive xanthomas suggesting a chylomicronaemia syndrome. Plasma postheparin LPL activity, however, was normal and no mutations were found in LPL and APOC2 genes. The sequence of APOA5 gene revealed that the patient was homozygous for a point mutation (c.289 C&gt;T) in exon 4, converting glutamine codon at position 97 into a termination codon (Q97X). Apo A-V was not detected in patient's plasma, indicating that he had complete apo A-V deficiency. The administration of a low-fat and low-oligosaccharide diet, either alone or supplemented with ω-3 fatty acids, started early in life, reduced plasma TG to a great extent but had a negligible effect on plasma HDL-C. Loss of function mutations of APOA5 gene may be the cause of severe HTG in patients without mutations in LPL and APOC2 genes.

2008 - Therapeutic management of a new case of LCAT deficiency with a multifactorial long-term approach based on high doses of angiotensin II receptor blockers (ARBs). [Articolo su rivista]
Aranda, P; Valdivielso, P; Pisciotta, L; Garcia, I; Garcã A., Arias C; Bertolini, S; Martã N., Reyes G; Gonzã Lez, Santos; CALANDRA BUONAURA, Sebastiano
abstract

Familial lecithin cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by the appearance of corneal opacity, anemia, proteinuria progressing to chronic renal failure and abnormalities in the composition of plasma lipoproteins. No established therapy currently exists for this condition. We report here a new case of FLD caused by two novel mutations in the LCAT gene in which, for the first time, aggressive therapy with angiotensin II receptor blockers and lipid-lowering drugs showed benefit in blood pressure, lipid abnormalities, proteinuria and also kidney function, probably delaying progression to renal failure

2008 - Variable phenotypic expression of homozygous familial hypobetalipoproteinaemia due to novel APOB gene mutations [Articolo su rivista]
E., Di Leo E; Magnolo, Antonia Lucia; Bertolotti, Marco; M., Bourbon; S., Carmo Pereira; M., Pirisi; CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria
abstract

Homozygous familial hypobetalipoproteinaemia (Ho-FHBL) is a rare co-dominant disorder characterized by extremely low levels of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB). Most patients with Ho-FHBL have mutations in APOB gene resulting in truncated apoBs. Some patients are asymptomatic, while others have fatty liver, intestinal fat malabsorption and neurological dysfunctions. We investigated three adult subjects with severe hypobetalipoproteinaemia and a family history of FHBL. Proband FHBL-47 had liver cirrhosis with hepatocarcinoma and a renal carcinoma but no clinical manifestations related to FHBL. He was a compound heterozygote for a 7-bp deletion in exon 21 and a base insertion in exon 26 resulting in truncated apoBs (apoB-22.46/apoB-66.51). Proband FHBL-53, with severe hepatic steatosis and fibrosis, had a nonsense mutation in exon 19 resulting in a truncated apoB (apoB-20.61) and a rare nucleotide substitution in intron 14 (c.2068-4T>A). The latter was also present in her daughter, found to have low plasma LDL-C and apoB. Proband FHBL-82 had chronic diarrhoea and steatorrhoea. She was found to be homozygous for a nonsense mutation in exon 24 resulting in a truncated apoB (apoB-26.65). In adult subjects, the presence of chronic liver disease and chronic diarrhoea, when associated with severe hypobetalipoproteinaemia, may lead to the diagnosis of Ho-FHBL.

2007 - Abnormal apolipoprotein B pre-mRNA splicing in patients with familial hypobetalipoproteinaemia [Articolo su rivista]
Di Leo, E; Magnolo, Antonia Lucia; Lancellotti, Sandra; Crocè, L; Visintin, L; Tiribelli, C; Bertolini, S; CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria
abstract

Background: Familial hypobetalipoproteinaemia (FHBL) is a codominant disorder characterised by fatty liver and reduced plasma levels of low-density lipoprotein (LDL) and its protein constituent apolipoprotein B ( apoB). FHBL is linked to the APOB gene in some but not all known cases. In a group of 59 patients with FHBL genotyped for APOB gene mutations, we found three novel splice-site mutations: c. 904+4AR -> G in intron 8, c. 3843-2A -> G in intron 24 and c. 4217-1G -> RT in intron 25. Objective: To assess the effects of these mutations on apoB prem-RNA splicing. Methods: ApoB mRNA was analysed in the liver of one proband and in cells expressing APOB minigenes harbouring the mutations found in the other probands. Results: In the liver of the c. 3843-2A -> RG carrier, an apoB mRNA devoid of exon 25 was identified, predicted to encode a truncated peptide of 1260 amino acids. The analysis of minigene transcripts in COS-1 cells showed that the c. 904+4A -> G mutation caused the formation of an mRNA devoid of exon 8, predicted to encode a short apoB of 247 amino acids. The minigene harbouring the c. 4217-1G -> T mutation in intron 25 generated an mRNA in which exon 25 joined to a partially deleted exon 26, resulting from the activation of an acceptor site in exon 26; this mRNA is predicted to encode a truncated protein of 1380 amino acids. All these truncated apoBs were not secreted as constituents of plasma lipoproteins. Conclusion: These findings demonstrate the pathogenic effect of rare splice- site mutations of the APOB gene found in FHBL.

2007 - Effect of ezetimibe coadministered with statins in genotype-confirmed heterozygous FH patients [Articolo su rivista]
Pisciotta, L; Fasano, Tommaso; Bellocchio, A; Bocchi, Letizia; Sallo, R; Fresa, R; Colangeli, I; Cantafora, A; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

We investigated the effect of statins and statins plus ezetimibe in 65 FH heterozygotes carrying LDLR-defective or LDLR-negative mutations as well as the effect of ezetimibe monotherapy in 50 hypercholesterolemic (HCH) patients intolerant to statins. PCSK9 and NPC1L1 genes were analysed to assess the role of genetic variants in response to therapy. In FH patients combined therapy reduced LDL-C by 57%, irrespective of the type of LDLR mutation. The additional decrease of plasma LDL-C induced by ezetimibe showed wide inter-individual variability (from −39% to −4.7%) and was negatively correlated with percent LDL-C decrease due to statin alone (r = −0.713, P < 0.001). The variable response to statins was not due to PCSK9 gene variants associated with statin hyper-sensitivity. The highest response to ezetimibe was observed in a carrier of R174H substitution in NPC1L1, which had been found to be associated with high cholesterol absorption.In HCH patients, ezetimibe monotherapy induced a variable decrease of plasma LDL-C (from −47.7% to −13.4%). To investigate this variability, we sequenced NPC1L1 gene in patients with the highest and the lowest response to ezetimibe. This analysis showed a higher prevalence of the G allele of the c.816 C>G polymorphism (L272L) in hyper-responders, an observation confirmed also in FH patients hyper-responders to ezetimibe. In both FH and HCH patients, the G allele carriers tended to have a higher LDL-C reduction in response to ezetimibe.These observations suggest that in FH heterozygotes LDL-C reduction following combined therapy reflects a complex interplay between hepatic synthesis and intestinal absorption of cholesterol.

2007 - MOLECULAR CHARACTERIZATION OF TWO PATIENTS WITH SEVERE LCAT DEFICIENCY [Articolo su rivista]
CHARLTON MENYS, V; Pisciotta, L; Neary, R; Short, Cd; Calabresi, L; CALANDRA BUONAURA, Sebastiano; Durrington, Pn; Bertolini, S.
abstract

Lecithin:cholesterol acyltransferase (LCAT; EC2.3.1.43) is the major enzyme responsible for theesterification of cholesterol in circulating plasmalipoproteins. This is important in the human becauseunlike many other animal species, cholesterol exportedinto the plasma from the liver is largely unesterified.LCAT associates in plasma preferentially with thediscoidal apo A-I containing high-density lipoproteins(preb1-HDL), where it esterifies the free cholesterol(FC) using apo A-I as co-factor [1]. Through thisaction, LCAT plays a central role in both HDLmaturation to a-migrating spherical HDL and inreverse transport of cholesterol from peripheral tissuesto the liver, directly by interaction of the mature HDLwith the hepatic SR-BI receptor, or indirectly bytransfer of CE by cholesteryl ester transfer protein(CETP) to the VLDL and then LDL, much of which isultimately cleared by the liver via the LDL receptor [2].In addition, LCAT protein seems to have a scavengereffect toward LDL oxidation products, which isindependent of its cholesterol-esterifying activity [3].Mutations of LCAT (chr. 16q22.1, 6 exons) inhomozygous or compound heterozygous form cancause two major phenotypes: FLD (familial LCATdeficiency) and FED (Fish Eye Disease). Patients withFLD (OMIM No, 245900) have a complete loss ofboth a-LCAT activity (i.e. LCAT activity exerted onHDL) and b-LCAT activity (i.e. LCAT activity exertedon LDL), an increased proportion (>80%) of unesterifiedcholesterol in plasma. Clinical manifestationsgenerally include corneal opacification, anaemia andrenal disease with proteinuria, which progresses toterminal renal insufficiency. In FED (OMIM No.131620), there is partial loss of a-LCAT activity withnormal or slightly elevated FC in plasma and cornealopacification without renal disease [4,5]. Intermediatephenotypes have also been described. To date, morethan 60 mutations of LCAT have been identified [4–6];they involve all regions of the coding sequence andproduce a variety of defects, including normal secretionof LCAT with total loss of catalytic activity,reduced secretion with a partial or total loss ofcatalytic activity, secretion of an unstable or rapidlycatabolized enzyme, and complete degradation of theenzyme at its site of synthesis [4]. All subjects withFED or FLD have greatly reduced plasma HDLcholesterol concentrations (usually <0.3 mmol/l) andplasma levels of apo A-I below 50 mg/dl [6,7]; however,premature coronary artery disease is absent in mostFLD cases, but sometimes present, for unclear reasons,in some patients with FED [4,6,8]. We investigated twounrelated patients with clinical features of LCATdeficiency. Two of the three LCAT mutations foundin these patients were novel.

2007 - Molecular diagnosis of hypobetalipoproteinemia: an ENID Review [Articolo su rivista]
Tarugi, Patrizia Maria; Averna, M; DI LEO, E; Cefalù, Ab; Noto, D; Magnolo, Antonia Lucia; Cattin, L; Bertolini, S; CALANDRA BUONAURA, Sebastiano
abstract

Primary hypobetalipoproteinemia (HBL) includes a group of genetic disorders: abetalipoproteinemia (ABL) and chylomicron retention disease (CRD), with a recessive transmission, and familial hypobetalipoproteinemia (FHBL) with a co-dominant transmission. ABL and CRD are rare disorders due to mutations in the MTP and SARA2 genes, respectively. Heterozygous FHBL is much more frequent. FHBL subjects often have fatty liver and, less frequently, intestinal fat malabsorption. FHBL may be linked or not to the APOB gene. Most mutations in APOB gene cause the formation of truncated forms of apoB which may or may be not secreted into the plasma. Truncated apoBs with a size below that of apoB-30 are not detectable in plasma; they are more frequent in patients with the most severe phenotype. Only a single amino acid substitution (R463W) has been reported as the cause of FHBL. Approximately 50% of FHBL subjects are carriers of pathogenic mutations in APOB gene; therefore, a large proportion of FHBL subjects have no apoB gene mutations or are carriers of rare amino acid substitutions in apoB with unknown effect. In some kindred FHBL is linked to a locus on chromosome 3 (3p21) but the candidate gene is unknown. Recently a FHBL plasma lipid phenotype was observed in carriers of mutations of the PCSK9 gene causing loss of function of the encoded protein, a proprotein convertase which regulates LDL-receptor number in the liver. Inactivation of this enzyme is associated with an increased LDL uptake and hypobetalipoproteinemia. HBL carriers of PCSK9 mutations do not develop fatty liver disease.

2006 - A novel sequence variant in APOA5 gene found in patients with severe hypertriglyceridemia [Articolo su rivista]
PRIORE OLIVA, Claudio; Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; L., Pisciotta; A., Bellocchio; S., Bertolini; O., Guardamagna; Fg, Schaap
abstract

The APOA5 gene encoding apolipoprotein A-V (a 366amino acid protein), present in minute amounts in VLDL andHDL, appears to be a modulator of plasma triglyceride (TG)homeostasis [1,2]. In apoA5 knock out mice plasma TG levelincreased almost four-fold, whereas in human APO5 transgenicmice it decreased 70% [1]. Some SNPs in the APOA5gene have been associated with differences in plasma TG levelsin the general population [1]. There is robust evidence thatapo A-V accelerates VLDL and chylomicron catabolism byactivating lipoprotein lipase (LPL)-mediated lipolysis [3–7].

2006 - APOA5 and trigliceride metabolism, lesson from human APOA5 deficiency. [Articolo su rivista]
Calandra Buonaura, Sebastiano; Priore Oliva, Claudio; Tarugi, Patrizia Maria; Bertolini, S.
abstract

Purpose of review: In this review we compare the phenotype and lipoprotein abnormalities of some patients who were found to carry mutations in the APOA5 gene predicted to result in apolipoprotein A-V deficiency. Recent findings: The sequencing of the APOA5 gene in patients with primary hypertriglyceridemia, in whom mutations of the LPL and APOC2 genes had been excluded, led to the identification of four families with two different mutations in this gene predicted to result in truncated apolipoprotein A-V. The first mutation (Q148X) was found in a homozygous state in a child with severe type V hyperlipidemia, some clinical manifestations of chylomicronemia syndrome and a slight reduction in plasma postheparin lipoprotein lipase activity. Carriers of a different mutation (Q139X) were recently reported. Four Q139X heterozygotes had type V hyperlipidemia and markedly reduced plasma postheparin lipoprotein lipase activity. The hypertriglyceridemic Q139X heterozygote had other factors that could have contributed to hypertriglyceridemia. ApoB-100 kinetic studies in hypertriglyceridemic Q139X heterozygotes revealed an impairment of very low-density lipoprotein catabolism. Summary: Mutations in the APOA5 gene, leading to truncated apolipoprotein A-V devoid of lipid-binding domains located in the carboxy-terminal end of the protein, if present in the homozygous state, are expected to cause severe type V hyperlipidemia in patients with no mutations in LPL or APOC2 genes. If present in the heterozygous state, these mutations predispose to hypertriglyceridemia in combination with other genetic factors or pathological conditions.

2006 - Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia [Articolo su rivista]
L., Pisciotta; PRIORE OLIVA, Claudio; Ab, Cefalu; D., Noto; A., Bellocchio; R., Fresa; A., Cantafora; D., Patel; M., Averna; Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; S., Bertolini
abstract

Patients homozygous or Compound heterozygous for LDLR mutations or double heterozygous for LDLR and apo B R3500Q mutation have higher LDL-C levels. more extensive xanthomatosis and more severe premature coronary disease (pCAD) than simple heterozygotes for mutations in either these genes or for missense mutations in PCSK9 gene. It is not known whether combined mutations in LDLR and PKCS9 are associated with such a severe phenotype. We sequenced Apo B and PCSK9 genes in two patients with the clinical diagnosis of homozygous FH who were heterozygous for LDLR gene mutations. Proband Z.P. (LDL-C 13.39 mmol/L and pCAD) was heterozygous for an LDLR mutation (p.E228K) inherited from her father (LDL-C 8.07 mmol/L) and a PCSK9 mutation (p.R496W) from her mother (LDL-C 5.58 mmol/L). Proband L.R. and her sister (LDL-C 11.51 and 10.47 mmol/L. xanthomatosis and carotid atherosclerosis) were heterozygous for all LDLR mutation (p.Y419X) inherited from their mother (LDL-C 6.54 mmol/L) and a PCSK9 mutation (p.N425S) probably from their deceased father. The LDL-C levels in double heterozygotes of these two families were 56 and 44% higher than those found in simple heterozygotes for the two LDLR mutations, respectively. The two PCSK9 Mutations are novel and were not found in I 10 controls and 80 patients with co-dominant hypercholesterolemia. These observations indicate that Fare missense Mutations of PCSK9 may worsen the clinical phenotype of patients carrying LDLR mutations.

2006 - Autosomal recessive hypercholesterolemia (ARH) and homozygous familial hypercholesterolemia (FH): a phenotypic comparison. [Articolo su rivista]
L., Pisciotta; PRIORE OLIVA, Claudio; Gm, Pes; L., Di Scala; A., Bellocchio; R., Fresa; A., Cantafora; M., Arca; CALANDRA BUONAURA, Sebastiano; S., Bertolini
abstract

Autosomal recessive hypercholesterolemia (ARH) is a rare disorder, due to complete loss of function of an adaptor protein (ARH protein) required for receptor-mediated hepatic uptake of LDL. ARH is a phenocopy of homozygous familial hypercholesterolemia (HoFH) due to mutations in LDL receptor (LDLR) gene; however, previous studies suggested that ARH phenotype is less severe than that of HoFH. To test this hypothesis we compared 42 HoFH and 42 ARH patients. LDLR and ARH genes were analysed by Southern blotting and sequencing. LDLR activity was measured in cultured fibroblasts. In ARH plasma LDL cholestrol (LDL-C) level (14.25 +/- 2.29 mmol/L) was lower than in receptor-negative HoFH (21.38 +/- 3.56 mmol/L) but similar to that found in receptor-defective HoFH (15.52 +/- 2.39 mmol/L). The risk of coronary artery disease (CAD) was 9-fold lower in ARH patients. No ARH patients < 20 years of age were found to have CAD as opposed to 43% of HoFH. The CAD prevalence was or tended to be lower in ARH also in the 21-40 (45% versus 86%) and 41-60 (78% versus 100%) age groups. Heterozygous ARH carriers showed higher level of LDL-C (+17%) than non-carrier family members. In conclusion the clinical phenotype of ARH is milder than that of receptor-negative HoFH and resembles that observed in receptor-defective HoFH. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

2005 - Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes [Articolo su rivista]
Mi, Sirinian; F., Belleudi; F., Campagna; M., Ceridono; T., Garofalo; F., Quagliarini; R., Verna; CALANDRA BUONAURA, Sebastiano; S., Bertolini; M., Sorice; Mr, Torrisi; M., Arca
abstract

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor ( LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.

2005 - Combined monogenic hypercholesterolemia and hypoalphalipoproteinemia caused by mutations in LDL-R and LCAT genes [Articolo su rivista]
Pisciotta, L; Calabresi, L; Luppattelli, G; Siepi, D; Mannarino, Mr; Moleri, E.; Bellocchio, A; Cantafora, A; Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; Bertolini, S.
abstract

We studied a three generation family with co-dominant monogenic hypercholesterolemia and hypoalphalipoproteinemia. The proband, a 48 year-old male, was found to be heterozygous for a previously reported mutation in LDL receptor (LDL-R) gene (IVS15–3 c>a) and a novel mutation in exon 6 of lecithin cholesterol acyltransferase (LCAT) gene (c.803 G>A) causing a non-synonymous amino acid substitution (p.R244H). These mutations segregated independently in the family. The LDL-R mutation was associated with high levels of LDL-C (6.20–9.85 mmol/L) and apo B (170–255 mg/dL), comparable to those previously reported in carriers of the same mutation. The LCAT mutation was associated with low levels of HDL-C (0.67–0.80 mmol/L) and apo A-I (96–110 mg/dL). The proband had reduced LCAT function, as measured by cholesterol esterification rate (29 nmol/(mL/h) versus 30–60 nmol/(mL/h)), LCAT activity (10 nmol/(mL/h) versus 20–55 nmol/(mL/h)) and LCAT mass (2.87 μg/mL versus 3.1–6.7 μg/mL). Carriers of LCAT mutation had lower LCAT activity and a tendency to reduced cholestrol esterification rate (CER) and LCAT mass as compared to non-carrier family members. The LCAT mutation was not found in 80 control subjects and 60 patients with primary hypoalphalipoproteinemia. Despite the unfavourable lipoprotein profile, the proband had only mild clinical signs of atherosclerosis. This unexpected finding is probably due to the intensive lipid lowering treatment the patient has been on over the last decade.

2005 - Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency [Articolo su rivista]
Fasano, Tommaso; Bocchi, Letizia; L., Pisciotta; S., Bertolini; CALANDRA BUONAURA, Sebastiano
abstract

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.

2005 - Inherited apolipoprotein A-V deficiency in severe hypertriglyceridemia [Articolo su rivista]
PRIORE OLIVA, Claudio; L., Pisciotta; G., Li Volti; M. P., Sambataro; A., Cantafora; A., Bellocchio; A., Catapano; Tarugi, Patrizia Maria; S., Bertolini; CALANDRA BUONAURA, Sebastiano
abstract

Objective - Mutations in LPL or APOC2 genes are recognized causes of inherited forms of severe hypertriglyceridemia. However, some hypertrigliceridemic patients do not have mutations in either of these genes. Because inactivation or hyperexpression of APOA5 gene, encoding apolipoprotein A-V (apoA-V), causes a marked increase or decrease of plasma triglycerides in mice, and because some common polymorphisms of this gene affect plasma triglycerides in humans, we have hypothesized that loss of function mutations in APOA5 gene might cause hypertriglyceridemia. Methods and Results - We sequenced APOA5 gene in 10 hypertriglyceridemic patients in whom mutations in LPL and APOC2 genes had been excluded. One of them was found to be homozygous for a mutation in APOA5 gene ( c. 433 C > T, Q145X), predicted to generate a truncated apoA-V devoid of key functional domains. The plasma of this patient was found to activate LPL in vitro less efficiently than control plasma, thus suggesting that apoA-V might be an activator of LPL. Ten carriers of Q145X mutation were found in the patient's family; 5 of them had mild hypertriglyceridemia. Conclusions - As predicted from animal studies, apoA-V deficiency is associated with severe hypertriglyceridemia in humans. This observation suggests that apoA-V regulates the secretion and/or catabolism of triglyceride-rich lipoproteins.

2005 - Lung involvement in Niemann-Pick disease type C1: improvement with bronchoalveolar lavage [Articolo su rivista]
S., Palmeri; Tarugi, Patrizia Maria; F., Sicurelli; R., Buccoliero; A., Malandrini; MM De, Santi; G., Marciano; C., Battisti; Mt, Dotti; CALANDRA BUONAURA, Sebastiano; A., Federico
abstract

Progressive lung infiltration is a major cause of death in Niemann-Pick disease type A and B (NPA, NPB) and in the recently defined type C2. In type C1 (NPC1), the main manifestations are neurological. We report a patient with a classic, neurological, late infantile form of NPC1 disease, carrying the mutation P474L and the variant 1642M in the NPC1 gene, who suffered recurrent respiratory manifestations. Bronchoalveolar lavage of a lung segment due to deteriorating respiratory condition revealed many foamy macrophages and was followed by an improvement in symptoms. Pneumopathy may therefore be considered a feature of NPC1 disease for which a partial bronchoalveolar lavage could be a useful treatment.

2005 - Mutations in MTP gene in abeta- and hypobeta-lipoproteinemia [Articolo su rivista]
Di Leo, E; Lancellotti, Sandra; Penacchioni, Jy; Cefalù, Ab; Averna, M; Pisciotta, L; Bertolini, S; CALANDRA BUONAURA, Sebastiano; Gabelli, C; Tarugi, Patrizia Maria
abstract

Familial hypobetalipoproteinemia (FHBL) and abetalipoproteinemia (ABL) are inherited disorders of apolipoprotein B (apo B)-containing lipoproteins that result from mutations in apo B and microsomal triglyceride transfer protein (MTP) genes, respectively. Here we report three patients with severe deficiency of plasma low-density lipoprotein (LDL) and apo B. Two of them (probands F.A. and P.E.) had clinical and biochemical phenotype consistent with ABL. Proband F.A. was homozygous for a minute deletion/insertion (c.1228delCCCinsT) in exon 9 of MTP gene predicted to cause a truncated MTP protein of 412 amino acids. Proband P. E. was heterozygous for a mutation in intron 9 (IVS9-1G>A), previously reported in an ABL patient. We failed to find the second pathogenic mutation in MTP gene of this patient. No mutations were found in apo B gene.The third proband (D.F.) had a less severe lipoprotein phenotype which was similar to that of heterozygous FHBL and appeared to be inherited as a co-dominant trait. However, he had no mutations in apo B gene. He was found to be a compound heterozygote for two missense mutations (D384A and G661A), involving highly conserved regions of MTP. Since this proband was also homozygous for 2 allele of apolipoprotein E (apo E), it is likely that his hypobetalipoproteinemia derives from a combined effect of a mild MTP deficiency and homozygosity for apo E2 isoform.

2005 - The molecular basis of lecithin: Cholesterol acyltransferase deficiency syndromes: A comprehensive study of molecular and biochemical findings in 13 unrelated Italian families [Articolo su rivista]
Calabresi, L; Pisciotta, L; Costantin, A; Frigerio, I; Eberini, I; Alessandrini, P; Arca, M; Bon, Gb; Boscutti, G; Busnach, G; Frascà, G; Gesualdo, L; Gigante, M; Lupattelli, G; Montali, A; Pizzolitto, S; Rabbone, I; Rolleri, M; Ruotolo, G; Sampietro, T; Sessa, A; Vaudo, G; Cantafora, A; Veglia, F; CALANDRA BUONAURA, Sebastiano; Bertolini, S; Franceschini, G.
abstract

To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. METHODS AND RESULTS: Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-beta high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene-dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. CONCLUSIONS: In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene-dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED

2004 - A point mutation in the lariat branch point of intron 6 of NPC1 as the cause of abnormal pre-mRNA splicing in Niemann-Pick type C disease. [Articolo su rivista]
Di Leo, E; Panico, Francesca; Tarugi, Patrizia Maria; Battisti, C; Federico, A; CALANDRA BUONAURA, Sebastiano
abstract

The lariat branch point sequence (BPS) is crucial for splicing pre-mRNA even if BPS mutations have infrequently been reported in human disease. In two siblings with Niemann-Pick type C (NPC) disease we identified two mutations of the NPC1 gene: i) one in exon 20 (c.2932C&gt;T) (p.R978C) previously reported in NPC patients; ii) the other (c.882-28A&gt;G) unreported, in the highly conserved adenosine of a putative lariat BPS of intron 6. Using RT-PCR we found that, besides the normally spliced mRNA, patients' fibroblasts contained minute amounts of an mRNA devoid of exon 7. The exon 6--exon 8 junction in this mRNA causes a frameshift and a premature stop codon, predicted to result in a truncated protein. To assess the effect of c.882-28A&gt;G mutation we constructed two minigenes (wild type and mutant), spanning from intron 5 to intron 8, which were inserted into a pTarget vector and transfected in COS1 cells. The wild type minigene generated an mRNA of the expected size and sequence; the mutant minigene generated only an mRNA devoid of exon 7. This is the first example of a splicing defect due to a mutation in the lariat BPS in an intron of NPC1 found in NPC patients.

2004 - Coronary heart disease in a patient with cerebrotendinous xanthomatosis [Articolo su rivista]
P., Valdivielso; CALANDRA BUONAURA, Sebastiano; Jc, Duran; Garuti, Rita; E., Herrera; P., Gonzalez
abstract

Coronary heart disease is a prevalent condition and a leading cause of death in developed countries. Most cases are due to the cluster of classical risk factors, such as smoking, diabetes, high blood pressure and dyslipidaemia. However, a few patients develop severe and premature arteriosclerosis in spite of absence of common risk factors. Here, we present the clinical, analytical and molecular features of a 36-years-old man who died from advanced ischaemic heart disease as a result of cerebrotendinous xanthomatosis (CTX), a rare condition characterized by elevation in plasma and most tissues of cholestanol and where neurological impairment is the hallmark of this disease. We discuss the relevance of heart disease and the mechanism leading to accelerate arteriosclerosis is CTX.

2004 - Familial HDL deficiency due to ABCA1 gene mutations with or without other genetic lipoprotein disorders [Articolo su rivista]
L., Pisciotta; I., Hamilton Craig; Tarugi, Patrizia Maria; A., Bellocchio; Fasano, Tommaso; P., Alessandrini; Gb, Bon; D., Siepi; E., Mannarino; L., Cattin; M., Averna; Ab, Cefalu; A., Cantafora; CALANDRA BUONAURA, Sebastiano; S., Bertolini
abstract

Mutations in ABCA1 have been shown to be the cause of Tangier disease (TD) and some forms of familial hypoalphalipoproteinemia (HA), two genetic disorders characterized by low plasma HDL levels. Here we report six subjects with low HDL, carrying seven ABCA1 mutations, six of which are previously unreported. Two mutations (R557X and H160FsX173) were predicted to generate short truncated proteins; two mutations (E284K and Y482C) were located in the first extracellular loop and two (R1901S and Q2196H) in the C-terminal cytoplasmic domain of ABCA1. Two subjects found to be compound heterozygotes for ABCA1 mutations did not have overt clinical manifestations of TD. Three subjects, all with premature coronary artery disease (pCAD), had a combination of genetic defects. Besides being heterozygotes for ABCA1 mutations, two of them were also carriers of the R3500Q substitution in apolipoprotein B and the third was a carrier of N291S substitution in lipoprotein lipase. By extending family studies we identified 17 heterozygotes for ABCA1 mutations. Plasma HDL-C and Apo A-I values in these subjects were 38.3 and 36.9% lower than in unaffected family members and similar to the values found in heterozygotes for Apo A-I gene mutations which prevent Apo A-I synthesis. This survey underlines the allelic heterogeneity of ABCA1 mutations and suggests that: (i) TD subjects, if asymptomatic, may be overlooked and (ii) there may be a selection bias in genotyping towards carriers of ABCA1 mutations who have pCAD possibly related to a combination of genetic and environmental cardiovascular risk factors.

2004 - Genetic polymorphisms affecting the phenotypic expression of familial hypercholesterolemia [Articolo su rivista]
S., Bertolini; L., Pisciotta; L., Di Scala; S., Langheim; Ab, Bellocchio; P., Masturzo; A., Cantafora; S., Martini; M., Averna; G., Pes; C., Stefanutti; CALANDRA BUONAURA, Sebastiano
abstract

The clinical expression of heterozygous familial hypercholesterolemia (FH) is highly variable even in patients carrying the same LDL receptor (LDL-R) gene mutation. This variability might be due to environmental factors as well as to modifying genes affecting lipoprotein metabolism. We investigated Apo E (epsilon2, epsilon3, epsilon4), MTP (-493G/T), Apo B (-516C/T), Apo A-V (-1131T/C), HL (-514C/T and -250G/A), FABP-2 (A54T), LPL (D9N, N291S, S447X) and ABCA1 (R219K) polymorphisms in 221 unrelated FH index cases and 349 FH relatives with defined LDL-R gene mutations. We found a significant and independent effect of the following polymorphisms on: (i) plasma LDL-C (Apo E, MTP and Apo 13); (ii) plasma HDL-C (HL, FABP-2 and LPL S447X); (iii) plasma triglycerides (Apo E and Apo AN). In subjects with coronary artery disease (CAD+), the prevalence of FABP-2 54TT genotype was higher (16.5% versus 5.2%) and that of ABCA1219RK and KK genotypes lower (33.0% versus 51.5%) than in subjects with no CAD. Independent predictors of increased risk of CAD were male sex, age, arterial hypertension, LDL-C level and FABP-2 54TT genotype, and of decreased risk the 219RK and KK genotypes of ABCA1. These findings show that several common genetic variants influence the lipid phenotype and the CAD risk in FH heterozygotes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

2004 - Hypobetalipoproteinemia with an apparently recessive inheritance due to a de novo mutation of apolipoprotein B [Articolo su rivista]
Lancellotti, S; Di Leo, E; Penacchioni, Jy; Balli, Fiorella; Viola, L; Bertolini, S; CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria
abstract

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder either linked or not linked to apolipoprotein (apo) B gene. Abetalipoproteinemia (ABL) is a recessive disorder due to mutations of microsomal triglyceride transfer protein (MTP) gene. We investigated a patient with apparently recessive hypobetalipoproteinemia consistent with symptomatic heterozygous FHBL or a mild form of ABL. The proband had fatty liver associated with LDL-cholesterol (LDL-C) and apo B levels &lt;5th percentile but no truncated apo B forms detectable in plasma. MTP gene sequence revealed that he was a carrier of the I128T polymorphism and an unreported amino acid substitution (V168I) unlikely to be the cause of hypobetalipoproteinemia. Apo B gene sequence showed that he was heterozygous for two single base substitutions in exon 9 and 22 resulting in a nonsense (Q294X) and a missense (R1101H) mutation, respectively. Neither of his parents carried the Q294X; his father and paternal grandmother carried the R 110 1 H mutation. Analysis of polymorphic genetic markers excluded non-paternity. In conclusion, the proband has a de novo mutation of apo B gene resulting in a short truncated apo B form (apo B-6.46). Sporadic cases of FHBL with an apparently recessive transmission may be caused by de novo mutations of apo B gene.

2004 - Quantitative polymerase chain reaction and microchip electrophoresis to detect major rearrangements of the low-density lipoprotein receptor gene causing familial hypercholesterolemia [Articolo su rivista]
A., Cantafora; I., Blotta; E., Pino; L., Pisciotta; CALANDRA BUONAURA, Sebastiano; S., Bertolini
abstract

A variety of rearrangements in the low-density lipoportein receptor (LDLR) gene cause severe forms of familial hypercholesterolemia (FH). However, current methods for searching these abnormalities in FH samples, e.g., Southern and Northern Blot, are labor-intensive and not routinely used by diagnostic laboratories. We developed a simpler approach based on the quantitative polymerase chain reaction (PCR) amplification of part or all gene's coding sequences by a series of multiplex amplifications comprising three nonadjacent gene sections plus a fourth section used as an internal reference. Thereafter, the analysis of these PCR products by microchip electrophoresis revealed either deletions or duplications in the investigated gene sections through the simple comparison of electropherograms obtained from mutant and control samples. This required primers leading to well-resolved peaks with minimal size differences among coamplified products and PCR conditions allowing a linear quantitative response to template amount variations as those caused by duplication or deletion of specific gene sections. Also, the inclusion of exon 17 amplification product as an internal reference in each multiplex PCR allowed the normalization of quantitative results by dividing the area of each amplified section by the area of exon 17. The comparison of these ratios calculated from 10 carriers of 6 LDLR known rearrangements with those obtained from 14 control samples showed that gross deletions roughly halved and duplications doubled the ratio values of exons involved in the mutation. This allowed to distinguish gross mutations from sample-to-sample differences that reached at maximum 8% variation over mean values.

2004 - Trascriptional regulation of human CYP27 integrates retinoid, peroxisome proliferator activated receptor, and liver X receptor signaling in macrophages [Articolo su rivista]
A., Szanto; S., Benko; I., Szatmari; Bl, Balint; I., Furtos; R., Ruhl; S., Molnar; L., Csiba; Garuti, Rita; CALANDRA BUONAURA, Sebastiano; H., Larsson; U., Diczfalusy; L., Nagy
abstract

Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARgamma, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARgamma-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARgamma-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.

2004 - β-thalassemia is a modifying factor of the clinical expression of familial hypercholesterolemia [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; S., Bertolini; Gm, Pes; L., Deiana; Tarugi, Patrizia Maria; L., Pisciotta; S., LI VOLTI; G., LI VOLTI; C., Maccarone
abstract

Familial hypercholesterolemia (FH) is a codominant disorder due to a variety of mutations of the low-density lipoprotein (LDL) receptor gene that result in an elevation of plasma LDL-cholesterol (LDL-C). Plasma levels of LDL-C show large interindividual variation even in subjects carrying the same mutation of the LDL receptor gene. This variability may be due to genetic factors (modifier genes). Several surveys indicate that the overall contribution of common polymorphisms of modifier genes (such as the genes encoding apolipoproteins E and B) to this variability is less than 10%. In contrast, beta-thalassemia has a profound LDL-lowering effect. This was documented in FH patients identified on the island of Sardinia, in Italy, where 12% of the inhabitants are carriers of beta-thalassemia due to a single mutation (Q39X) of the beta-globin gene that abolishes the synthesis of beta-globin chain of hemoglobin (beta(o)-thalassemia). Plasma LDL-C in FH heterozygotes carrying the beta(o)-thalassemia trait is 25% lower than in noncarriers, regardless of the LDL receptor gene mutation. It is likely that this effect is due to two main mechanisms: (1) increased uptake of LDL by the bone marrow to provide cholesterol for the increased proliferation of erythroid progenitor cells and (2) increased production of inflammatory cytokines that reduce the hepatic secretion and increase the catabolism of LDL. In view of its LDL-C-lowering effect, beta-thalassemia trait may protect FH heterozygotes against premature coronary atherosclerosis.

2003 - Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2+5G > C mutation in ABCA1 gene [Articolo su rivista]
S., Altilia; L., Pisciotta; R., Garuti; Tarugi, Patrizia Maria; A., Cantafora; L., Calabresi; J., Tagliabue; S., Maccari; F., Bernini; I., Zanotti; C., Vergani; S., Bertolini; CALANDRA BUONAURA, Sebastiano
abstract

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G&gt;C in intron 2 (IVS2 +5G&gt;C) and ii) c.844 C&gt;T in exon 9 (R282X). The IVS2 +5G&gt;C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G&gt;C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2(-)/mRNA), exon 4 (Ex4(-)/mRNA), or both these exons (Ex2(-)/Ex4(-)/ mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G&gt;C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.

2003 - Adult-onset Niemann-Pick type C disease: A clinical, neuroimaging, and molecular genetic study [Articolo su rivista]
C., Battisti; Tarugi, Patrizia Maria; Mt, Dotti; N., De Stefano; A., Vattimo; F., Chierichetti; CALANDRA BUONAURA, Sebastiano; A., Federico
abstract

We report on a patient with adult-onset Niemann-Pick type C (NPC) disease, carrying the mutations P1007 and I1061T in the NPC1 gene, presenting with marked psychiatric changes followed by dystonia and cognitive impairment. Filipin staining, single photon emission computed tomography perfusional, positron emission tomography metabolic, conventional magnetic resonance imaging, and magnetic resonance spectroscopy findings suggested a pathophysiological correlation with phenotype expression. This case expands the clinical and genetic spectrum of the rare adult-onset NPC disease phenotype

2003 - Apolipoprotein C-II deficiency presenting as a lipid encephalopathy in infancy [Articolo su rivista]
Cj, Wilson; Cp, Oliva; F., Maggi; Al, Catapano; CALANDRA BUONAURA, Sebastiano
abstract

An infant presented with massive hyperchylomicronemia. and a severe encephalopathy. MRI showed marked lipid deposition throughout the brain. Despite the normalization of the biochemistry, there was little clinical improvement, and at 18 months of age she has severe developmental delay, a strikingly abnormal MRI. Apolipoprotein C-II, the lipoprotein on chylomicrons responsible for the activation of lipoprotein lipase, was not detectable in blood. Analysis of the APO C-II gene revealed a novel homozygous point mutation, 1118C--&gt;A. Subsequently, another sibling has been born with the same homozygous mutation and similar biochemistry but, perhaps because of early treatment, a normal neurological outcome.

2003 - Recurrent mutations of the apolipoprotein A-I gene in three kindreds with severe HDL deficiency [Articolo su rivista]
L., Pisciotta; R., Miccoli; A., Cantafora; L., Calabresi; Tarugi, Patrizia Maria; P., Alessandrini; G., Bittolo Bon; G., Franceschini; C., Cortese; CALANDRA BUONAURA, Sebastiano; S., Bertolini
abstract

Two siblings with high density lipoprotein (HDL) deficiency and no plasma apolipoprotein A-I (Apo A-I) were found to be homozygous for a cytosine deletion in exon 3 of Apo A-I gene (c.85 del C, Q5FsX11). This mutation causes a frameshift leading to a premature stop codon and abolishes the synthesis of Apo A-I. Although both siblings had corneal opacifications and planar xanthomas, only one of them had premature coronary artery disease, probably as the result of mildly elevated LDL levels. In two other unrelated subjects HDL deficiency was due to heterozygosity for a nucleotide substitution in exon 4 of Apo A-I gene (c.494 T > G, L141R). Both Apo A-I mutations were reported previously in an Italian kindred which included compound heterozygotes and simple heterozygotes. We investigated all carriers of these mutations in the three kindreds and in the one previously reported. Plasma Apo A-I and HDL-C levels were lower in the mutation carriers than in non-carrier family members. These levels, however, were lower in L141R carriers than in carriers of c.85 del C. Haplotype analysis performed using several polymorphisms suggested that both the c.85 del C and L141R are likely to be recurrent mutations. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

2002 - Niemann Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts [Articolo su rivista]
Tarugi, Patrizia Maria; Ballarini, ; G. Bembi, B.; Battisti, C.; Palmeri, ; S. Panzani, F.; Di Leo, E.; Martini, C.; Federico, A.; CALANDRA BUONAURA, Sebastiano
abstract

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T--&gt;G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.

2001 - A point mutation in ABC1 gene in a patient with severe premature coronary heart disease and mild clinical phenotype of Tangier disease [Articolo su rivista]
S., Bertolini; L., Pisciotta; M., Seri; R., Cusano; A., Cantafora; L., Calabresi; G., Franceschini; R., Ravazzolo; CALANDRA BUONAURA, Sebastiano
abstract

The proband is a 50 year-old woman born from a consanguineous marriage. She has been suffering from angina pectoris since the age of 38 and underwent coronary bypass surgery for three-vessel disease at 48. The presence of low plasma levels of total cholesterol and high density lipoprotein (HDL) cholesterol (2.4 and 0.1 mmol/l) and apo AI (< 15 mg/dl) associated with corneal lesions and a mild splenomegaly suggested the diagnosis of Tangier disease. However, none of the other features of Tangier disease, including hepatomegaly anemia and peripheral neuropathy, were present. The analysis of the dinucleotide microsatellites located in chromosome 9q31 region demonstrated that the proband was homozygous for the alleles of D9S53, D9S1784 and D9S1832. The mother and son of the proband, both with low levels of HDL. cholesterol, shared one of the proband's haplotypes, whereas neither of these haplotypes was present in the normolipidemic proband's sister. The sequence of ATP-binding cassette transporter 1 (ABC1-1) cDNA obtained by reverse transcription-PCR (RT-PCR) of total RNA isolated from cultured fibroblasts showed that the proband was homozygous for a C > T transition in tron 13, which caused a tryptophane for arginine substitution (R527W). This mutation was confirmed by direct sequencing of exon 13 amplified from genomic DNA. It can be easily screened, as the nucleotide change introduces a restriction sits for the enzyme All III. R527W substitution occurs in a highly conserved region of the NH2 cytoplasmic domain of ABC1 protein. R527W co-segregates with the low HDL phenotype in the family and was not found in 200 chromosomes from normolipidemic individuals. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

2001 - Identification of an alternative transcript of ABCA1 gene in different human cell types [Articolo su rivista]
L., Bellincampi; Ml, Simone; C., Motti; C., Cortese; S., Bernardini; S., Bertolini; CALANDRA BUONAURA, Sebastiano
abstract

We have observed two ABCA1 gene transcripts in human skin fibroblasts, The RT-PCR amplification of the exon 3-exon 8 region generated a normal fragment (740 bp) and an abnormal fragment (600 bp) in a ratio ranging from 3:1 to 8/9:1. These two transcripts were present in other cells such as leukemia T-cells, endothelial and smooth muscle cells as well human hepatoma cells (HepG2). Restriction enzyme analysis and sequencing indicated that in the abnormal fragment exon 3 was followed by exon 5. The complete skipping of exon 4 leads to a premature stop and a predicted translation product of 74 amino acids. The ratio between the normal and alternative transcript is not affected by variation in ABCA1 gene expression induced by incubating cells in serum-free medium and in the presence of cholesterol. It is possible that this alternative splicing represents as mechanism that regulates the ABCA1 content in tissues.

2001 - Phenotypic expression of heterozygous familial hypobetalipoproteinemia in three kindreds with novel mutations of apolipoprotein B gene [Articolo su rivista]
Tarugi, Patrizia Maria; Lonardo, A.; Gabelli, C.; Sala, F.; Ballarini, G.; Cortella, I.; Previato, L.; Bertolini, S.; Cordera, R.; CALANDRA BUONAURA, Sebastiano
abstract

We report the clinical phenotype in three kindreds with familial heterozygous hypobetalipoproteinemia (FHBL) carrying novel truncated apolipoprotein Bs (apoBs) of different sizes (apoB-8.15, apoB-33.4 and apoB-75.7). In D.A. kindred, we found three carriers of a C-deletion in exon 10 leading to the synthesis of apoB-8.15 not detectable in plasma. They showed steatorrhea and fatty liver. In N.L. kindred, the proband is heterozygous for a nonsense mutation in exon 26, leading to the formation of apoB-33.4. He had premature cerebrovascular disease and fatty liver; two apoB-33.4 carriers in this kindred showed only fatty liver. In B.E. kindred, the proband is heterozygous for a T-deletion in exon 26, which converts tyrosine at codon 3435 into a stop codon, resulting in apoB-75.7. The proband, a heavy alcohol drinker, had steatohepatitis, whereas his teetotaller daughter, an apoB-75.7 carrier, had no detectable fatty liver. This study suggests that: i) fatty liver invariably develops in FHBL carriers of short and medium-size truncated apoBs (&lt; apoB-48), but its occurrence needs additional environmental factors in carriers of longer apoB forms; ii) intestinal lipid malabsorption develops only in carriers of short truncated apoBs, which are not secreted into the plasma; and iii) cerebrovascular disease due to premature atherosclerosis may occur even in FHBL subjects.

2000 - A study of fatty liver disease and plasma lipoproteins in a kindred with familial hypobetalipoproteinemia due to a novel truncated form of apolipoprotein B (apo B-54.5) [Articolo su rivista]
Tarugi, Patrizia Maria; Lonardo, A.; Ballarini, G.; Erspamer, L.; Tondelli, E.; Bertolini, S.; CALANDRA BUONAURA, Sebastiano
abstract

BACKGROUND/AIMS: Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of low-density lipoproteins. It can be caused by mutations in the gene encoding apolipoprotein B-100 (apo B), leading to the formation of truncated apo Bs which have a reduced capacity to export lipids from the hepatocytes as lipoprotein constituents. Case reports suggest the occurrence of liver disease in FHBL, but there are no studies of liver involvement in FHBL with defined apo B gene mutations. The presence of fatty liver disease was investigated in a large FHBL kindred. METHODS: Plasma lipoprotein and apolipoprotein analysis, liver function tests, and apo B gene sequence were performed in 16 members of a FHBL kindred. The presence of fatty liver was assessed by ultrasound and computed tomography scanning. RESULTS: The proband, a non-obese heavy drinker male with hypobetalipoproteinemia, had steatohepatitis with fibrosis. He was heterozygous for a novel non-sense mutation of apo B gene producing a truncated apo B of 2745 amino acids (designated apo B-54.5, having half the size of normal apo B-100). Seven other members of his kindred carried apo B-54.5. Although all of them were hypolipidemic, their lipid levels showed a large inter-individual variability not accounted for by polymorphisms of genes involved in apo B metabolism. Four carriers (two heavy drinkers and two teetotallers), irrespective of their plasma lipid levels, had ultrasonographic evidence of fatty liver. In the other four carriers no evidence of fatty liver was found. CONCLUSIONS: In this kindred apo B-54.5 predisposes to fatty liver, which however may require some additional factors to become clinically relevant.

2000 - Cerebrotendinous xanthomatosis with predominant Parkinsonian syndrome: Further confirmation of the clinical heterogeneity [Articolo su rivista]
Mt, Dotti; A., Federico; R., Garuti; CALANDRA BUONAURA, Sebastiano
abstract

2000 - Influence of beta(0)-thalassemia on the phenotypic expression of heterozygous familial hypercholesterolemia - A study of patients with familial hypercholesterolemia from Sardinia [Articolo su rivista]
L., Deiana; R., Garuti; G. M., Pes; C., Carru; A., Errigo; M., Rolleri; L., Pisciotta; P., Masturzo; A., Cantafora; CALANDRA BUONAURA, Sebastiano; S., Bertolini
abstract

One of the genetic features of the Sardinian population is the high prevalence of hemoglobin disorders. It has been estimated that 13% to 33% of Sardinians carry a mutant allele of the alpha-globin gene (alpha-thalassemia trait) and that 6% to 17% are beta-thalassemia carriers. In this population, a single mutation of beta-globin gene (Q39X, beta(0) 39) accounts for &gt;95% of beta-thalassemia cases. Because previous studies have shown that Sardinian beta-thalassemia carriers have lower total and low density lipoprotein (LDL) cholesterol than noncarriers, we wondered whether this LDL-lowering effect of the beta-thalassemia trait was also present in subjects with familial hypercholesterolemia (FPI). In a group of 63 Sardinian patients with the clinical diagnosis of FH, we identified 21 unrelated probands carrying 7 different mutations of the LDL receptor gene, 2 already known (313+1 g&gt;a and C95R) and 5 not previously reported (D118N, C255W, A378T, T413R, and Fs572). The 313+1 g&gt;a and Fs572 mutations were found in several families. In cluster Fs572, the plasma LDL cholesterol level was 5.76+/-1.08 mmol/L in subjects with beta(0)-thalassemia trait and 8.25+/-1.66 mmol/L in subjects without this trait (P&lt;0.001). This LDL-lowering effect was confirmed in an FH heterozygote of the same cluster who had beta(0)-thalassemia major and whose LDL cholesterol level was below the 50th percentile of the distribution in the normal Sardinian population. The hypocholesterolemic effect of beta(0)-thalassemia trait emerged also when we pooled the data from all FH subjects with and without beta(0)-thalassemia trait, regardless of the type of mutation in the LDL receptor gene. The LDL-lowering effect of beta(0)-thalassemia may be related to (1) the mild erythroid hyperplasia, which would increase the LDL removal by the bone marrow, and (2) the chronic activation of the monocyte-macrophage system, causing an increased secretion of some cytokines (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) known to affect the hepatic secretion and the receptor-mediated removal of apolipoprotein B-containing lipoproteins, The observation that our FH subjects with beta(0)-thalassemia trait (compared with noncarriers) have an increase of blood reticulocytes (40%) and plasma levels of interleukin-6 (+60%) supports these hypotheses. The lifelong LDL-lowering effect of beta(0)-thalassemia trait might slow the development and progression of coronary atherosclerosis in FH.

1999 - Analysis of LDL receptor gene mutations in Italian patients with homozygous familial hypercholesterolemia [Articolo su rivista]
S., Bertolini; Cassanelli, Stefano; R., Garuti; Ghisellini, Margherita; Ml, Simone; M., Rolleri; CALANDRA BUONAURA, Sebastiano; P., Masturzo
abstract

The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996, The age of the patients ranged from 1 to 64 years; one third of them were older than 30, Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L, The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from <2% to 30% of normal and was inversely correlated with the plasma LDL cholesterol level (r = -0.665; P < 0.003). The most severe coronary atherosclerosis was observed in those patients with the lowest residual LDL receptor activity (less than or equal to 5% of normal) and the highest plasma LDL cholesterol levels. Twenty-nine patients (23 of whom were unrelated) were found to be homozygotes at the LDL receptor locus. In this group we discovered 2 major rearrangements and 12 different point mutations (9 in the coding region and 3 in splice sites). Some mutations (D200G, C358R, V502M, G528D, and P664L) were found in 3 or more unrelated patients. Patients with the same mutation shared the same haplotype at the LDL receptor gene locus and came from the same geographic area. Ten patients (9 of whom were unrelated) were found to be compound heterozygotes. The mutations found in this group consisted of one large deletion and 12 point mutations (11 in the coding sequence and one in a splice site). In 3 compound heterozygotes we failed to identify the second mutant allele at the LDL receptor locus. These observations confirm the allelic heterogeneity underlying familial hypercholesterolemia in the Italian population and indicate that the variability of phenotypic expression of homozygous familial hypercholesterolemia is, to a large extent, related to the type of mutation of the LDL receptor gene.

1999 - Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia [Articolo su rivista]
F., Parise; L., Simone; M. A., Croce; Ghisellini, Margherita; Battini, Renata; S., Borghi; Tiozzo, Roberta; Ferrari, Sergio; CALANDRA BUONAURA, Sebastiano; Ferrari, Stefano
abstract

A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.

1998 - A 'de novo' point mutation of the low-density lipoprotein receptor gene in an Italian subject with primary hypercholesterolemia [Articolo su rivista]
Cassanelli, Stefano; S., Bertolini; M., Rolleri; F., De Stefano; L., Casarino; N., Elicio; A., Naselli; CALANDRA BUONAURA, Sebastiano
abstract

Severe hypercholesterolemia was found in an Ii-year-old boy with no family history of familial hypercholesterolemia. The reduced LDL-receptor activity in cultured skill fibroblasts (40% I-125-LDL degradation as compared with a control cell line) indicated the presence of an LDL-receptor defect. The analysis of the promoter region and the exons of LDL-receptor gene by single strand conformation polymorphism revealed an abnormal migration pattern in exon 1, which was due to a T-->A transversion at nucleotide 28 of the cDNA. This novel mutation causes an arginine for tryptophane substitution at position - 12 of the signal peptide (W-12R) and introduces an AviII restriction site in exon 1. Screening of the mutation by polymerase chain reaction (PCR) amplification of exon 1 and AviII digestion revealed that none of the proband's family members carried the mutation. Non-paternity was excluded after the analysis of a battery of 14 short tandem repeats located in 13 different chromosomes. These results are consistent with the hypothesis that the proband is heterozygous for a 'de novo' mutation of the LDL-receptor gene producing a non-conservative amino acid substitution. We suggest that the change in the net charge of the signal peptide, caused by the addition of a positively charged amino acid, impairs the co-translational translocation of the nascent receptor protein across the endoplasmic reticulum membrane.

1998 - Analysis of two duplications of the LDL receptor gene affecting intracellular transport, catabolism, and surface binding of the LDL receptor [Articolo su rivista]
Dd, Patel; N., Lelli; R., Garuti; Sl, Volti; S., Bertolini; Bl, Knight; CALANDRA BUONAURA, Sebastiano
abstract

Two novel mutations of the lo tv density lipoprotein (LDL)-receptor gene were found in two Italian familial hypercholesterolemia (FH)-heterozygotes. The first mutation was an 18 nucleotide duplication in exon 8 which is preceded by an A--&gt;T transversion, The translation product of the mutant allele tvas predicted to be a receptor with an in-frame insertion of 6 amino acids in repeat B of the epidermal growth factor precursor homology domain. Analysis of LDL-receptor activity in the proband's fibroblasts showed a 50% reduction of I-125-labeled LDL binding and pulse-chase studies suggested that Little, if an); of the mutant protein was processed to the mature form. The second mutation was a 7 kb duplication (from intron 2 to intron 6) of exons 3 through 6, predicted to encode an elongated receptor with the duplication of repeats 2-7 of the ligand binding domain. The elongated receptor was processed slightly more slowly than the normal receptor, but was converted to a mature form of the expected size. This mature, mutant receptor was degraded more rapidly than the normal receptor, On ligand blotting the elongated receptor bound twice as much LDL or beta-very low density lipoproteiu (beta VLDL) as the normal receptor. In contrast, maximum binding of LDL to proband's cells was decreased to approximately 70% of the normal cells with a significant increase in apparent affinity. Cell association at 37 degrees C, internalization, and degradation showed a similar reduced maximum. Thus these mutations demonstrate that duplications of amino acid sequences in the low density lipoprotein LDL-receptor may disrupt the LDL-receptor pathway at different levels.

1998 - Secretion of apoB- and apoA-I-containing lipoproteins by chick kidney [Articolo su rivista]
Tarugi, Patrizia Maria; G., Ballarini; B., Pinotti; Franchini, Antonella; Ottaviani, Enzo; CALANDRA BUONAURA, Sebastiano
abstract

Previous studies showed that chick kidney is a site of synthesis of apolipoprotein (ape) B(B-100) and A-I. Aims of the present study were: a) to compare apoB and apoA-I production in chick kidney and liver; b) to investigate whether kidney apolipoproteins were secreted as constituents of lipoproteins; and c) to define the cellular sites of renal apolipoprotein synthesis. Kidney and liver slices taken from the same animals were incubated with S-35-labeled amino acids and radioactive apoB and apoA-I were immunoprecipitated from cell homogenate and incubation medium. The percentage of total protein radioactivity incorporated into cell plus medium apoB and apoA-I was 0.23 +/- 0.08 and 0.19 +/- 0.11 in kidney and 0.38 +/- 0.05 and 0.38 +/- 0.07 in liver, respectively (P &lt; 0.05 kidney vs. liver). S-35-labeled medium lipoproteins were separated by density gradient ultracentrifugation and three major classes corresponding to VLDL + IDL, LDL, and HDL were identified. Most of the apoB secreted by the liver was found in VLDL, IDL, and LDL whereas kidney apoB was found in VLDL, LDL and light HDL (d 1.070-1.130 g/ml). In both hepatic and renal lipoproteins apoA-I was found not only in HDL but also in the other lipoproteins. Immunohistochemical analysis of kidney sections showed that apoB and apoA-I were present almost exclusively in the epithelial cells of proximal and distal convoluted tubules. Thus apoB and apoA-I synthesized by the epithelial cells of the proximal and dis tal convoluted tubules of chick kidneys are secreted as constituents of lipoprotein particles floating within the density range of plasma lipoproteins. These observations suggest that in the chick, the kidneys may contribute to the plasma lipoprotein pool.

1997 - Effect of heparin on cell proliferation: Lack of correlation with heparin binding sites on cell membrane [Articolo su rivista]
Tiozzo, Roberta; D., Reggiani; Ma, Croce; CALANDRA BUONAURA, Sebastiano; B., Osima; P., Bianchini
abstract

In this study an attempt was made to correlate the in-vitro anti-proliferative effect of heparin with the heparin binding on the cell surface. Cells with different sensitivities to the anti-proliferative effect of heparin (BHK-21, FAO, SMC, BAEC, A-431, V-79, and skin fibroblasts) were incubated with [H-3]heparin either in the presence or in the absence of unlabelled heparin. A saturable binding was found only in BHK-21, FAG, SMC, BAEC and V-79. Scatchard analysis revealed the presence of a single class of binding sites. The binding of [H-3]heparin was efficiently displaced by unlabelled heparin, pentosan polysulfate and low-molecular-weight heparin, but not by dermatan sulfate. Although the sensitivity to the anti-proliferative effect of heparin varied considerably among the cell types (BHK-21 &gt;SMC, FAO &gt;BAEC &gt;V-79), there was no correlation between the reduction of proliferation of these cells and either their heparin binding capacity or the number of binding sites per cell.

1997 - Four novel mutations of sterol 27-hydroxylase gene in Italian patients with cerebrotendinous xanthomatosis [Articolo su rivista]
R., Garuti; M. A., Croce; Tiozzo, Roberta; M. T., Dotti; A., Federico; S., Bertolini; CALANDRA BUONAURA, Sebastiano
abstract

We report the characterization of eight mutations of sterol 27-hydroxylase gene (CYP27) in five Italian patients with cerebrotendinous xanthomatosis, who were found to be compound heterozygotes. Four mutations (C --&gt; T at nt 45 of exon 4, G(+1) --&gt; A in intron 6, G(+5) --&gt; T in intron 7, and G(-1) --&gt; A in intron 7) are novel. The C --&gt; T at nt 45 of exon 4 converts the arginine codon into a stop codon thus generating a truncated protein of 198 amino acids. The three splice site mutations reduced the content of CYP27 mRNA in skill fibroblasts to very low or undetectable levels and generated minute amounts of abnormal mRNAs. The G(+1) --&gt; A transition in intron 6 produced three abnormal mRNAs. In the first, the 5' half of exon 6 joins to exon 7, skipping 89 bp of exon 6, and in the second, exon 5 joins directly to exon 7. The predicted translation products of these mRNAs are truncated proteins. In the third abnormal mRNA, exon 5 joins to exon 8 with an in-frame deletion of 246 bp. The G(+5) --&gt; T transversion in intron 7 generates a single abnormal mRNA in which exon 6 joins directly to exon 8, with a frameshift and a premature stop codon. In the G(-1) --&gt; A transition in intron 7, two mRNAs are generated. In the first, the retention of the whole intron 7 causes a frameshift and a premature stop codon; in the second, the joining of exon 7 to exon 8 is associated with an in-frame deletion of the first 6 nucleotides. All these novel mutations are predicted to produce structurally abnormal enzymatic proteins with no measurable biological activity.

1996 - Fatty liver in heterozygous hypobetalipoproteinemia caused by a novel truncated form of apolipoprotein B [Articolo su rivista]
Tarugi, Patrizia Maria; A., Lonardo; G., Ballarini; A., Grisendi; M., Pulvirenti; A., Bagni; CALANDRA BUONAURA, Sebastiano
abstract

Fatty liver has been anecdotally associated with heterozygous hypobetalipoproteinemia. The aim of this study was to characterize the molecular defect in a subject with heterozygous hypobetalipoproteinemia (low-density lipoprotein cholesterol, 52 mg/dL; apolipoprotein [apo] B, 15 mg/dL) and otherwise unexplained fatty liver. Plasma lipoproteins were separated by ultracentrifugation, and apo B was analyzed by electrophoresis and immunoblotting. A fragment of genomic DNA corresponding to the 5' end of exon 26 of the apo B gene was amplified by polymerase chain reaction and sequenced. The plasma lipoproteins of the proband contained, besides normal apo B-100, a 200-kilodalton truncated apo B whose size suggested the presence of a mutation in exon 26 of the apo B gene. The nucleotide sequence of a fragment of the 5' end of exon 26 revealed that the proband was a heterozygote for a 14-nucleotide deletion, producing a frameshift resulting in a premature stop codon at residue 1768. This truncated apo B was named apo B-38.95. The proband's father was a carrier of the same mutation. Fatty liver in this subject with familiar heterozygous hypobetalipoproteinemia most likely results from the inability of apo B-38.95 to export lipids from hepatocytes into the blood stream. Heterozygous hypobetalipoproteinemia should be considered in a hypolipidemic subject with an otherwise unexplained fatty liver.

1996 - Synthesis and secretion of B-100 and A-I apolipoproteins in response to the changes of intracellular cholesteryl ester content in chick liver [Articolo su rivista]
Tarugi, Patrizia Maria; S., Nicolini; G., Ballarini; L., Marchi; C., Duvigneau; P., Tartoni; CALANDRA BUONAURA, Sebastiano
abstract

We investigated in the chick whether the diet-induced changes of the hepatic content of cholesteryl esters (CE) influence the synthesis and the secretion of apoB- and apoA-I-containing lipoproteins. Control chicks received a low cholesterol diet for 2 (SD-1), 4 (SD-2), or 7 (SD-3) weeks; the chicks in the experimental groups received a cholesterol-rich diet for 2 weeks and were killed at the end of the cholesterol feeding (CH-F), and after 2 (CH-D) or 5 (CH-DD) weeks of a low cholesterol diet. Hepatic CE content in CH-F chicks was 30-fold that observed in controls, but returned to the control level after 5 weeks of cholesterol depletion (CH-DD). The incorporation of S-35-labeled amino acids into cell and medium apoB and apoA-I was measured in liver slices. Intracellular S-35-labeled apoB was similar in all groups whereas medium S-35-labeled apoB was 2-fold higher in CH-F than in controls (SD-I). Pulse-chase experiments showed that radioactive apoB secreted by CH-F chicks at 120 min of chase was 2 times that of SD-1 chicks. This increased secretion of apoB was not found in CH-D chicks. In H-F chicks, the intracellular and medium S-35-labeled apoA-I were 2-fold the values found in controls (SD-1); apoA-I production returned to the control level only after 5 weeks of cholesterol depletion (CH-DD). The increased secretion of apoB and apoA-I in CH-F chicks was associated with an increased secretion of very low, intermediate, and low density lipoproteins containing newly synthesized apoB and apoA-I and of high density lipoproteins containing predominantly apoA-I. Thus, in response to hepatic CE accumulation induced by cholesterol feeding, a larger proportion of newly synthesized apoB is driven to the secretory pathway and more apoA-I is synthesized. This promotes an increased secretion of plasma lipoproteins that contribute to the removal of CE from the liver.

1995 - Four novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia. [Articolo su rivista]
S., Bertolini; R., Garuti; W., Lelli; M., Rolleri; Tiozzo, Roberta; Ghisellini, Margherita; Ml, Simone; P., Masturzo; Nc, Elicio; C., Stefanutti; D., Coviello; C., Carabbio; G., Orecchini; CALANDRA BUONAURA, Sebastiano
abstract

In this study, we report four new partial deletions of the LDL-receptor (LDL-R) gene discovered during a survey of 326 Italian patients with familial hypercholesterolemia (FH). All deletions were found in FH heterozygotes whose LDL-R activity in skin fibroblasts ranged from 52% to 43% of the values found in control cells. The size and boundaries of the deletions were defined by Southern blotting and, in some cases, by polymerase chain reaction (PCR) amplification of genomic DNA. The sequence of the deletion joint was performed after the reverse transcription and PCR amplification of the appropriate regions of LDL-R mRNA. FHMassa is a 12-kilobase deletion spanning from intron 2 to intron 10. RT-PCR showed that the mutant allele is transcribed into one major and two minor mRNAs. In the most abundant mRNA species, exon 2 joins exon 11, as expected from DNA analysis. In one minor mRNA, which was sequenced, exon 2 joins exon 13, with exons 11 and 12 skipped as a result of an alternative splicing. FHGenova is a 4-kb deletion spanning from intron 10 to intron 12 and eliminating exons 11 and 12. FHRoma is a 4.7-kb deletion spanning from the 5' end of intron 12 to the middle of intron 14 and eliminating exons 13 and 14. This deletion differs in size from the previously described deletion (FHChieti/Macerata), which is located in the same region of the LDL-R gene but is smaller (3.7 kb). In both FHRoma and FHChieti/Macerata, the mutant LDL-R mRNA is present in a minute amount, suggesting that the deletion of exons 13 and 14 may increase mRNA degradation. FHPadova-2 is a 2-kb deletion spanning from intron 15 to intron 16 and eliminating the sole exon 16. All deletions except FHPadova-2 produce a shift in the reading frame, leading to either a very short peptide or a truncated protein. In FHPadova-2, elimination of exon 16 does not change the reading frame but is predicted to produce a receptor protein of 513 amino acids, lacking 18 amino acids of the O-linked sugar and 8 amino acids of the transmembrane domain. Ligand blot experiments with rabbit I-125-beta VLDL indicate that half the amount of LDL-receptor is present in FHPadova-2 fibroblasts, suggesting that the in-frame deletion of 26 amino acids may disrupt the intracellular transport and/or the insertion of the receptor in the plasma membrane or may increase its degradation.

1994 - Apolipoprotein B-100 production and cholesteryl ester content in the liver of developing chick [Articolo su rivista]
Tarugi, Patrizia Maria; Nicolini, S; Marchi, L; Ballarini, G; CALANDRA BUONAURA, Sebastiano
abstract

In the chick, the large cholesteryl ester (CE) store present in the liver during the last period of embryonic life increases at hatching and is rapidly depleted after 2-7 days of postnatal life. In this study we asked whether these changes were associated with variations in the hepatic production of apoB-containing lipoproteins. Liver slices taken from chicks at -3, 0 (hatching), 2, 4, 7, and 10 days of development were incubated with [35S]methionine in steady state incubations. ApoB production (cell + medium radioactivity) decreased from day -3 to day 0 (40%), increased at day 4 (54%), and decreased afterwards (45%). At day 4 the amount of 35S-labeled apoB-containing lipoproteins (VLDL-LDL) secreted into the medium was 1.7- and 1.5-times that found at days 0 and 7, respectively; the radioactivity incorporated into medium HDL (containing predominantly apoA-I) was 1.7-times that found at days 0 and 7. The incubation of liver slices with [3H]oleate showed that CE production at days 4 and 7 was 58% and 33%, respectively, of that found at day 0. The percentage of newly synthesized hepatic CE secreted into medium lipoproteins was 2.4%, 3.1%, and 2.2% at days 0, 4, and 7, respectively. The percentage of lipoprotein CE present in VLDL-LDL ranged from 38% at day 0 to 21% at day 7, and that present in HDL ranged from 62% at day 0 to 79% at day 7. To define whether the changes in the production of apoA-I- and apoB-containing lipoproteins were due to variations in apoB and apoA-I synthesis, the initial synthetic rate (pulse-labeling) and the mRNA content of these apolipoproteins were investigated. The initial apoB synthetic rate decreased 1.5-fold from day -3 to day 0, remained stable up to day 7, and decreased at day 10. Hepatic apoB mRNA followed a similar trend. The synthesis of apoA-I increased 2-fold from days -3/2 up to day 4 and did not change afterwards. In conclusion the increased hepatic CE content at hatching reflects a decreased production of apoB, while the depletion of CE observed from day 2 to day 7 is associated with an increased production of both apoB- and apoA-I-containing lipoproteins. The decreased apoB production at hatching is due to a decreased apoB synthesis whereas the increased apoB production at day 4 appears to be related to a post-translational event.

1994 - Effect of a thromboxane A2 synthase inhibitor on the dyslipoproteinemia of an inbred rat strain with spontaneous age-related nephrotic syndrome [Articolo su rivista]
Tarugi, Patrizia Maria; Nicolini, S.; Albertazzi, L.; Marchi, L.; CALANDRA BUONAURA, Sebastiano; Salvati, P.
abstract

We have previously shown that the administration of a thromboxane A(2) (TXA(2)) synthase inhibitor (FCE 22178) reduced the progression of glomerular lesions and proteinuria in MNS rats, an inbred strain which develops an age-related nephrotic syndrome. In the present study we investigated the effect of FCE 22178 on the plasma lipoproteins of MNS rats at 28 weeks of age (with mild proteinuria and moderate dyslipoproteinemia) and at 48 weeks of age (with heavy proteinuria and severe dyslipoproteinemia). Drug treatment reduced proteinuria (by 70% and 36% at 28 and 48 weeks of age, respectively) plasma cholesterol (by 36% and 27% at 28 and 48 weeks of age, respectively) and prevented the decrease of plasma albumin observed in untreated rats (C-MNS) 48 weeks old. In treated rats (T-MNS), the decrease of proteinuria wets positively correlated with that of plasma cholesterol. FCE 22178 reduced the elevation in plasma HDL(1) (by 17.4%) and HDL(2) levels (by 30%), a key feature of nephrotic dyslipoproteinemia in the rat. From 28 to 48 weeks of age plasma apo A-I and apo E increased 217% and 128%, respectively, in C-MNS rats and 191% and 121%, respectively, in T-MNS-rats. A significant increase of apo A-I/apo E ratio was found in C-MNS rats from 28 (2.28+/-0.36) to 48 weeks of age (3.84+/-0.9) but not in T-MNS rats. FCE 22178 altered the lipid composition of VLDL and HDL(2) by reducing the content of cholesteryl esters and increasing that of free cholesterol and phospholipids. These findings suggest that the beneficial effect of FCE 22178 on the dyslipoproteinemia of nephrotic MNS rats is secondary to the amelioration in kidney function and to the reduction of proteinuria produced by this drug.

1994 - Partial duplication of the EGF precursor homology domain of the LDL receptor protein causing familial hypercholesterolemia (FH-Salerno) [Articolo su rivista]
Bertolini, S; Patel, Dd; Coviello, Da; Lelli, N; Ghisellini, Margherita; Tiozzo, Roberta; Masturzo, P; Elicio, N; Knight, Bl; CALANDRA BUONAURA, Sebastiano
abstract

Abstract A novel mutation of low density lipoprotein (LDL)-receptor gene was found in an Italian familial hypercholesterolemia(FH) patient during a screening of 300 FH patients. Theproband as well as her daughter were found to be heterozygotesfor the mutation. Binding, internalization, and degradation of‘25I-labeled LDL by the proband’s fibroblasts were reduced toapproximately 50% compared to values found in control cells.DNA analysis by Southern blotting showed that the mutant allelewas characterized by an insertion of about 10 kb, whichresulted from a duplication of exons 9-14 of the LDL-receptorgene. In addition, Northern blot analysis of the proband’s RNAshowed, besides the normal-sized LDL-receptor mRNA (5.3kb), an additional mRNA of about 6.2 kb. The junction betweenexon 14 and the duplicated exon 9 was amplified by polymerasechain reaction (PCR) from the cDNA. The sequence of the amplifiedfragment showed that exon 14 joined the duplicated exon9 without changing the reading frame. The derived amino acidsequence indicated that the mutated receptor protein had a partialduplication of the EGF precursor homology domain. Ligandand immunoblotting revealed that proband’s fibroblasts containedone-half of the normal amount of LDL-receptor protein(molecular mass 130 kDa) and an abnormally large receptor ofapproximately 160 kDa. The amount of this abnormal receptoras detected by two monoclonal antibodies (10A2 and 4B3) wasfound to be approximately 30% that of the normal LDLreceptorpresent in the same cells. Treatment of the proband’scells with pronase greatly reduced the amount of both normaland abnormal receptors detected, indicating that both receptorswere present on the cell surface. Pulse-chase experiments using[35S]methionine indicated that the receptor was processed to themature form (195 kDa), although at a rate slightly slower thanthe normal receptor (160 kDa) present in the same cells.However, the mature abnormal receptor was degraded muchmore rapidly (half life 4.6 h) than the normal receptor presentin the proband’s cells (half life 11.9 h) or in normal cells (half life12.4 h). I In conclusion, the mutant allele present in our probandproduces an abnormally large receptor protein that is normallyprocessed to the mature form but is degraded morerapidly than the normal counterpart. As the proband‘s familyoriginated from the city of Salerno, in southern Italy, the mutationwas named FH-Salerno.

1993 - Effect of the desulfation of heparin on its anticoagulant and anti-proliferative activity [Articolo su rivista]
Tiozzo, Roberta; Cingi, Mr; Reggiani, D; Andreoli, T; CALANDRA BUONAURA, Sebastiano; Milani, Mr; Piani, S; Marchi, E; Barbanti, M.
abstract

Some glycosaminoglycans (GAGs), such as heparin and heparin-like compounds inhibit the proliferation of several cell types, including smooth muscle cells, cervical epithelial cells and fibroblasts (1-3). In order to establish which domain of the hepar-in molecule is specifically responsible for the anti-proliferative activity, several strategies have been adopted such as: chemical modification or fractionation of the heparin molecule into low molecular weight fragments or synthesis of oligosaccharides with a defined chemical structure (4-6). In the present study we attempted to determine the role of N- and 0- linked sulfate groups on the anti-proliferative and anticoagulant effect of heparin. To that purpose we modified the molecule to produce N-desulfated, 0-desulfated compounds. The anti-proliferative activity of these modified heparins was compared to that of low molecular weight heparins obtained by depolymerization, heparan sulfate as N-acetylated compound with glucuronic acid and heparin. Since the anti-proliferative effect of heparin depends also on the cell type, we used two different cell types: BHK-21 (hamster fibroblasts) and human arterial SMC (smooth muscle cells), that were found to exhibit a high or intermediate sensitivity to heparin (3,7).

1993 - SYNTHESIS AND SECRETION OF APOLIPOPROTEIN-A-I [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria
abstract

1992 - A large deletion in the LDL receptor gene--the cause of familial hypercholesterolemia in three Italian families: a study that dates back to the 17th century (FH-Pavia [Articolo su rivista]
Bertolini, S; Lelli, N; Coviello, Da; Ghisellini, Margherita; Masturzo, P; Tiozzo, Roberta; Elicio, N; Gaddi, A; CALANDRA BUONAURA, Sebastiano
abstract

In the LDL-receptor gene, a large rearrangement causing hypercholesterolemia was detected in three apparently unrelated families living in northern Italy. In all probands, binding, internalization, and degradation of 125I-LDL measured in skin fibroblasts were found to be 40%-50% of control values, indicative of heterozygous familial hypercholesterolemia (FH). Southern blot analysis revealed that the probands were heterozygous for a large (25-kb) deletion of the LDL-receptor gene eliminating exons 2-12. The affected subjects possessed two LDL-receptor mRNA species: one of normal size (5.3 kb) and one of smaller size (3.5 kb). In the latter mRNA, the coding sequence of exon 1 is joined to the coding sequence of exon 13, causing a change in the reading frame and thereby giving rise to a premature stop codon. The receptor protein deduced from the sequence of the defective mRNA is a short polypeptide of 29 amino acids, devoid of any function. Tracing these three families back to the 17th century, we found both their common ancestor and the possible origin of the mutation, in a region which is called "Lomellina" and which is located in southwest Lombardy, near the old city of Pavia. Therefore we named the mutation "FH-Pavia."

1992 - Sequential expression during postnatal development of specific markers of junctional and free sarcoplasmic reticulum in chicken pectoralis muscle. [Articolo su rivista]
Damiani, E; Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; Margreth, A.
abstract

Skeletal muscle sarcoplasmic reticulum comprises two distinct membrane domains, i.e., the Ca(2+)-pump membrane, corresponding mainly to longitudinal tubules, and the junctional membrane of the terminal cisternae containing the ryanodine receptor/Ca(2+)-release channel. Additional minor proteins previously shown in rabbit fast-twitch skeletal muscle to fractionate selectively to each membrane domain comprise 160- and 53-kDa glycoproteins and 170-kDa low-density lipoprotein (LDL)-binding protein, respectively (Damiani and Margreth, 1991, Biochem. J. 277, 825-832). We report evidence in chicken pectoralis, a predominantly fast muscle, on two closely immunologically related glycoproteins, a minor component of 130-kDa and a major 53-kDa protein. In contrast to the seemingly highly conserved structure of this protein, our results show marked differences in mobilities for chicken 125I-LDL that were detected as a 130- to 116-kDa protein doublet after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although being otherwise indistinguishable from rabbit 170-kDa protein in LDL-binding characteristics, as well as for preferential association to junctional terminal cisternae. Chicken Ca(2+)-ATPase, although being extensively homologous to rabbit Ca(2+)-ATPase, is shown to be less active and to differ slightly in electrophoretic properties. We have investigated the time course of expression of the specific protein components of longitudinal and of junctional sarcoplasmic reticulum in chick pectoralis muscle from late embryonic development up to 2 months after hatching. Coincident with the posthatching increase in membrane density of high-affinity [3H]ryanodine-binding sites in muscle, both calsequestrin and the species-specific LDL-binding protein(s) are detected in increasing amounts, using ligand blot techniques. In contrast, the appearance and steady accumulation in muscle of Ca(2+)-ATPase, like the time-correlated increase of sarcoplasmic reticulum glycoproteins, are relatively delayed, the most striking changes occurring from 1 week after hatching onward. The sequential expression in chick developing muscle of proteins selectively associated with the junctional terminal cisternae and with longitudinal sarcoplasmic reticulum, respectively, argues for a similar morphogenetic program in avian and mammalian species and, to account for that, for the existence of common epigenetic differentiating influences on the expression of sarcoplasmic reticulum protein genes.

1991 - Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia. [Articolo su rivista]
Lelli, N; Ghisellini, Margherita; Gualdi, R; Tiozzo, Roberta; CALANDRA BUONAURA, Sebastiano; Gaddi, A; Ciarrocchi, A; Arca, M; Fazio, S; Coviello, Da; Bertolini, S.
abstract

Three gross rearrangements of the low density lipoprotein receptor (LDL-R) gene were recognized during a survey of 23 unrelated Italian subjects with familial hypercholesterolemia (FH). Restriction endonuclease data were obtained by Southern blotting and hybridization with exon-specific probes. Proband FH-29 is heterozygous for a 4-kb deletion, which eliminates exons 13 and 14. This mutation is similar to that previously reported by other investigators in one Italian homozygous and two British and Canadian heterozygous patients. Proband FH-30 is homozygous for a 5.5-kb insertion caused by a duplication of exons 16 and 17 of the LDL-R gene. LDL-R mRNA isolated from skin fibroblasts of FH-30 was found to be larger than normal mRNA (5.6 versus 5.3 kb), in concordance with the insertion of the 236 nucleotides corresponding to exons 16 and 17. Proband FH-44 was found to have greater than 25-kb deletion, which eliminates the first six exons and the promoter region of the gene. This is the first example of a deletion that eliminates the promoter as well as the ligand-binding domain of the LDL-R gene. In the skin fibroblasts of this patient, the level of LDL-R mRNA was approximately half that found in control fibroblasts. We designate the new mutations found in FH-30 and FH-44 as FHviterbo and FHBologna-1, respectively, after the names of the Italian cities where the two patients were born.

1991 - Duplication of exons 13, 14 and 15 of the LDL-receptor gene in a patients with heterozygous familial hypercholesterolemia [Articolo su rivista]
Lelli, N; Ghisellini, Margherita; CALANDRA BUONAURA, Sebastiano; Gaddi, A; Ciarrocchi, A; Coviello, Da; Bertolini, S.
abstract

During a survey of Italian patients with familial hypercholesterolemia (FH), we identified an FH heterozygous patient with a gross rearrangement of the low density lipoprotein (LDL) receptor gene. Southern blot analysis of the proband's DNA digested with restriction enzymes PvuII, BamHI, BglII and XbaI and hybridization with cDNA probes complementary to the 3' end of the gene revealed the presence of abnormal fragments that were approximately 7 kb larger than their normal counterparts. DNA digestion with other enzymes (EcoRV, NcoI, KpnI and StuI) and hybridization with probes complementary to exons 13-17 generated normal fragments and an abnormal fragment of 6.3-6.8 kb. These results are consistent with the presence of an insertion of approximately 7 kb caused by a duplication of exons 13, 14 and 15. This is a novel mutation that is most probably the result of an unequal crossing-over between repetitive sequences located in intron 12 and intron 15. This novel mutation has been designated FHBologna

1991 - Effect of heparin derived fractions on the proliferation and protein synthesis of cells in culture. [Articolo su rivista]
Tiozzo, Roberta; Reggiani, D; Cingi, Mr; Bianchini, P; Osima, B; CALANDRA BUONAURA, Sebastiano
abstract

We investigated the effect of sulfated oligosaccharides derived from depolymerization of heparin on the proliferation and protein synthesis of smooth muscle cells (SMC), hamster kidney (BHK-21) and lung (V-79) fibroblasts, rat hepatoma cells (FAO) and human promyelocytes (HL-60). BHK-21 and FAO showed the highest sensitivity to heparin; V-79 and HL-60 cells were completely resistant. LMWH (Low Molecular Weight Heparin) (MW 4.5 kD) was as effective as unfractionated heparin in reducing cell proliferation. The oligo-derivative 381/1 (MW 2 kD) was effective only on FAO and BHK-21 cells; oligo-derivative 381/2 (MW 1KD) had a negligible effect. The anti-proliferative effect was associated with an increased secretion of some protein classes. This effect was not present in heparin-resistant cells. In conclusion when the molecular size of heparin derivative is reduced below 2 kD (i.e. the size of a hexasaccharide) the anti-proliferative activity decreases dramatically.

1991 - Influence of age-related nephropathy on plasma lipoproteins of the aging rat [Articolo su rivista]
Calandra, S.; Tarugi, P.
abstract

1991 - Pravastatin in heterozygous familial hypercholesterolemia: low-density lipoprotein (LDL) cholesterol-lowering effect and LDL receptor activity on skin fibroblastS. [Articolo su rivista]
Gaddi, A; Arca, M; Ciarrocchi, A; Fazio, S; D'Alò, G; Tiozzo, Roberta; Descovich, Gc; CALANDRA BUONAURA, Sebastiano
abstract

The cholesterol-lowering effect of provastatin, a new competitive inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, was studied in 10 patients with heterozygous familial hypercholesterolemia (FH). Residual low-density lipoprotein receptor (LDL-R) activity was also evaluated in cultured skin fibroblasts prior to treatment, and showed a wide range of reduction from 30% to 70% of the normal value. Treatment with pravastatin 40 mg once daily reduced total and LDL cholesterol (LDL-C) after 6 months by 19.7% and 25.4%, respectively (P less than .001). Serum apolipoprotein (apo) B levels decreased significantly by 29.1% (P less than .001). No significant changes were observed in mean serum total triglycerides or high-density lipoprotein cholesterol (HDL-C) levels. A positive correlation between residual LDL-R activity and maximum percent reduction of LDL-C levels was observed (r = .676, P less than .05). No clinically important side effects were recorded and the treatment was well tolerated. Thus, pravastatin effectively reduces LDL in heterozygous FH, and this effect appears to be related to LDL-R status.

1991 - Synthesis and secretion of apolipoprotein A-I by chick skin. [Articolo su rivista]
Tarugi, Patrizia Maria; Albertazzi, L; Nicolini, S; Ottaviani, Enzo; CALANDRA BUONAURA, Sebastiano
abstract

Chick skin slices were incubated with [35S]methionine and labeled apoA-I was immunoprecipitated from incubation medium and tissue homogenate. ApoA-I accounted for approximately 13 and 2.5% of radioactive medium and cell proteins, respectively. After ultracentrifugation of the medium, 55% of labeled apoA-I was found as a constituent of lipoproteins (d less than 1.210 g/ml) and 45% in a lipid-poor form (1.210-1.260 g/ml). To ascertain whether this large proportion of lipid-poor apoA-I was due to a dissociation of this peptide from medium lipoproteins during ultracentrifugation, labeled incubation medium was applied to an anti-chick apoA-I immunoaffinity column. The material bound to the column was analyzed by nondenaturing polyacrylamide gradient gel electrophoresis and found to contain three subpopulations of lipoproteins with a particle size of 12, 11, and 9 nm, respectively. The radioactivity of these subpopulations accounted for 82% of total radioactive medium apoA-I. ApoA-I was localized by immunohistochemistry in the viable cells of the epidermis and in the stratum corneum. Rat skin slices were found to synthesize and secrete apoE but no apoA-I. ApoA-I and apoE secreted by chick and rat skin, respectively, may play a role in the secretion of lipids from the differentiating keratinocytes and thus contribute to the formation of the hydrophobic barrier of the skin.

1990 - Absence of apolipoprotein B-48 in the chick, Gallus domesticus [Articolo su rivista]
Tarugi, Patrizia Maria; Albertazzi, L; Nicolini, S; CALANDRA BUONAURA, Sebastiano
abstract

This study was designed to investigate: a) whether multiple forms of apoB are present in chick plasma lipoproteins; and b) which forms of apoB are produced in vitro by liver and intestine at various stages of pre- and post-natal development. Plasma lipoproteins of d less than 1.019 g/ml, isolated from fasted and nonfasted chicks, contained exclusively the high molecular weight apoB form (apoB-100) that comigrated with human and rat apoB-100 on SDS-PAGE gel. No apoB-48 was detected either in overloaded Coomassie blue-stained gels or after immunoblotting. ApoB-100 but no apoB-48 was found in portomicrons, the triglyceride-rich lipoproteins equivalent to chylomicrons, that in the chick are transported via the porto-mesenteric venous system. To ascertain whether a minute amount of apoB-48 was present in chick plasma, [35S]methionine was injected intraduodenally and the 35S-labeled d less than 1.019 g/ml plasma lipoproteins were isolated 45 min later from the systemic and the porto-mesenteric circulation. Only apoB-100 was found to be labeled in these lipoproteins. Cholesterol feeding did not induce the appearance of apoB-48 in plasma despite a marked accumulation of cholesterol-rich d less than 1.040 g/ml lipoproteins in the plasma. In vitro synthesis of apoB forms was studied in liver and intestinal slices isolated from chick embryos (8 and 5 days before hatching), newly hatched chicks (2 and 7 days after hatching), and young chicks (21 days old) that were incubated in the presence of [35S]methionine. At each stage of development, liver slices secreted predominantly apoB-100. Intestinal slices of newly hatched and young chicks secreted two forms of apoB: apoB-100 and an additional form with an electrophoretic mobility similar to rat plasma apoB-95. No apoB-48 was synthesized or secreted by the intestine. Our results indicate that the absence of apoB-48 in chick plasma reflects the lack of synthesis of this peptide in the intestine. It is conceivable that in chick intestine the recently described molecular mechanism responsible for the co/posttranscriptional modification of apoB mRNA leading to the formation of apoB-48 is lacking or defective.

1990 - Biochemistry of lipids and lipoproteins [Articolo su rivista]
Calandra, S.; Tarugi, P.
abstract

1989 - Effect of development on apo A-I gene expression in the chick [Relazione in Atti di Convegno]
Calandra, S.; Tarugi, P.
abstract

1989 - Plasma lipoproteins, tissue cholesterol overload, and skeletal muscle apolipoprotein A-I synthesis in the developing chick. [Articolo su rivista]
Tarugi, Patrizia Maria; Reggiani, D; Ottaviani, Enzo; Ferrari, S; Tiozzo, Roberta; CALANDRA BUONAURA, Sebastiano
abstract

In the present study we investigated the changes of plasma lipids, lipoproteins, and tissue lipids that occur during the late embryonic life (5 days before hatching) and the postnatal period (0, 2, 7, 14, and 30 days after hatching) of the chick. The chick emerges from the egg with extreme hypercholesterolemia associated with a high level of cholesterol-rich VLDL + IDL. The density gradient profile of plasma lipoproteins showed that the concentrations of VLDL + IDL and LDL decreased during the first week of postnatal life, whereas HDL concentration increased sharply around hatching and remained stable afterwards. All plasma lipoprotein classes of the newborn chick (2 days from hatching) were enriched in cholesterol and cholesteryl esters; 2 weeks after hatching, the relative amount of cholesterol and cholesteryl esters decreased. In the newborn chick, plasma VLDL + IDL consisted of two populations of cholesteryl ester-rich lipoproteins: the main one (designated apoB-VLDL) contained apoB and no apoA-I; the other (designated apoA-I-VLDL) contained predominantly apoA-I. In the newborn chick there was an accumulation of free and esterified cholesterol in the liver and, to a lesser extent, in the skeletal muscle. These cholesterol deposits were depleted 2 to 7 days after hatching. The depletion in skeletal muscle was preceded by and associated with a striking increase in the synthesis of apoA-I in this tissue, as demonstrated by immunological methods and apoA-I mRNA measurements. In addition, apoA-I-containing HDL were secreted in vitro by explants of skeletal muscle of the newborn chick. The synthesis of apoA-I in the skeletal muscle decreased to the level found in the adult animal 1 week after hatching. It is likely that the rise of HDL and apoA-I in plasma observed 1-2 days after hatching reflects the production of apoA-I-containing HDL by skeletal muscle. We suggest that the cholesterol overload in skeletal muscle might stimulate the production of apoA-I which, in turn, would promote the removal of cholesterol from this tissue. The hypothesis that metabolic stimuli play a role in inducing apoA-I synthesis in skeletal muscle is supported by the observation that feeding the newborn chick a diet rich in proteins and lipids and free of carbohydrates delays the fall of apoA-I mRNA which normally occurs 1 week after hatching

1988 - Modulation of the synthesis of apolipoproteins in rat hepatoma cells. [Articolo su rivista]
Pietrangelo, Antonello; Tiozzo, Roberta; Ghisellini, Margherita; Cingi, Mr; Albertazzi, L; Ventura, E; CALANDRA BUONAURA, Sebastiano
abstract

The present study was designed to investigate whether plasma lipoproteins and albumin can affect the basal synthetic rate of apolipoproteins in differentiated rat hepatoma cells (Fao) incubated in serum-free medium. The synthesis of apolipoproteins was measured by the incorporation of [35S]methionine into medium lipoproteins isolated by density gradient ultracentrifugation. Under all the experimental conditions used, Fao cells synthesized almost exclusively apolipoprotein E. When cells were incubated in the presence of 5-10% rat plasma the synthesis of apolipoprotein E increased 2-3-fold; lipoprotein-deficient serum had a negligible effect. Fatty acid-poor bovine serum albumin (BSA), which had been found to reduce very-low-density lipoprotein secretion in isolated rat hepatocytes, did not modify the synthesis of apolipoprotein E. When Fao cells were incubated in medium containing rat plasma lipoprotein fractions, the synthesis of apolipoprotein E increased. The d less than 1.090 g/ml plasma lipoprotein fraction had the major stimulatory effect. Increased apolipoprotein E synthesis was observed when cells were incubated in the presence of lipids extracted from rat plasma lipoproteins. These results suggest that the intracellular accumulation of lipoprotein-lipids plays an important role in regulating apolipoprotein E synthesis in Fao cells.

1987 - The complete sequence of chick apolipoprotein AI mRNA and its expression in the developing chick [Articolo su rivista]
Ferrari, Stefano; Tarugi, Patrizia Maria; Drusiani, E; CALANDRA BUONAURA, Sebastiano; Fregni, M.
abstract

The nucleotide (nt) sequence analysis of a full-length cDNA for chick apolipoprotein AI (Apo-AI) shows an open reading frame (ORF) of 792 nt, coding for a 264-aa protein. RNase mapping and sequence analysis of the 3' end show that apo-AI mRNA consists of at least two different species of 985 and 996 nt, respectively. During the embryonic life of the chick apo-AI mRNA is found in high concentration only in the liver, while its level in the intestine, the major Apo-AI producing organ in the adult, becomes significant only after hatching. This switch from liver to intestine, as primary site of apo-AI mRNA synthesis, takes place about ten days after hatching. The developmental control of the tissue levels of apo-AI mRNA is particularly evident in the skeletal muscle, where this mRNA species is present at high level only immediately after hatching. Preliminary evidence suggests that the time-limited rise in muscle apo-AI mRNA might be due to an increased rate of transcription.

1986 - Changes in apolipoprotein A-I mRNA level in the liver of rats with experimental nephrotic syndrome [Articolo su rivista]
Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; Chan, L.
abstract

In previous studies we had shown that: one of the most specific feature of hyperlipoproteinemia found in rats with experimental nephrotic syndrome is the accumulation of apolipoprotein A-I-rich HDL in plasma and this disorder is associated with an overproduction of apolipoprotein A-I by the liver. The present study was designed to investigate whether the increased hepatic synthesis of apolipoprotein A-I was due to an accumulation of functionally active apolipoprotein A-I mRNA in liver of nephrotic rats. Hepatic mRNA was translated in vitro by rabbit reticulocyte lysate in the presence of [35S]methionine and in vitro synthesized apolipoprotein A-I, albumin and apolipoprotein E were immunoprecipitated by specific rabbit IgG. In nephrotic rats the amount of in vitro synthesized apolipoprotein A-I was almost twice that found in the controls, suggesting that functionally active apolipoprotein A-I mRNA was increased in liver of nephrotic rats. To confirm that this difference in apolipoprotein A-I mRNA activity was due to an actual increase of hepatic apolipoprotein A-I mRNA sequences, we performed nucleic acid hybridization experiments (northern blot) using several cloned cDNA probes (rat and human apolipoprotein A-I, rat apolipoprotein E and apolipoprotein A-II). The results indicate that in nephrotic rats the amount of hybridizable apolipoprotein A-I mRNA sequences was about 3-fold higher than that in controls. In contrast, there was no difference in the amount of hybridizable apolipoprotein A-II and apolipoprotein E mRNA sequences, indicating that the change in apolipoprotein A-I mRNA induced by the nephrotic state was specific for this mRNA.

1986 - Changes of the main isoform of human apolipoprotein A-I following incubation of plasma. [Articolo su rivista]
Ghisellini, Margherita; Pecorari, M; CALANDRA BUONAURA, Sebastiano
abstract

AbstractThis study was aimed to ascertain whether the more acidic isoforms of plasma apo A-I (A-I-1 and A-I-2) could originate in vitro from the main plasma isoform (A-I0). Apo A-I isoforms were separated by two-dimensional gel electrophoresis before and after a prolonged incubation of serum or EDTA-plasma at 37 degrees C. Incubated plasma there was a marked decrease of apo A-I0 and a concomitant linear increase of apo A-I-1 + and A-I-2. The relative content of the latter raised from 22 +/- 7% before the incubation to 60 +/- 7% after 48 h of incubation. This conversion of A-I0 was not inhibited by either pre-heating of plasma at 60 degrees C for 1 h or the addition of protease inhibitors, EDTA and p-chloromercuriphenylsulfonic acid. The conversion of apo A-I0 was not observed in isolated HDL, incubated either in the absence or in the presence of d less than 1.063 g/ml lipoproteins and lipoprotein deficient plasma, nor in plasma which had been dialyzed before being incubated at 37 degrees C. This suggests that plasma contains a low molecular weight factor capable of promoting the conversion of A-I0. The increase of the relative content of apo A-I-1 and apo A-I-2 in incubated plasma was not due to glucosylation or carbamylation of A-I0 as no radioactive glucose and urea were found to be bound to A-I. Since the conversion of apo A-I0 was prevented by the addition of an antioxidant (butylated hydroxytoluene, BHT) to the incubated plasma it is conceivable that some product of lipid peroxidation renders apo A-I0 more electronegative by reacting with some free amino groups of this peptide

1986 - Isolation of a cDNA clone for chick intestinal apolipoprotein AI (Apo-AI) and its use for detecting apo-AI mRNA expression in several chick tissues. [Articolo su rivista]
Ferrari, Stefano; Drusiani, E; CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria
abstract

Three cDNA clones for chick apolipoprotein AI (Apo-AI), the major protein component of plasma high-density lipoproteins, have been isolated. The identity of the clones has been established first by screening a cDNA library in the pEX1 expression vector with anti-Apo-AI antibodies, second by Western blot analysis of the proteins expressed by positive clones. The use of the clone containing the largest, presumably full-size, cDNA insert (apo5C12) in molecular hybridization experiments confirms that apo-AI mRNA is expressed mainly in chick small intestine and liver. Furthermore, we provide evidence that brain, heart and skeletal muscle also synthesize significant amounts of apo-AI mRNA. The Southern-blot hybridization pattern of the restriction-enzyme-digested chick DNA with the apo5C12 DNA is consistent with there being a single copy of the apo-AI gene.

1985 - Isoforms of rat apolipoprotein A-I isolated from the lipoproteins of hepatic Golgi apparatus and plasma. [Articolo su rivista]
Tarugi, Patrizia Maria; Ghisellini, Margherita; Pecorari, M; Brugni, N; CALANDRA BUONAURA, Sebastiano
abstract

We compared apo A-I isolated from the lipoproteins of the Golgi apparatus of rat liver with apo A-I found in plasma lipoproteins. Golgi apo A-I consists of 3 main isoforms with a molecular weight of approximately 28000 and isoelectric points (pI) of 5.97, 5.88 and 5.76, respectively. Plasma apo A-I consists of 4 major and 3 minor isoforms with a molecular weight of 27000. The pI of the major isoforms (numbered 4-7) is 5.88, 5.80, 5.70 and 5.60, respectively. In order to investigate which of the plasma isoforms derived directly from Golgi apo A-I, [35S]methionine was injected into the portal vein and Golgi and plasma apo A-I were isolated shortly thereafter. While all Golgi isoforms were labelled only 3 isoforms of plasma apo A-I (namely isoforms 5, 6 and 7) were found to be labelled. The major plasma isoform (isoform 4 which accounts for more than 60% of apo A-I mass of plasma HDL) was found to be unlabelled. However, when 35S plasma lipoproteins newly secreted by the liver were incubated in vitro in the presence of heparinized plasma, labelled isoform 4 appeared suggesting that heparinized plasma contained some factor capable of converting isoforms 5-7 into isoform 4. This plasma factor appears to be a protease as the in vitro formation of isoform 4 is prevented by protease inhibitors.

1984 - Separation of the isoprotein forms of apoprotein A-I of rat, rabbit and human HDL by combined isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Ghisellini, Margherita
abstract

The distribution and the relative content of the isoprotein forms (isoforms) of apoprotein A-I (apo A-I) of HDL isolated from rat, rabbit and human plasma were studied by combined isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Rat apo A-I consists of seven isoforms having the same molecular weight (27,000) and moving in the 6.44-5.58 pH range. Isoforms 4, 5 and 6 are the major ones. Both rat HDL2 (1.090-1.210 g/ml) and purified rat apo A-I contain additional minor bands (designated 4a, 5a and 6a) which have the same isoelectric point as isoforms 4-6 but higher molecular weight (27,900). It is suggested that they might represent precursors of the main apo A-I isoforms. Rabbit apo A-I contains five isoforms focusing in the 5.69-5.34 pH range. Isoform 4 accounts for about 90% of apo A-I mass. Human apo A-I consists of five isoforms focusing in the pH range 5.91-5.0. Isoforms 3 and 4 are the main ones: their respective contents show high degrees of individual variation.

1983 - Plasma and urine lipoproteins during the development of nephrotic syndrome induced in the rat by adriamycin. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Ghisellini, Margherita; Gherardi, E.
abstract

The changes of plasma lipoproteins which occur during the development of nephrotic syndrome induced in the rat were investigated by the administration of the antineoplastic drug adriamycin. Rats received a single intravenous injection of the drug (7.5 mg/Kg) and were sacrificed 5, 10, 15, 20, 25, and 30 days after treatment. By monitoring plasma and urine albumin, four stages in the development of nephrosis were identified: (1) a prenephrotic stage, (2) a mild nephrosis with moderate albuminuria and hypoalbuminemia; (3) a severe nephrosis with massive albuminuria and severe hypoalbuminemia; and (4) a recovery stage in which plasma albumin showed the tendency to increase. Apart from a mild elevation of plasma triacylglycerols and VLDL observed as early as Day 5, no changes in plasma cholesterol and in the other lipoprotein classes were observed at the stage of mild nephrosis (Day 10). However, as the disease became more severe (Day 15-25) there was a striking increase of HDL1 (1.050-1.090 g/ml) and, above all, of HDL2 (1.090-1.210 g/ml). VLDL and LDL also increased but at a later stage. The elevation of HDL1 and HDL2 was associated with an increase of apolipoprotein A-I in plasma (fourfold increase). Moreover, the relative content of this apolipoprotein in HDL1 and HDL2 increased as the disease progressed from mild to severe, so that in severely nephrotic rats HDL1 and HDL2 contained almost exclusively A-I and C apolipoproteins. HDL enriched in apolipoprotein A-I were also found in urine of severely nephrotic animals. Since these findings are similar to those previously described in nephrotic syndrome induced by puromycin aminonucleoside (Gherardi, E., and Calandra, S. (1982). Biochim. Biophys. Acta 710, 188.) the following conclusions can be drawn: (1) the key signs of nephrotic syndrome (albuminuria and hypoalbuminemia) precede the elevation of plasma lipoproteins; (2) the pattern of nephrotic hyperlipoproteinemia evolves as a function of the severity of the disease; (3) the accumulation of HDL enriched in apolipoprotein A-I represents an early and specific feature of nephrotic hyperlipoproteinemia in the rat

1983 - Plasma post-heparin lipolytic activity in rats with nephrotic syndrome [Articolo su rivista]
Calandra, S.; Gottardi, E.; Tarugi, P.
abstract

1982 - Heavy metals and experimental atherosclerosis. Effect of lead intoxication on rabbit plasma lipoproteins. [Articolo su rivista]
Tarugi, Patrizia Maria; CALANDRA BUONAURA, Sebastiano; Borella, Paola; Vivoli, Gianfranco
abstract

Previous studies have suggested that exposure to heavy metals may be a risk factor in coronary atherosclerotic heart disease in humans as well as in experimental animals. Little is known however on the mechanism underlying the effect of heavy metals on the development of atherosclerosis. In this study we tried to ascertain whether exposure to lead might: (a) alter plasma lipoprotein in normally fed rabbits; and (b) aggravate the hyperlipidemia usually found in cholesterol-fed animals. Rabbits were fed a normal diet or a diet containing 1% cholesterol in the presence or in the absence of 0.5% of lead subacetate for 45 days. This produced an accumulation of lead in plasma and bone. While in cholesterol-fed rabbits, lead exposure did not modify the plasma lipoprotein pattern, in normally fed animals it induced a striking elevation of cholesterol esters. This was associated with an increased concentration of VLDL (1.006 g/ml), LDL1 (1.006-1.020 g/ml), LDL2 (1.020-1.050 g/ml) and HDL1 (1.050-1.210 g/ml). These lipoproteins had an elevated content of cholesterol esters and apolipoprotein B. It is suggested that some of these lipoproteins may be important in the development of atherosclerosis in subjects chronically exposed to lead.

1982 - Plasma and urinary lipids and lipoproteins during the development of nephrotic syndrome induced in the rat by puromycin aminonucleoside. [Articolo su rivista]
Gherardi, E; CALANDRA BUONAURA, Sebastiano
abstract

This study was undertaken to ascertain whether the alterations of plasma lipoproteins found in nephrotic syndrome induced by puromycin aminonucleoside were due to nephrotic syndrome per se, or, at least in part, to the aminonucleoside. The purpose of the present study was to investigate the changes in plasma and urinary lipoproteins during the administration of puromycin aminonucleoside (20 mg/kg for 7 days) and the subsequent development of nephrotic syndrome. Since massive albuminuria occurred after 6 days of treatment, the time-course study was divided into two stages: pre-nephrotic stage (day 1-5) and nephrotic stage (day 6-11). In pre-nephrotic stage the plasma level of fatty acids, triacylglycerol and VLDL decreased while that of phospholipid, cholesteryl esters and HDL remained constant. Plasma apolipoprotein A-I tended to increase (40% increase at day 5). At the beginning of nephrotic stage (day 6) the concentration of plasma albumin dropped to a very low level, while that of apolipoprotein A-I increased abruptly (4-fold increase) and continued to rise, although less steeply, in the following days. The plasma concentration of HDL followed the same pattern. Plasma VLDL and LDL increased at a later stage (day 9). Plasma apolipoprotein A-I was found not only in HDL (1.063-1.210 g/ml) but also in the LDL density class (1.025-1.050 g/ml). In the pre-nephrotic stage lipoproteinuria was negligible, while in the early nephrotic stage the urinary loss of plasma lipoproteins consisted mainly of HDL. These observations indicate that puromycin aminonucleoside alters plasma lipoproteins by lowering VLDL and increasing HDL. It is likely that the early and striking increase of plasma HDL found in nephrotic rats is related to a direct effect of the drug on HDL metabolism.

1981 - Secretion of lipoproteins, apolipoprotein A-I and apolipoprotein E by isolated and perfused liver of rat with experimental nephrotic syndrome. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Gherardi, E; Fainaru, M; Guaitani, A; Bartosek, I.
abstract

Nephrotic syndrome induced in the rat by the administration of puromycin aminonucleoside is accompanied by a hyperlipoproteinemia characterized by an elevation of all plasma lipoproteins, particularly of VLDL (1.006 g/ml) and HDL1 (1.050-1.090 g/ml). The increase of HDL1 is due to the accumulation of a lipoprotein species floating mainly in the density interval 1.050-1.090 g/ml, in which apolipoprotein A-I replaces apolipoprotein E as the major constituent peptide. This lipoprotein has been designated nephrotic HDL. The present study was conducted to establish whether nephrotic liver secreted more lipoproteins than the control liver and, in addition, produced a lipoprotein similar to nephrotic HDL found in plasma. Isolated livers from control and nephrotic rats were perfused with a lipoprotein-free medium for 3 h in a recirculating system. Lipoproteins were isolated by ultracentrifugation; apolipoprotein A-I and apolipoprotein E were measured in the whole perfusate at various time intervals. Nephrotic liver secreted twice as much VLDL and HDL2 and 30% more LDL and HDL1 than the control liver. This was accompanied by an increased secretion of both apolipoprotein A-I and apolipoprotein E, the levels of which were 6.5- and 2-fold, respectively, of those found in the control perfusates at the end of the perfusion. In view of the increased secretion of apolipoprotein A-I, the apolipoprotein A-I to apolipoprotein E ratio was much higher in the perfusates of nephrotic livers than in those of the controls. The concentration of apolipoproteins A-I and E in plasma of nephrotic rats was 7- and 2-fold, respectively, of that found in the plasma of the controls. In the perfusates of the nephrotic livers, we could not find a HDL1 (1.050-1.090 g/ml) rich in apolipoprotein A-I similar to that isolated from plasma (nephrotic HDL). We suggest that the latter is formed in the circulation from the intravascular modification of HDL2 secreted in excess by the liv

1980 - Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside. Plasma and urinary lipoproteins [Articolo su rivista]
E., Gherardi; L., Vecchia; CALANDRA BUONAURA, Sebastiano
abstract

Nephrotic syndrome (ascites, proteinuria, hypoalbuminemia, and hyperlipidemia) was induced in male Wistar rats by daily administration of puromycin aminonucleoside (20 mg/Kg). Hyperlipidemia was the result of a marked increase of all plasma lipids particularly triacylglycerols (7 to 8-fold the values found in the pair fed control rats). All plasma lipoprotein fractions were increased in nephrotic rats. VLDL increased 14-fold; LDL and HDL were 7- and 6.5-fold respectively above the pairfed control values. The apoprotein pattern of nephrotic VLDL was characterized by a loss of apoC-1 peptide and the presence of two bands in the region of ARP and apoA-1 peptides. SDS polyacrylamide gel electrophoresis indicated an increased content of ARP in nephrotic VLDL. Nephrotic LDL but not control LDL contained peptides entering the running gel in 8 M-urea gels in the region of ARP, A-1 and C peptides. Nephrotic HDL were almost devoided of ARP and apoA-IV peptides whereas control HDL contained significant amounts of these peptides. The urine of nephrotic rats contained a large amount of lipids (21.9 vs 1.5 M × 10−6/day). Fatty acids, cholesteryl esters, and phospholipids were the major lipids of nephrotic urine. More than 72% of these lipids were isolated by preparative ultracentrifugation into three fractions: Total Low Density Lipoproteins (< 1.050 g/ml), High Density Lipoproteins (1.063 to 1.210 g/ml) and Ultracentrifugal Residue (> 1.210 g/ml) which contained respectively 7.4, 44.4, and 48.2% of the total recovered lipids. Urinary excretion of HDL was much greater than that of TLDL indicating that lipoproteinuria of aminonucleoside induced nephrotic syndrome was selective. The relationship between these findings, previously reported data and the alterations of plasma and urinary lipoproteins in human nephrotic syndrome is discussed

1980 - Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside: hepatic synthesis of lipoproteins and apolipoproteins. [Articolo su rivista]
Gherardi, E; Messori, M; Rozzi, R; CALANDRA BUONAURA, Sebastiano
abstract

Hepatic synthesis of lipoproteins and apolipoproteins was investigated in male Wistar rats with severe nephrotic syndrome induced by puromycin aminonucleoside by incubating liver slices with a mixture of 14C-amino acids. Labeled lipoproteins were separated by preparative ultracentrifugation from the incubation medium after the addition of carrier plasma. The incorporation of 14C-amino acids into very low density lipoproteins (VLDL) (1.006 g/ml), low density lipoproteins (LDL) (1.006-1.063 g/ml) and high density lipoproteins (HDL) (1.063-1.210 g/ml) was increased in nephrotic liver 6.1-, 5.7- and 5.0-fold, respectively. The measurement of radioactivity associated to apolipoproteins isolated by SDS-PAGE documented an increased incorporation into apolipoprotein E (apoE) of nephrotic VLDL (33.1% vs 20% of the total radioactivity incorporated into VLDL apoproteins) and a markedly increased incorporation into apolipoprotein A-I (apo-A-I) of nephrotic HDL (44.3% vs 16.3% of the total radioactivity incorporated into HDL apoproteins). In nephrotic liver, the total incorporation of amino acids into apolipoproteins (apoVLDL + apoLDL + apoHDL) was increased 12.6 times for apo-A-I, 6,4 times for apoB, 5.0 times for apoE, 4.2 times for apoC + apoA-II and 2.5 times for apoA-IV. We suggest that, in nephrotic liver: (a) the synthesis of VLDL, LDL and HDL is increased, and (b) the total synthesis of apoA-I is selectively increased when compared to that of the other apolipoproteins

1980 - Experimental nephrotic syndrome induced in the rat by puromycin aminonucleoside: hepatic synthesis of neutral lipids and phospholipids from 3H-water and 3H-palmitate. [Articolo su rivista]
Gherardi, E; CALANDRA BUONAURA, Sebastiano
abstract

Abstract not available

1979 - Cholesterol synthesis in isolated rat hepatocytes: effect of homologous and heterologous serum lipoproteins. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Tarugi, Patrizia Maria; Battistini, Nino Carlo; Ferrari, Renata
abstract

The present study investigated the effect of serum lipoproteins on sterol synthesis by isolated rat hepatocytes. These cells were maintained in culture medium for 24 hr and incubated for the same period of time with increasing concentrations of serum lipoproteins (5-150 microgram of lipoprotein-protein per ml) isolated from different animal species. The viability of the cells was ascertained by their ability to synthesize cholesterol and protein and to secrete serum proteins into the medium. Rat VLDL and LDL did not alter sterol synthesis, which was stimulated instead by HDL. Rat serum chylomicrons were also ineffective. Human LDL significantly reduced the synthesis of sterols from both acetate and tritiated water; this effect was also induced by human VLDL to a reduced extent. VLDL isolated from hypercholesterolemic rabbit (VLDLC) strongly inhibited sterol synthesis from acetate but not from mevalonate. Cholesteryl-ester-rich VLDL isolated from a patient with type III hyperlipidemia (type III VLDL) were more effective than normal VLDL in suppressing sterol synthesis from acetate. The implications of these findings are discussed with regard to the possible role of cholesteryl-ester-rich lipoproteins on the in vivo regulation of sterol synthesis in the liver.

1979 - Failure of LP-X to suppress sterol synthesis in rat hepatocytes [Articolo su rivista]
Battistini, N.; Tarugi, P.; Calandra, S.
abstract

1977 - Chemical and morphological changes of rat plasma lipoproteins after a prolonged administration of diets containing olive oil and cholesterol. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Pasquali Ronchetti, I; Gherardi, E; Fornieri, C; Tarugi, Patrizia Maria
abstract

No abstract available

1977 - Cholesterol synthesis in freshly isolated human leukocytes. [Articolo su rivista]
Tarugi, Patrizia Maria; Romoli, V; Crovetti, F; CALANDRA BUONAURA, Sebastiano
abstract

Cholesterol synthesis has been studied in human leukocytes shortly after the isolation from healthy subjects. Not delipidated human serum reduced the cholesterol synthesis when added to the incubation medium. A similar effect was obtained when the leukocytes were incubated in the presence of physiological concentrations of low density lipoproteins.

1977 - nship among the concentrations of serum lipoproteins and changes in their chemical composition in patients with untreated nephrotic syndrome [Articolo su rivista]
Gherardi, E; Rota, E; CALANDRA BUONAURA, Sebastiano; Genova, R; Tamborino, A.
abstract

No abstract available

1975 - Effect of cholesterol feeding on cholesterol biosynthesis in maternal and foetal rat liver. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano
abstract

Hepatic cholesterol biosynthesis has been studied in rat foetuses whose mothers had been fed on a cholesterol rich diet during the last week of gestation. Foetal liver was found to be capable of synthesizing cholesterol from acetate in vitro. The rate of incorporation of labelled acetate into digitonin precipitable sterols, fatty acids and CO(2) in foetal liver was much higher than that found in maternal liver. Cholesterol feeding reduced the rate of sterol synthesis in maternal liver but it did not have any appreciable effect on foetal liver. In order to investigate whether this lack of feed-back control in foetal liver could be attributable to an obstacle to the placental transfer of dietary cholesterol. 14-C-cholesterol was administered to the pregnant rats and its distribution in maternal and foetal liver and plasma was studied. Our results indicate that placental transfer of cholesterol from mother to foetus occurs very slowly so that only a small proportion of labelled cholesterol is found in foetal plasma over a 48 hour period following the administration of radioactive cholesterol. Cholesterol transferred from the mother into the foetal plasma is efficiently taken up by the foetal liver. These findings would suggest that the low amount of dietary cholesterol transferred from the mother into the foetal plasma is not sufficient to activate the control mechanism of the cholesterol biosynthetic pathway in the foetal liver.

1975 - Plasma lipoproteins in rats with experimental biliary obstruction. I. A chemical study. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Montaguti, M; Cartoni, V; Pasquali Ronchetti, I.
abstract

AbstractAcute biliary obstruction in the rat is associated with striking alterations of the plasma level and the physico-chemical properties of plasma lipoproteins. 1.1. The level of very low density lipoproteins (VLDL) in plasma increases from 2 to 3 fold. The chemical composition of VLDL is characterized by a high content of phospholipids and cholesterol and by a diminution of the relative content of triacylglycerols and protein. On cellulose acetate electrophoresis, VLDL show a β-mobility.2.2. The plasma concentration of low density lipoproteins (1.019–1.063 g/ml, LDL2) increases several fold above the control level. Phospholipids and unesterified cholesterol are the major components of this fraction which contains only a minute amount of cholesteryl ester (4%) and triacylglycerols (10%). LDL2 contain a component which migrates to the cathode in 1% agar gel electrophoresis. Separation of LDL2 by gel filtration on 2% agarose column results in the identification of three subfractions. Subfraction I contains a large proportion of cholesterol and triacylglycerols, subfraction II is rich in unesterified cholesterol and phospholipids whereas subfraction III has a chemical composition fairly similar to that of the control LDL2.3.3. The level of high density lipoproteins (HDL) also increases after bile duct ligation. The chemical composition of HDL2 (1.063–1.125 g/ml) is characterized by a high content of unesterified cholesterol and phospholipids and by a remarkable reduction in the content of cholesteryl esters and protein

1975 - Plasma lipoproteins in rats with experimental biliary obstruction. II. An ultrastructural study. [Articolo su rivista]
Pasquali Ronchetti, I; CALANDRA BUONAURA, Sebastiano; Baccarani Contri, M; Montaguti, M.
abstract

Acute biliary obstruction in the rat is associated with abnormalities of the ultrastructure of plasma lipoproteins. 1.1. The d < 1.006 g/ml fraction contains two types of particles: (a) vesicles having a mean diameter of 42.0 nm and (b) spheroidal particles which have a mean diameter of 80.0 nm. The latter show surface irregularities which make these particles appear as spherical bodies with holes.2.2. The d 1.019–1.063 g/ml fraction is heterogeneous. Gel filtration on 2% agarose allows the characterization of three components designated subfraction I, II and III respectively. Subfraction I is composed of large aggregates of spherical and vesicular particles. Subfraction II consists of 50.0–80.0 nm vesicles which show strong similarities to artificially prepared emulsions of phospholipids in water. Subfraction III contains particles which have a mean diameter of 20.0 nm and resemble low density lipoproteins of normal rats.3.3. The d 1.063–1.125 g/ml fraction contains 35.0 nm discoidal particles which tend to form ruleaux with a periodicity of 4.9 nm. This fraction also contains a few vesicles and 10.0 nm spherical particles similar to those found in the corresponding fraction of normal rat.

1975 - The ultrastructure of rat plasma lipoproteins. [Articolo su rivista]
Pasquali Ronchetti, I; CALANDRA BUONAURA, Sebastiano; Baccarani Contri, M; Montaguti, M.
abstract

No abstract available

1973 - Effect of prostaglandin E1 on cholesterol biosynthesis in rat liver. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Montaguti, M.
abstract

No abstract available

1973 - Regulation of cholesterol biosynthesis in rat liver after the administration of N-2-fluorenylacetamide [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Guariento, D; Rivasi, Francesco
abstract

No abstract available

1973 - The relation between plasma cholesterol and cholesterol synthesis in rats with experimental biliary obstruction. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano
abstract

No abstract available

1972 - The effect of experimental biliary obstruction on the structure and lipid content of rat erythrocytes [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Martin, Mj; O'Shea, Mj; Mcintyre, N.
abstract

No abstract available

1971 - Plasma lecithin: cholesterol acyltransferase activity in liver disease. [Articolo su rivista]
CALANDRA BUONAURA, Sebastiano; Martin, Mj; Mcintyre, N.
abstract

No abstract available