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Rossella TUPLER

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

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2022 - Counseling and prenatal diagnosis in facioscapulohumeral muscular dystrophy: A retrospective study on a 13‐year multidisciplinary approach [Articolo su rivista]
Di Feo, Maria Francesca; Bettio, Cinzia; Salsi, Valentina; Bertucci, Emma; Tupler, Rossella

2022 - De novo variants and recombination at 4q35: hints for preimplantation genetic testing in facioscapulohumeral muscular dystrophy [Articolo su rivista]
Pini, Sara; Napoli, Floriana Maria; Tagliafico, Enrico; Marca, Antonio La; Bertucci, Emma; Salsi, Valentina; Tupler, Rossella

2021 - A 5-year clinical follow-up study from the Italian National Registry for FSHD [Articolo su rivista]
Vercelli, L.; Mele, F.; Ruggiero, L.; Sera, F.; Tripodi, S.; Ricci, G.; Vallarola, A.; Villa, L.; Govi, M.; Maranda, L.; Di Muzio, A.; Scarlato, M.; Bucci, E.; Maggi, L.; Rodolico, C.; Moggio, M.; Filosto, M.; Antonini, G.; Previtali, S.; Angelini, C.; Berardinelli, A.; Pegoraro, E.; Siciliano, G.; Tomelleri, G.; Santoro, L.; Mongini, T.; Tupler, R.

Background: The natural history of facioscapulohumeral muscular dystrophy (FSHD) is undefined. Methods: An observational cohort study was conducted in 246 FSHD1 patients. We split the analysis between index cases and carrier relatives and we classified all patients using the Comprehensive Clinical Evaluation Form (CCEF). The disease progression was measured as a variation of the FSHD score performed at baseline and at the end of 5-year follow-up (ΔFSHD score). Findings: Disease worsened in 79.4% (112/141) of index cases versus 38.1% (40/105) of carrier relatives and advanced more rapidly in index cases (ΔFSHD score 2.3 versus 1.2). The 79.1% (38/48) of asymptomatic carriers remained asymptomatic. The highest ΔFSHD score (1.7) was found in subject with facial and scapular weakness at baseline (category A), whereas in subjects with incomplete phenotype (facial or scapular weakness, category B) had lower ΔFSHD score (0.6) p < 0.0001. Conclusions: The progression of disease is different between index cases and carrier relatives and the assessment of the CCEF categories has strong prognostic effect in FSHD1 patients.

2021 - Facioscapulohumeral Muscular Dystrophy and Poliomyelitis followed by Multiple Sclerosis: A “triple trouble” case report and review of the literature on the association of MS and muscle disorders [Articolo su rivista]
Ziccone, V.; Rodolico, C.; Rizzo, V.; Tupler, R.; Buccafusca, M.; Toscano, A.

We describe herein a “triple trouble” case of a patient affected by Facioscapulohumeral Muscular Dystrophy type 1 (FSHD1), with a previous history of poliomyelitis, who later developed Multiple Sclerosis (MS). Association of muscle disorders and MS is uncommon; in fact, there are only three case reports of this unusual co-occurrence. As regard as this combination, some hypotheses have been raised about the role of immunological factors. Genetic basis of FSHD1 is a deletion of a critical number of macrosatellite repeats (D4Z4) in the subtelomeric region of chromosome 4q35, resulting in transcriptional de-repression of a gene DUX4. This molecular change could induce an alteration of immune responses, likely conferring susceptibility to both diseases. In this case, poliomyelitis could have delayed the FSHD1 diagnosis and likely acted as a trigger for MS onset. Association of multiple neurological disorders has to be kept in mind to avoid misinterpretation of symptoms and diagnostic delays.

2021 - Increased resistance towards fatigability in patients with facioscapulohumeral muscular dystrophy [Articolo su rivista]
Beretta-Piccoli, Matteo; Calanni, Luca; Negro, Massimo; Ricci, Giulia; Bettio, Cinzia; Barbero, Marco; Berardinelli, Angela; Siciliano, Gabriele; Tupler, Rossella; Soldini, Emiliano; Cescon, Corrado; D'Antona, Giuseppe

Purpose In facioscapulohumeral muscular dystrophy (FSHD) fatigue is a major complaint. We aimed to investigate whether during isometric sustained elbow flexions, performance fatigability indexes differ in patients with FSHD with respect to healthy controls. Methods Seventeen patients with FSHD and seventeen healthy controls performed two isometric flexions of the dominant biceps brachii at 20% of their maximal voluntary contraction (MVC) for 2 min and then at 60% MVC until exhaustion. Muscle weakness was characterized as a percentage of predicted values. Maximal voluntary strength, endurance time and performance fatigability indices (mean frequency of the power spectrum (MNF), muscle fiber conduction velocity (CV) and fractal dimension (FD)), extracted from the surface electromyogram signal (sEMG) were compared between the two groups. Results In patients with FSHD, maximal voluntary strength was 68.7% of predicted value (p < 0.01). Compared to healthy controls, FSHD patients showed reduced MVC (p < 0.001; r = 0.62) and lower levels of performance fatigability, characterized by reduced rate of changes in MNF (p < 0.01; r = 0.56), CV (p < 0.05; 0.37) and FD (p < 0.001; r = 0.51) and increased endurance time (p < 0.001; r = 0.63), during the isometric contraction at 60% MVC. Conclusion A decreased reduction in the slopes of all the considered sEMG parameters during sustained isometric elbow flexions suggests that patients with FSHD experience lower levels of performance fatigability compared to healthy controls.

2021 - Muscle Fiber Conduction Velocity Correlates With the Age at Onset in Mild FSHD Cases [Articolo su rivista]
Beretta-Piccoli, M.; Negro, M.; Calanni, L.; Berardinelli, A.; Siciliano, G.; Tupler, R.; Soldini, E.; Cescon, C.; D'Antona, G.

A majority of patients with facioscapulohumeral muscular dystrophy (FSHD) report severe fatigue. The aim of this study was to explore whether fatigability during a performance task is related to the main clinical features of the disease in mildly affected patients. A total of 19 individuals with a molecular genetic-based diagnosis of FSHD (median D4Z4 deletion length of 27 kb) performed two isometric flexions of the dominant biceps brachii at 20% of their maximal voluntary contraction (MVC) for 2 min, and then at 60% MVC until exhaustion. Fatigability indices (average rectified value, mean frequency, conduction velocity, and fractal dimension) were extracted from the surface electromyogram (sEMG) signal, and their correlations with age, age at onset, disease duration, D4Z4 contraction length, perceived fatigability, and clinical disability score were analyzed. The conduction velocity during the low level contraction showed a significant negative correlation with the age at onset (p < 0.05). This finding suggest the assessment of conduction velocity at low isometric contraction intensities, as a potential useful tool to highlight differences in muscle involvement in FSHD patients.

2021 - Role of PD-L1 in licensing immunoregulatory function of dental pulp mesenchymal stem cells [Articolo su rivista]
Di Tinco, R.; Bertani, G.; Pisciotta, A.; Bertoni, L.; Pignatti, E.; Maccaferri, M.; Bertacchini, J.; Sena, P.; Vallarola, A.; Tupler, R.; Croci, S.; Bonacini, M.; Salvarani, C.; Carnevale, G.

Background: Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods: Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results: Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions: Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties.

2021 - The Italian National Registry for FSHD: an enhanced data integration and an analytics framework towards Smart Health Care and Precision Medicine for a rare disease [Articolo su rivista]
Bettio, C.; Salsi, V.; Orsini, M.; Calanchi, E.; Magnotta, L.; Gagliardelli, L.; Kinoshita, J.; Bergamaschi, S.; Tupler, R.

Background: The Italian Clinical network for FSHD (ICNF) has established the Italian National Registry for FSHD (INRF), collecting data from patients affected by Facioscapulohumeral dystrophy (FSHD) and their relatives. The INRF has gathered data from molecular analysis, clinical evaluation, anamnestic information, and family history from more than 3500 participants. Methods: A data management framework, called Mediator Environment for Multiple Information Sources (MOMIS) FSHD Web Platform, has been developed to provide charts, maps and search tools customized for specific needs. Patients’ samples and their clinical information derives from the Italian Clinical network for FSHD (ICNF), a consortium consisting of fourteen neuromuscular clinics distributed across Italy. The tools used to collect, integrate, and visualize clinical, molecular and natural history information about patients affected by FSHD and their relatives are described. Results: The INRF collected the molecular data regarding FSHD diagnosis conducted on 7197 subjects and identified 3362 individuals carrying a D4Z4 Reduced Allele (DRA): 1634 were unrelated index cases. In 1032 cases the molecular testing has been extended to 3747 relatives, 1728 carrying a DRA. Since 2009 molecular analysis has been accompanied by clinical evaluation based standardized evaluation protocols. In the period 2009–2020, 3577 clinical forms have been collected, 2059 follow the Comprehensive Clinical Evaluation form (CCEF). The integration of standardized clinical information and molecular data has made possible to demonstrate the wide phenotypic variability of FSHD. The MOMIS (Mediator Environment for Multiple Information Sources) data integration framework allowed performing genotype–phenotype correlation studies, and generated information of medical importance either for clinical practice or genetic counseling. Conclusion: The platform implemented for the FSHD Registry data collection based on OpenClinica meets the requirement to integrate patient/disease information, as well as the need to adapt dynamically to security and privacy concerns. Our results indicate that the quality of data collection in a multi-integrated approach is fundamental for clinical and epidemiological research in a rare disease and may have great value in allowing us to redefine diagnostic criteria and disease markers for FSHD. By extending the use of the MOMIS data integration framework to other countries and the longitudinal systematic collection of standardized clinical data will facilitate the understanding of disease natural history and offer valuable inputs towards trial readiness. This approach is of high significance to FSHD medical community and also to rare disease research in general.

2020 - Cochlear Dysfunction Is a Frequent Feature of Facioscapulohumeral Muscular Dystrophy Type 1 (FSHD1) [Articolo su rivista]
Frezza, Erica; Fuccillo, Emanuela; Petrucci, Antonio; Greco, Giulia; Nucera, Gabriele; Bruno, Ernesto; Giardina, Emiliano; Tupler, Rossella; Di Mauro, Roberta; Di Girolamo, Stefano; Massa, Roberto

INTRODUCTION: Facioscapulohumeral muscular dystrophy type 1 (FSHD) represents one of the most common forms of muscular hereditary diseases and it is characterized by a great clinical variability with the typical muscular symptoms and other clinical features, including hearing impairment. However, etiopathogenetic mechanisms of auditory dysfunction are still not completely understood and it has been suggested that it could be assigned to a cochlear alteration that is present even in those subjects with a normal pure tonal audiometry (PTA) examination.METHODS: We found out the cochlear function in 26 patients with molecular diagnosis of FSHD1 and in healthy controls. All patients underwent complete neurological and audiological examinations, including FSHD clinical score, pure-tone audiometry (PTA), and otoacoustic emissions (OAEs), in particular transient evoked otoacoustic emissions (TEOAEs) and distortion product evoked otoacoustic emissions (DPOAEs).RESULTS: All FSHD1 patients showed significantly reduced DPOAEs and TEOAEs, bilaterally and at all frequencies, even when considering only subjects with a normal PTA or a mild muscular involvement (FSHD score ≤ 2). No correlation between OAEs and FSHD clinical score was found.DISCUSSION: Cochlear echoes represent a sensitive tool in detecting subclinical cochlear dysfunction in FSHD1 even in subjects with normal hearing and/or subtle muscle involvement. Our study is focused on the importance of evaluating the cochlear alteration through OAEs and, in particular, by performing TEOAEs and DPOAEs sequentially, to evaluate more frequent specificities of cochlear dysfunction with a wider spectrum of analysis.

2020 - Deletion of the Williams Beuren syndrome critical region unmasks facioscapulohumeral muscular dystrophy [Articolo su rivista]
Rodolico, C.; Politano, L.; Portaro, S.; Murru, S.; Boccone, L.; Sera, F.; Passamano, L.; Brizzi, T.; Tupler, R.

Among 1339 unrelated cases accrued by the Italian National Registry for facioscapulohumeral muscular dystrophy (FSHD), we found three unrelated cases who presented signs of Williams-Beuren Syndrome (WBS) in early childhood and later developed FSHD. All three cases carry the molecular defects associated with the two disorders. The rarity of WBS and FSHD, 1 in 7500 and 1 in 20,000 respectively, makes a random association of the two diseases unlikely. These cases open novel and unexpected interpretation of genetic findings. The nonrandom association of both FSHD and WBS points at a gene co-expression network providing hints for the identification of modules and functionally enriched pathways in the two conditions.

2020 - Does DNA Methylation Matter in FSHD? [Articolo su rivista]
Salsi, Valentina; Magdinier, Frédérique; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD) has been associated with the genetic and epigenetic molecular features of the CpG-rich D4Z4 repeat tandem array at 4q35. Reduced DNA methylation of D4Z4 repeats is considered part of the FSHD mechanism and has been proposed as a reliable marker in the FSHD diagnostic procedure. We considered the assessment of D4Z4 DNA methylation status conducted on distinct cohorts using different methodologies. On the basis of the reported results we conclude that the percentage of DNA methylation detected at D4Z4 does not correlate with the disease status. Overall, data suggest that in the case of FSHD1, D4Z4 hypomethylation is a consequence of the chromatin structure present in the contracted allele, rather than a proxy of its function. Besides, CpG methylation at D4Z4 DNA is reduced in patients presenting diseases unrelated to muscle progressive wasting, like Bosma Arhinia and Microphthalmia syndrome, a developmental disorder, as well as ICF syndrome. Consistent with these observations, the analysis of epigenetic reprogramming at the D4Z4 locus in human embryonic and induced pluripotent stem cells indicate that other mechanisms, independent from the repeat number, are involved in the control of the epigenetic structure at D4Z4.

2020 - Interpretation of the epigenetic signature of facioscapulohumeral muscular dystrophy in light of genotype-phenotype studies [Articolo su rivista]
Nikolic, A.; Jones, T. I.; Govi, M.; Mele, F.; Maranda, L.; Sera, F.; Ricci, G.; Ruggiero, L.; Vercelli, L.; Portaro, S.; Villa, L.; Fiorillo, C.; Maggi, L.; Santoro, L.; Antonini, G.; Filosto, M.; Moggio, M.; Angelini, C.; Pegoraro, E.; Berardinelli, A.; Maioli, M. A.; D'Angelo, G.; Di Muzio, A.; Siciliano, G.; Tomelleri, G.; D'Esposito, M.; Ragione, F. D.; Brancaccio, A.; Piras, R.; Rodolico, C.; Mongini, T.; Magdinier, F.; Salsi, V.; Jones, P. L.; Tupler, R.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

2020 - Large genotype-phenotype study in carriers of D4Z4 borderline alleles provides guidance for facioscapulohumeral muscular dystrophy diagnosis [Articolo su rivista]
Ricci, Giulia; Mele, Fabiano; Govi, Monica; Ruggiero, Lucia; Sera, Francesco; Vercelli, Liliana; Bettio, Cinzia; Santoro, Lucio; Mongini, Tiziana; Villa, Luisa; Moggio, Maurizio; Filosto, Massimiliano; Scarlato, Marina; Previtali, Stefano C; Tripodi, Silvia Maria; Pegoraro, Elena; Telese, Roberta; Di Muzio, Antonio; Rodolico, Carmelo; Bucci, Elisabetta; Antonini, Giovanni; D'Angelo, Maria Grazia; Berardinelli, Angela; Maggi, Lorenzo; Piras, Rachele; Maioli, Maria Antonietta; Siciliano, Gabriele; Tomelleri, Giuliano; Angelini, Corrado; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9-10 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9-10 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9-10 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9-10 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9-10 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype-phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases.

2020 - Modulation of Cell Death and Promotion of Chondrogenic Differentiation by Fas/FasL in Human Dental Pulp Stem Cells (hDPSCs) [Articolo su rivista]
Pisciotta, Alessandra; Bertani, Giulia; Bertoni, Laura; Di Tinco, Rosanna; De Biasi, Sara; Vallarola, Antonio; Pignatti, Elisa; Tupler, Rossella; Salvarani, Carlo; de Pol, Anto; Carnevale, Gianluca

2020 - Mosaicism in Human Health and Disease [Articolo su rivista]
Thorpe, Jeremy; Osei-Owusu, Ikeoluwa A; Avigdor, Bracha Erlanger; Tupler, Rossella; Pevsner, Jonathan

Mosaicism refers to the occurrence of two or more genomes in an individual derived from a single zygote. Germline mosaicism is a mutation that is limited to the gonads and can be transmitted to offspring. Somatic mosaicism is a postzygotic mutation that occurs in the soma, and it may occur at any developmental stage or in adult tissues. Mosaic variation may be classified in six ways: (a) germline or somatic origin, (b) class of DNA mutation (ranging in scale from single base pairs to multiple chromosomes), (c) developmental context, (d) body location(s), (e) functional consequence (including deleterious, neutral, or advantageous), and ( f ) additional sources of mosaicism, including mitochondrial heteroplasmy, exogenous DNA sources such as vectors, and epigenetic changes such as imprinting and X-chromosome inactivation. Technological advances, including single-cell and other next-generation sequencing, have facilitated improved sensitivity and specificity to detect mosaicism in a variety of biological contexts. Expected final online publication date for the Annual Review of Genetics, Volume 54 is November 23, 2020. Please see for revised estimates.

2020 - Phenotypic Variability Among Patients With D4Z4 Reduced Allele Facioscapulohumeral Muscular Dystrophy [Articolo su rivista]
Ruggiero, Lucia; Mele, Fabiano; Manganelli, Fiore; Bruzzese, Dario; Ricci, Giulia; Vercelli, Liliana; Govi, Monica; Vallarola, Antonio; Tripodi, Silvia; Villa, Luisa; Di Muzio, Antonio; Scarlato, Marina; Bucci, Elisabetta; Antonini, Giovanni; Maggi, Lorenzo; Rodolico, Carmelo; Tomelleri, Giuliano; Filosto, Massimiliano; Previtali, Stefano; Angelini, Corrado; Berardinelli, Angela; Pegoraro, Elena; Moggio, Maurizio; Mongini, Tiziana; Siciliano, Gabriele; Santoro, Lucio; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disorder, associated with the deletion of tandemly arrayed D4Z4 repetitive elements. The extensive use of molecular analysis of the D4Z4 locus for FSHD diagnosis has revealed wide clinical variability, suggesting that subgroups of patients exist among carriers of the D4Z4 reduced allele (DRA).

2019 - Phenotype may predict the clinical course of facioscapolohumeral muscular dystrophy [Articolo su rivista]
Ricci, G.; Cammish, P.; Siciliano, G.; Tupler, R.; Lochmuller, H.; Evangelista, T.

Introduction: The correct phenotypic classification of patients with facioscapulohumeral muscular dystrophy (FSHD) is crucial for directing genetic diagnosis and for the definition of outcome measures in clinical trials. Methods: Our objective was to ascertain the utility of the Comprehensive Clinical Evaluation Form (CCEF), the clinical classification proposed by the Italian Clinical Network for FSHD, in an independent FSHD patient population from the UK FSHD Patient Registry. We subdivided the patients into group 1, classic FSHD phenotype/category A of CCEF, and group 2, facial sparing phenotypes/category B1 of CCEF. Results: Among 642 patients with FSHD1, 68.1% reported facial and shoulder weakness, whereas 24.1% reported shoulder weakness without facial impairment. The phenotype in group 2 was milder, with a higher mean age at onset (P < 0.0001) and less severe motor disability. Discussion: Patients with different FSHD phenotypes may have different disease courses. Muscle Nerve 59:711–713, 2019.

2018 - Genotype-phenotype correlation: The ultimate challenge in facioscapolohumeral muscular dystrophy [Articolo su rivista]
Tupler, Rossella

Comment on a 22-year follow-up reveals a variable disease severity in early-onset facioscapulohumeral dystrophy. This is the analysis of the clinical significance of molecular findings in subjects affected by early onset FSHD following a 22 year follow up.

2017 - 225th ENMC international workshop:: A global FSHD registry framework, 18–20 November 2016, Heemskerk, The Netherlands [Articolo su rivista]
Mul, Karlien; Kinoshita, June; Dawkins, Hugh; Van Engelen, Baziel; Tupler, Rossella; Ferreira, Verã²nica Alonso; Attarian, Sharam; Berardinelli, Angela; Bogard, Betsy; Evangelista, Teresinha; Van Der Graaf, Kees; Heatwole, Chad; Van Der Maarel, Silvãre; Mah, Jean; Van Rens, Jacqui; Richiardi, Armelle; Roxburgh, Richard; Sacconi, Sabrina; Tawil, Rabi; Van Der Meij-kim, Diana; Voet, Nicole; Vohã¡nka, Stanislav

On 18–20 November 2016, the 225th ENMC Workshop on ‘A global FSHD Registry framework’ took place in Heemskerk, the Netherlands. Twenty-two participants from 11 different countries gathered, including clinicians, researchers, policy makers and representatives from patient advocacy groups and industry. Facioscapulohumeral muscular dystrophy (FSHD) is an inherited muscle disorder. Expansion of our knowledge on the (epi)genetic mechanism underlying FSHD has led to advances in identifying (targeted) therapeutic strategies. Consequently it is now important to develop a ‘clinical trial toolbox’, consisting of patient registries, biomarkers and clinical outcome measures to ensure resources are utilized effectively The wide phenotypic expression in rare diseases, such as FSHD, means that patient registries are particularly important for clinical trial readiness. The aims of this workshop were to analyze the experience and results of the existing FSHD patient registries, update the Treat-NMD recommended dataset for FSHD, increase collaboration among established research groups and patient advocacy organizations and create the foundation on which to establish a global registry for FSHD.

2017 - Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function [Articolo su rivista]
Morelli F., F.; Verbeek D., S.; Bertacchini, Jessika; Vinet, Jonathan; Mediani, Laura; Marmiroli, Sandra; Cenacchi, G.; Nasi, Milena; DE BIASI, Sara; Brunsting J., F.; Lammerding, J.; Pegoraro, E.; Angelini, C.; Tupler, Rossella; Alberti, S.; Carra, Serena

Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy.

2017 - Early onset facioscapulohumeral dystrophy - a systematic review using individual patient data [Articolo su rivista]
Goselink, Rianne J. M.; Voermans, Nicol C.; Okkersen, Kees; Brouwer, Oebele F.; Padberg, George W.; Nikolic, Ana; Tupler, Rossella; Dorobek, Malgorzata; Mah, Jean K.; Van Engelen, Baziel G. M.; Schreuder, Tim H. A.; Erasmus, Corrie E.

Infantile or early onset is estimated to occur in around 10% of all facioscapulohumeral dystrophy (FSHD) patients. Although small series of early onset FSHD patients have been reported, comprehensive data on the clinical phenotype is missing. We performed a systematic literature search on the clinical features of early onset FSHD comprising a total of 43 articles with individual data on 227 patients. Additional data from four cohorts was provided by the authors. Mean age at reporting was 18.8 years, and 40% of patients were wheelchair-dependent at that age. Half of the patients had systemic features, including hearing loss (40%), retinal abnormalities (37%) and developmental delay (8%). We found an inverse correlation between repeat size and disease severity, similar to adult-onset FSHD. De novo FSHD1 mutations were more prevalent than in adult-onset FSHD. Compared to adult FSHD, our findings indicate that early onset FSHD is overall characterized by a more severe muscle phenotype and a higher prevalence of systemic features. However, similar as in adults, a significant clinical heterogeneity was observed. Based on this, we consider early onset FSHD to be on the severe end of the FSHD disease spectrum. We found natural history studies and treatment studies to be very scarce in early onset FSHD, therefore longitudinal studies are needed to improve prognostication, clinical management and trial-readiness.

2017 - The Italian FSHD registry: An enhanced data integration and analytics framework for smart health care [Relazione in Atti di Convegno]
Orsini, Mirko; Calanchi, Enrico; Magnotta, Luca; Gagliardelli, Luca; Govi, Monica; Mele, Fabiano; Tupler, Rossella

Facioscapulohumeral dystrophy (FSHD) is a rare genetic disease that has been described more than a hundred years ago. The Miogen Lab has been able to collect a large amount of data on patients affected by FSHD and their relatives over the years, also extending the research to their ancestors. Collected data include molecular analysis, clinical information on health status, family pedigree and geographic origin. The challenge of FSHD Registry is to investigate these large amount of information, discover additional elements related to disease onset and better understand the clinical progression and genetic inheritance of the disease, exploiting data integration capabilities and Big Data techniques. In this paper we describe the tools we used to collect, integrate and display these data in a framework that allows users to search among clinical records to elaborate brief reports and discover new relations on collected data. The solution provides charts, maps and search tools customized on the specific needs that came to light during the collaboration between DataRiver and Miogen Lab, joining the clinical knowledge of the latter with the information technology expertise of the former. The framework offers a single entry point for all genomic and therapeutic studies.

2016 - A novel clinical tool to classify facioscapulohumeral muscular dystrophy phenotypes [Articolo su rivista]
Ricci, Giulia; Ruggiero, Lucia; Vercelli, Liliana; Sera, Francesco; Nikolic, Ana; Govi, Monica; Mele, Fabiano; Daolio, Jessica; Angelini, Corrado; Antonini, Giovanni; Berardinelli, Angela; Bucci, Elisabetta; Cao, Michelangelo; D’Amico, Maria Chiara; D’Angelo, Grazia; Di Muzio, Antonio; Filosto, Massimiliano; Maggi, Lorenzo; Moggio, Maurizio; Mongini, Tiziana; Morandi, Lucia; Pegoraro, Elena; Rodolico, Carmelo; Santoro, Lucio; Siciliano, Gabriele; Tomelleri, Giuliano; Villa, Luisa; Tupler, Rossella

Based on the 7-year experience of the Italian Clinical Network for FSHD, we revised the FSHD clinical form to describe, in a harmonized manner, the phenotypic spectrum observed in FSHD. The new Comprehensive Clinical Evaluation Form (CCEF) defines various clinical categories by the combination of different features. The inter-rater reproducibility of the CCEF was assessed between two examiners using kappa statistics by evaluating 56 subjects carrying the molecular marker used for FSHD diagnosis. The CCEF classifies: (1) subjects presenting facial and scapular girdle muscle weakness typical of FSHD (category A, subcategories A1–A3), (2) subjects with muscle weakness limited to scapular girdle or facial muscles (category B subcategories B1, B2), (3) asymptomatic/healthy subjects (category C, subcategories C1, C2), (4) subjects with myopathic phenotype presenting clinical features not consistent with FSHD canonical phenotype (D, subcategories D1, D2). The inter-rater reliability study showed an excellent concordance of the final four CCEF categories with a κ equal to 0.90; 95 % CI (0.71; 0.97). Absolute agreement was observed for categories C and D, an excellent agreement for categories A [κ = 0.88; 95 % CI (0.75; 1.00)], and a good agreement for categories B [κ = 0.79; 95 % CI (0.57; 1.00)]. The CCEF supports the harmonized phenotypic classification of patients and families. The categories outlined by the CCEF may assist diagnosis, genetic counseling and natural history studies. Furthermore, the CCEF categories could support selection of patients in randomized clinical trials. This precise categorization might also promote the search of genetic factor(s) contributing to the phenotypic spectrum of disease.

2016 - Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1-3 D4Z4 reduced alleles: experience of the FSHD Italian National Registry [Articolo su rivista]
Nikolic, Ana; Ricci, Giulia; Sera, Francesco; Bucci, Elisabetta; Govi, Monica; Mele, Fabiano; Rossi, Marta; Ruggiero, Lucia; Vercelli, Liliana; Ravaglia, Sabrina; Brisca, Giacomo; Fiorillo, Chiara; Villa, Luisa; Maggi, Lorenzo; Cao, Michelangelo; D'Amico, Maria Chiara; Siciliano, Gabriele; Antonini, Giovanni; Santoro, Lucio; Mongini, Tiziana; Moggio, Maurizio; Morandi, Lucia; Pegoraro, Elena; Angelini, Corrado; Di Muzio, Antonio; Rodolico, Carmelo; Tomelleri, Giuliano; Grazia D'Angelo, Maria; Bruno, Claudio; Berardinelli, Angela; Tupler, Rossella

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1-3 repeats (1-3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1-3 DRA.

2016 - The genetic basis of undiagnosed muscular dystrophies and myopathies [Articolo su rivista]
Savarese, Marco; Di Fruscio, Giuseppina; Torella, Annalaura; Fiorillo, Chiara; Magri, Francesca; Fanin, Marina; Ruggiero, Lucia; Ricci, Giulia; Astrea, Guja; Passamano, Luigia; Ruggieri, Alessandra; Ronchi, Dario; Tasca, Giorgio; D'Amico, Adele; Janssens, Sandra; Farina, Olimpia; Mutarelli, Margherita; Marwah, Veer Singh; Garofalo, Arcomaria; Giugliano, Teresa; Sanpaolo, Simone; Del Vecchio Blanco, Francesca; Esposito, Gaia; Piluso, Giulio; D'Ambrosio, Paola; Petillo, Roberta; Musumeci, Olimpia; Rodolico, Carmelo; Messina, Sonia; Evilä, Anni; Hackman, Peter; Filosto, Massimiliano; Di Iorio, Giuseppe; Siciliano, Gabriele; Mora, Marina; Maggi, Lorenzo; Minetti, Carlo; Sacconi, Sabrina; Santoro, Lucio; Claes, Kathleen; Vercelli, Liliana; Mongini, Tiziana; Ricci, Enzo; Gualandi, Francesca; Tupler, Rossella; De Bleecker, Jan; Udd, Bjarne; Toscano, Antonio; Moggio, Maurizio; Pegoraro, Elena; Bertini, Enrico; Mercuri, Eugenio; Angelini, Corrado; Santorelli, Filippo Maria; Politano, Luisa; Bruno, Claudio; Comi, Giacomo Pietro; Nigro, Vincenzo

Objective: To apply next-generation sequencing (NGS) for the investigation of the genetic basis of undiagnosed muscular dystrophies and myopathies in a very large cohort of patients. Methods: We applied an NGS-based platform namedMotorPlex to our diagnostic workflow to test muscle disease genes with a high sensitivity and specificity for small DNA variants. We analyzed 504 undiagnosed patients mostly referred as being affected by limb-girdle muscular dystrophy or congenital myopathy. Results: MotorPlex provided a complete molecular diagnosis in 218 cases (43.3%). A further 160 patients (31.7%) showed as yet unproven candidate variants. Pathogenic variants were found in 47 of 93 genes, and in more than 30%of cases, the phenotype was nonconventional, broadening the spectrum of disease presentation in at least 10 genes. Conclusions: Our large DNA study of patients with undiagnosed myopathy is an example of the ongoing revolution in molecular diagnostics, highlighting the advantages in using NGS as a first-tier approach for heterogeneous genetic conditions.

Morelli, FEDERICA FRANCESCA; Heldens, Lonneke; Verbeek, Dineke; Angelini, Corrado; Cenacchi, Giovanna; Tupler, Rossella; Carra, Serena

HSPB3 is a poorly characterized member of the small HSPB family that forms a complex with HSPB2. The complex is induced in differentiated muscle cells and might play a role in muscle maintenance. R7S mutation was associated with distal hereditary motor neuropathy type 2C. We identified in myopathic patients two novel mutations of HSPB3: a) R116P, affecting a key amino acid in the alpha-crystallin domain, whose mutation in other HSPBs causes neuromuscular diseases; b) p.A33AfsX50-HSPB3, a missense mutation, which leads to a premature stop codon. We characterized the subcellular localization of HSPB2-HSPB3 and the effects of the HSPB3 mutants on protein localization, stability and complex formation. While p.A33AfsX50-HSPB3 is degraded after synthesis, R7S and R116P are stable. Unlike of R116P, R7S still interacts with HSPB2. HSPB2 and HSPB3 are enriched in the nuclei, where they form intranuclear (IN) and perinuclear (PN) aggregates. Aggregation tendency of HSPB3 is increased by its mutants. These IN and PN aggregates influence nuclear envelope and lamin A/C distribution. Lamins regulate not only nuclear shape, but also transcription. Moreover, mutations on lamin A/C are associated with neuromuscular disease. In addition, lamin A/C is recruited in nuclear speckles that contain splicing factors such as SC35. HSPB2-HSPB3 do not colocalize with speckles and do not alter the recruitment in speckles of lamin A/C; instead, they alter speckles shape, which became round (mimicking transcription inhibition). Combined our results show that HSPB2-HSPBB3 affect nuclear structure; this in turn may deregulate remodeling of nuclear lamina and RNA transcription. Intriguingly, the muscle biopsy from patient with R116P mutation shows nuclear aggregation and morphological alterations. This further suggests that HSPB3 (in complex with HSPB2) may modulate nuclear structure/functions and that alteration thereof may contribute to disease.

2015 - Characterization of the R7S mutation of Heat Shock Protein HSPB3 and of two novel mutations found in patients suffering of myopathy: understanding the mechanisms leading to disease. [Poster]
Morelli, FEDERICA FRANCESCA; Heldens, Lonneke; Verbeek, Dineke; Angelini, Corrado; Cenacchi, Giovanna; Tupler, Rossella; Carra, Serena

HSPB3 is a poorly characterized member of the small HSP/HSPB family (HSPB1-HSPB10) that forms a complex with HSPB2. Expression of HSPB2 and HSPB3 is restricted to differentiated skeletal and cardiac muscle cells, where HSPB2-HSPB3 participates in muscle maintenance. Recently the R7S mutation in HSPB3 was associated with distal hereditary motor neuropathy type 2C (dHMN 2C). We identified in myopathic patients two novel mutations of HSPB3: 1) R116P, affecting a key amino acid in the alpha-crystallin domain, whose mutation in other HSPBs also causes neuromuscular diseases; 2) p.A33AfsX50-HSPB3, which disrupts the reading frame leading to a premature stop codon at amino acid 50. Here we first characterized in cells the subcellular distribution of HSPB2 and HSPB3. Next, we studied whether/how HSPB3 mutations affect: a) HSPB3 stability and subcellular localization; b) complex formation. While p.A33AfsX50-HSPB3 is immediately degraded after synthesis and cannot be detected, R7S and R116P are rather stable. Concerning interaction with HSPB2, R7S still interacted with HSPB2, while R116 disrupted complex formation. Concerning subcellular distribution, in two cell types (HEK293T and HeLa cells), HSPB2 and HSPB3 were enriched in the nuclei, where they formed intranuclear and perinuclear aggregates. Aggregation propensity was increased by R7S and R116P mutations. Intriguingly, nuclear aggregation and alteration of nuclear morphology were also found in the muscle biopsy from the patient with the R116P mutation. Next, we found in cells that HSPB2 and HSPB3 (wt and mutants) alter nuclear envelope (NE) integrity and lamin A/C distribution. Interestingly, DMPK, which has been shown to interact with HSPB2, also affects NE. Lamins regulate not only nuclear shape, but also gene expression, transcription and mutations in NE components cause neuromuscular and muscular diseases. Lamin A/C has been associated with intranuclear speckles, where it can colocalize with HSPB1 or HSPB5. Speckles contain splicing factors such as SC35. We thus analyzed speckles in HSPB2-B3 expressing cells. HSPB2-B3 did not inhibit Lamin A/C recruitment into speckles. Also, we did not find any major colocalization of HSPB2 and HSPB3 with speckles. Instead, HSPB2 and HSPB3 altered the shape of speckles, which became round (mimicking a treatment with the RNA transcription inhibitor actinomycin D). In summary, our results show that HSPB2-HSPB3 affect nuclear architecture, which may in turn deregulate RNA transcription. Future studies will identify the molecular mechanisms leading to the observed effects on NE and speckles. Since remodeling of nuclear lamina is required for muscle differentiation, it is tempting to speculate that HSPB2-B3 may modulate muscle differentiation/maintenance by regulating lamin functions and NE stability. Alteration of such functions due to HSPB3 mutations may be detrimental for motor neuron and muscle cells, contributing to disease progression.

2015 - FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1) [Articolo su rivista]
Feeney, Sandra J.; Mcgrath, Meagan J.; Sriratana, Absorn; Gehrig, Stefan M.; Lynch, Gordon S.; D'Arcy, Colleen E.; Price, John T.; Mclean, Catriona A.; Tupler, Rossella; Mitchell, Christina A.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.

2014 - Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1). [Articolo su rivista]
Sancisi, Valentina; Germinario, E; Esposito, A; Morini, Elisabetta; Peron, S; Moggio, M; Tomelleri, G; Danieli Betto, D; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca2+. We demonstrate that the decrease of Ca2+ sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.

2014 - An integrated approach in a case of facioscapulohumeral dystrophy [Articolo su rivista]
Pasotti, Stefano; Magnani, Bruno; Longa, Emanuela; Giovanetti, Giuseppe; Rossi, Albino; Berardinelli, Angela; Tupler, Rossella; D'Antona, Giuseppe

BACKGROUND: Muscle fatigue, weakness and atrophy are basilar clinical features that accompany facioscapulohumeral dystrophy (FSHD) the third most common muscular dystrophy.No therapy is available for FSHD. CASE PRESENTATION: We describe the effects of 6mo exercise therapy and nutritional supplementation in a 43-year-old woman severely affected by FSHD. CONCLUSION: A mixed exercise program combined with nutritional supplementation can be safely used with beneficial effects in selected patients with FSHD.

2014 - Facioscapulohumeral muscular dystrophy: more complex than it appears [Articolo su rivista]
Ricci, Giulia; Zatz, M; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD) has been classified as an autosomal dominant myopathy, linked to rearrangements in an array of 3.3 kb tandemly repeated DNA elements (D4Z4) located at the 4q subtelomere (4q35). For the last 20 years, the diagnosis of FSHD has been confirmed in clinical practice by the detection of one D4Z4 allele with a reduced number (≤8) of repeats at 4q35. Although wide inter- and intra-familial clinical variability was found in subjects carrying D4Z4 alleles of reduced size, this DNA testing has been considered highly sensitive and specific. However, several exceptions to this general rule have been reported. Specifically, FSHD families with asymptomatic relatives carrying D4Z4 reduced alleles, FSHD genealogies with subjects affected with other neuromuscular disorders and FSHD affected patients carrying D4Z4 alleles of normal size have been described. In order to explain these findings, it has been proposed that the reduction of D4Z4 repeats at 4q35 could be pathogenic only in certain chromosomal backgrounds, defined as "permissive" specific haplotypes. However, our most recent studies show that the current DNA signature of FSHD is a common polymorphism and that in FSHD families the risk of developing FSHD for carriers of D4Z4 reduced alleles (DRA) depends on additional factors besides the 4q35 locus. These findings highlight the necessity to re-evaluate the significance and the predictive value of DRA, not only for research but also in clinical practice. Further clinical and genetic analysis of FSHD families will be extremely important for studies aiming at dissecting the complexity of FSHD.

2013 - Characterization of the R7S mutation of Heat Shock Protein HSPB3 and of two novel mutations found in patients suffering of myopathy: understanding the mechanisms leading to disease. [Poster]
Heldens, Lonneke; Morelli, FEDERICA FRANCESCA; Verbeek, Dineke; Vinet, Jonathan; Angelini, Corrado; Boelens, Wilbert; Tupler, Rossella; Carra, Serena

HSPB3 is a poorly characterized member of the small HSP/HSPB family (HSPB1-HSPB10) that forms a complex with HSPB2 with a defined 1:3 ratio. The HSPB2/HSPB3 complex is induced during muscle differentiation and plays a role in muscle maintenance. Recently the R7S mutation in HSPB3 has been associated with distal hereditary motor neuropathy type 2C (dHMN 2C). Here we report the identification in myopathic patients of two novel mutations in HSPB3: 1) one mutation affects the R116 residue, which corresponds to a key amino acid in the alpha-crystallin domain, whose mutation in other members of the HSPB family also causes disease (it is equivalent to e.g. R120 in HSPB5, whose mutation into G causes MFM and to K141 in HSPB8, whose mutation into E or N causes dHMN); 2) the other mutation disrupts the reading frame leading to a premature stop codon at amino acid 50. Both mutations were not found in more than 400 normal alleles. Expression studies allowed us to confirm that the mutation causing a premature stop codon leads to the generation of an unstable protein that is likely immediately degraded after synthesis and cannot be detected. Also, while both expressed, the R7S mutant was more stable than the R116 one. We next characterized in cells and in vitro the ability of these HSPB3 mutants to interact with HSPB2 and form the HSPB2/HSPB3 complex. We found that while the R7S mutant of HSPB3 was still able to interact with HSPB2, the R116 mutant was not. Future studies will allow us to better characterize how these HSPB3 mutants affect HSPB3 and, indirectly, HSPB2 stability, subcellular localization and function. They will also elucidate on HSPB3 and HSPB2 function in both motor neurons and myoblasts and will shed light on how mechanistically the mutations in HSPB3 affect the function and viability of these cell types, contributing to disease.

2013 - D4Z4 reduced allele in myopathic subjects with no FSHD phenotype: why inconsistency between molecular and clinical data should prompt us to further investigations. [Poster]
Daolio, Jessica; Nikolic, Ana; Ricci, Giulia; Govi, Monica; Mele, Fabiano; Carra, Serena; Vercelli, L.; Antonini, G.; Berardinelli, Maria Angela; Mongini, T.; Servida, M.; Ruggiero, L.; Angelini, C.; Cao, M.; D’Angelo, G.; Muzio, A. Di; Moggio, M.; Morandi, L.; Ricci, Eusebio; Rodolico, C.; Santoro, Luisa; Siciliano, G.; Tomelleri, G.; Tupler, Rossella

Aim of the study. Chacacterization of different FSHD clinical subsets and/or more complex myopathic conditions among carriers of D4Z4 reduced allele (DRA) associated with atypical myopathic phenotypes. Materials and methods. Out of 751 consecutive index cases recruited from the Italian National Registry for FSHD, we identified 298 myopathic patients carrying DRA with 4-8 repats who did not present obvious FSHD phenotype. Re-evaluation of clinical data and depth molecular characterization of 4q35 region were performed. Results. Twenty-three carriers of 4-8 DRA presented myopathic features related to non-FSHD conditions. In this group we observed high clinical heterogeneity. In particular subjects displayed different combinations of clinical signs that were considered atypical for FSHD: 1) involvement of muscles that are not usually affected in FSHD, 2) sparing of muscles that are typically affected in FSHD, 3) additional clinical, genetic and instrumental features that are not included in the FSHD phenotype. In addition, Molecular analysis of the 4q35 subtelomeric haplotype of myopathic DRA patients did not reveal any molecular element enabling us to differentiate these patients from classical FSHD patients. In two patients, heterozygous mutations were found in CAV-3 and HSPB3 genes. Interestingly, these patients were sporadic and no other myopathic subjects were reported in the two families. Discussion and conclusions. Our study shows that not all myopathic patients carrying 4-8 DRA are affected by FSHD. We conclude that in myopathic carriers of 4q short allele who do not display a typical autosomal dominant FSHD additional molecular and clinical investigations should be performed for a more precise characterization of these patients. This approach will favor clinical diagnosis and genetic counseling.

2013 - Large scale genotype-phenotype analyses indicate that novel prognostic tools are required for families with facioscapulohumeral muscular dystrophy. [Articolo su rivista]
Ricci, Giulia; Scionti, Isabella; Sera, F; Govi, Monica; D'Amico, Roberto; Frambolli, Ilaria; Mele, Fabiano; Filosto, M; Vercelli, L; Ruggiero, L; Berardinelli, A; Angelini, C; Antonini, G; Bucci, E; Cao, M; Daolio, Jessica; Di Muzio, A; Di Leo, R; Galluzzi, G; Iannaccone, E; Maggi, L; Maruotti, V; Moggio, M; Mongini, T; Morandi, L; Nikolic, Ana; Pastorello, E; Ricci, E; Rodolico, C; Santoro, L; Servida, M; Siciliano, G; Tomelleri, G; Tupler, Rossella

Facioscapulohumeral muscular dystrophy has been genetically linked to reduced numbers (<8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4 polyadenylation signal haplotype. However, we have recently reported that 1.3% of healthy individuals carry this molecular signature and 19% of subjects affected by facioscapulohumeral muscular dystrophy do not carry alleles with eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at risk of carrying D4Z4 reduced alleles has become more complicated. To test for additional prognostic factors, we measured the degree of motor impairment in a large group of patients affected by facioscapulohumeral muscular dystrophy and their relatives who are carrying D4Z4 reduced alleles. The clinical expression of motor impairment was assessed in 530 subjects, 163 probands and 367 relatives, from 176 unrelated families according to a standardized clinical score. The associations between clinical severity and size of D4Z4 allele, degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2% of relatives did not display any muscle functional impairment. This phenotype was influenced by the degree of relation with proband, because 47.1% of secondthrough fifth-degree relatives were unaffected, whereas only 27.5% of first-degree family members did not show motor impairment. The estimated risk of developing motor impairment by age 50 for relatives carrying a D4Z4 reduced allele with 1-3 repeats or 4-8 repeats was 88.7% and 55%, respectively. Male relatives had a mean score significantly higher than females (5.4 versus 4.0, P = 0.003). No 4q haplotype was exclusively associated with the presence of disease. In 13% of families in which D4Z4 alleles with 4-8 repeats segregate, the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one generation. In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 4-8 D4Z4 repeats segregates. In addition, the reduced expression of disease observed in distant relatives suggests that a family's genetic background plays a role in the occurrence of facioscapulohumeral muscular dystrophy. These results indicate that the identification of new susceptibility factors for this disease will require an accurate classification of families.

2012 - Effects of Creatine and Exercise on Skeletal Muscle of FRG1-Transgenic Mice. [Articolo su rivista]
D. I., Ogborn; Kj, Smith; Jd, Crane; A., Safdar; Bp, Hettinga; Tupler, Rossella; Ma, Tarnopolsky

Background: The FRG1-transgenic mouse displays muscle dysfunction and atrophy reminiscent of fascioscapulohumeral muscular dystrophy (FSHD) and could provide a model to determine potential therapeutic interventions. Methods: To determine if FRG1 mice benefit from treatments that improve muscle mass and function, mice were treated with creatine alone (Cr) or in combination with treadmill exercise (CrEX). Results: The CrEx treatment increased quadriceps weight, mitochondrial content (cytochome c oxidase (COX) activity, COX subunit one and four protein), and induced greater improvements in grip strength and rotarod fall speed. While Cr increased COX subunits one and four protein, no effect on muscle mass or performance was found. Since Cr resulted in no functional improvements, the benefits of CrEx may be mediated by exercise; however, the potential synergistic action of the combined treatment cannot be excluded. Conclusion: Treatment with CrEx attenuates atrophy and muscle dysfunction associated with FRG1 overexpression. These data suggest exercise and creatine supplementation may benefit individuals with FSHD.

2012 - Facioscapulohumeral muscular dystrophy: new insights from compound heterozygotes and implication for prenatal genetic counselling. [Articolo su rivista]
I., Scionti; G., Fabbri; C., Fiorillo; G., Ricci; F., Greco; D'Amico, Roberto; A., Termanini; L., Vercelli; G., Tomelleri; M., Cao; L., Santoro; Percesepe, Antonio; Tupler, Rossella

AbstractBackground Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disease with a prevalence of 1 in 20 000. Almost all patients with FSHD carry deletions of integral copies of tandem 3.3 kb repeats (D4Z4) located on chromosome 4q35. However, FSHD families have been reported in which individuals carrying a D4Z4-reduced allele remain asymptomatic. Recently, it has been proposed that the D4Z4-reduced allele is pathogenic only in association with the permissive haplotype, 4APAS. Methods and results Through the Italian National Registry for FSHD (INRF), genotype-phenotype correlations were extensively studied in 11 non-consanguineous families in which two D4Z4-reduced alleles segregate. Overall, 68 subjects carrying D4Z4-reduced alleles were examined, including 15 compound heterozygotes. It was found that in four families the only FSHD-affected subject was the compound heterozygote for the D4Z4-reduced allele, and 52.6% of subjects carrying a single D4Z4-reduced 4A161PAS haplotype were non-penetrant carriers; moreover, the population frequency of the 4A161PAS haplotype associated with a D4Z4-reduced allele was found to be as high as 1.2%. Conclusions This study reveals a high frequency of compound heterozygotes in the Italian population and the presence of D4Z4-reduced alleles with the 4A161PAS pathogenic haplotype in the majority of non-penetrant subjects in FSHD families with compound heterozygosity. These data suggest that carriers of FSHD-sized alleles with 4A161PAS haplotype are more common in the general population than expected on the basis of FSHD prevalence. These findings challenge the notion that FSHD is a fully penetrant autosomal dominant disorder uniquely associated with the 4A161PAS haplotype, with relevant repercussions for genetic counselling and prenatal diagnosis.

2012 - Large scale population analysis challenges the current criteria for the molecular diagnosis of fascioscapulohumeral muscular dystrophy (FSHD) [Articolo su rivista]
Scionti, Isabella; Greco, Francesca; G., Ricci; Govi, Monica; P., Arashiro; L., Vercelli; A., Berardinelli; A., Angelini; G., Antonini; M., Cao; A., Di Muzio; M., Moggio; L., Morandi; E., Ricci; C., Rodolico; L., Ruggiero; L., Santoro; G., Siciliano; G., Tomelleri; C. P., Trevisan; G., Galluzzi; W. E., Wright; M., Zatz; Tupler, Rossella

ABSTRACT Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤ 8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, since individuals carrying D4Z4 reduced alleles and no FSHD and patients with FSHD and no short allele have been observed additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with reduced number (4-8) of D4Z4 repeats on chromosome 4q and one third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited since this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

2012 - Response [NEUROMUSCULAR DISORDERS] [Articolo su rivista]
Ricci, Giulia; Scionti, Isabella; Tupler, Rossella; Siciliano, Gabriele

This is a letter to the Editor in response to comments from Spatafora et al. Neuromuscul DisThe letter by Spadafora and coworkers questions the pathogenic role of heterozygous CAV3 T78M variant in a patient we recently described (Ricci et al, 2012) because the same heterozygous variant has been found in 3, out of 195, healthy unrelated controls. As an alternative hypothesis to explain our patient carrying both heterozygous D4Z4 reduction and CAV3 T78M variant the authors suggests that the dramatic reduction of caveolin-3 we observed in the muscle biopsy might be due to mutations in other genes. This interesting hypothesis is consistent with several reports suggesting that other genetic modifiers may be implied in Cav-3 deficiency, so that the same CAV3 mutation can lead to heterogeneous clinical phenotypes and muscle histopathological changes (REF). In particular the study by Traverso et al. (2008) suggested that the T78M mutation might results pathogenic only in homozygous feature through a loss-of-function mechanism. In our view the finding that CAV3 T78M mutation is a common variant with a frequency of 1.5% might disclose a different scenario. In fact by analyzing DNA elements at 4q35 in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients, we have recently discovered that 1.3 % of healthy unrelated subjects carry alleles of 21-35 kb in size on chromosome 4q associated with the 4APAS161 permissive haplotype (Scionti et al 2012). This discovery points at the possibility that in the heterozygous state D4Z4 reduction might produce a subclinical sensitized condition that requires other epigenetic mechanisms or a contributing factor to cause overt myopathy. In some rare cases, it could be by becoming homozygous (Scionti et al 2012b) while in others, it might be by the simultaneous heterozygosity for a different and recessive myopathy, as suggested by many reports in which the FSHD contractions are found in association with a second molecular defect (REF). The clinical case, we have recently reported, seems to fall in this complex picture. The observation of Spatafora et al supports the idea that D4Z4 reduced allele as well CAV3 T78M variant, are not per se sufficient to cause a disease phenotype. In our patient, whose geographical origin is Calabria, the simultaneous mutations (heterozygous CAV3 T78M and 35 kb 4A161PAS D4Z4 allele) could play a synergistic effect in reaching disease threshold and determining overlapping phenotype.In light of this finding particular attention should be paid to the geographical origin of similar cases. As suggested by Spatafora et al, the frequency of the CAV3 T78M variant on the whole Italian territory should be established. In conclusion these population studies could have important relevance for prenatal genetic counseling of healthy carriers who are at risk of transmitting the mutation to next generation.

2012 - Rippling muscle disease and facioscapulohumeral dystrophy-like phenotype in a patient carrying heterozygous CAV3 T78M mutation and D4Z4 partial deletion: further evidence for “double trouble” overlapping syndromes [Articolo su rivista]
Ricci, G; Scionti, Isabella; Alì, G; Volpi, L; Zampa, V; Fanin, M; Angelini, C; Politano, L; Tupler, Rossella; Siciliano, G.

We report the first case of a heterozygous T78M mutation in the caveolin-3 gene (CAV3) associated with rippling muscle disease and proximal myopathy. The patient displayed also bilateral winged scapula with limited abduction of upper arms and marked asymmetric atrophy of leg muscles shown by magnetic resonance imaging. Immunohistochemistry on the patient's muscle biopsy demonstrated a reduction of caveolin-3 staining, compatible with the diagnosis of caveolinopathy. Interestingly, consistent with the possible diagnosis of FSHD, the patient carried a 35 kb D4Z4 allele on chromosome 4q35. We discuss the hypothesis that the two genetic mutations may exert a synergistic effect in determining the phenotype observed in this patient.

2011 - Interference Improves Myopathic Phenotypes in Mice Over-expressing FSHD Region Gene 1 (FRG1). [Articolo su rivista]
Wallace, Lm; Garwick Coppens, Se; Tupler, Rossella; Harper, S. Q.

Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders.

2011 - New molecular findings in congenital myopathies due to selenoprotein N gene mutations. [Articolo su rivista]
Cagliani, R; Fruguglietti, Me; Berardinelli, A; D'Angelo, Mg; Prelle, A; Riva, S; Gorni, K; Orcesi, S; Lamperti, C; Pichiecchio, A; Signaroldi, E; Tupler, Rossella; Magri, F; Govoni, A; Corti, S; Bresolin, N; Moggio, M; Comi, G. P.

Selenoprotein N-related myopathy (SEPN1-RM) is an early-onset muscle disorder that can manifest clinically as congenital muscular dystrophy with spinal rigidity and can result in specific pathological entities such as multiminicore disease, desmin-related myopathy with Mallory body-like inclusions, and congenital fiber-type disproportion. Here we describe the clinical, histopathological, muscle magnetic resonance imaging (MRI) and genetic findings of three Italian SEPN1-RM families. Proband 1 is a 31-year-old female who was floppy at birth and developed axial and mild lower limb-girdle weakness. The second proband is a 13-year-old boy with RSMD1. Probands 3 and 4 were brothers showing clinical phenotype of congenital myopathy. Muscle MRI demonstrated selective involvement of sartorius, gluteal muscles and distal gastrocnemius and sparing of rectus femoris and gracilis. Muscle histopathology showed in proband 1 myopathic changes with mild connective tissue increase and some fibres lacking the Z-line, while probands 2 and 3 had multiminicores. SEPN1 gene analysis revealed five mutations, three of which are novel. Proband 1 was a compound heterozygote for a 92-bp (exon 1) and a 1-bp deletion (exon 9); proband 2 had a 99-bp deletion and a 10-bp duplication in exon 1, and proband 3 presented a novel homozygous mutation in intron 10 acceptor splice site.

2010 - A standardized clinical evaluation of patients affected by facioscapulohumeral muscular dystrophy: The FSHD clinical score [Articolo su rivista]
Costanza, Lamperti; Greta, Fabbri; Liliana, Vercelli; D'Amico, Roberto; Roberto, Frusciante; Bonifazi, Emanuela; Chiara, Fiorillo; Carlo, Borsato; Michelangelo, Cao; Maura, Servida; Greco, Francesca; Rita Di, Leo; Leda, Volpi; Claudia, Manzoli; Paola, Cudia; Ebe, Pastorello; Leopoldo, Ricciardi; Gabriele, Siciliano; Giuliana, Galluzzi; Carmelo, Rodolico; Lucio, Santoro; Giuliano, Tomelleri; Corrado, Angelini; Enzo, Ricci; Laura, Palmucci; Maurizio, Moggio; Tupler, Rossella

To define numerically the clinical severity of facioscapulohumeral muscular dystrophy (FSHD), we developed a protocol that quantifies muscle weakness by combining the functional evaluation of six muscle groups affected in this disease. To validate reproducibility of the protocol, 69 patients were recruited. Each patient was evaluated by at least five neurologists, and an FSHD severity score was given by each examiner. The degree of agreement among clinicians' evaluations was measured by kappa-statistics. Nineteen subjects received a score between 0 and 1, 9 had a score between 2 and 4, 20 received a score between 5 and 10, and 8 had a score between 11 and 15. Of the 13 subjects with D4Z4 alleles within the normal range (ranging from 10 to 150 repeats), 12 obtained a score of 0 and only 1 had a score of 1. Kappa-statistics showed a very high concordance for all muscle groups. We developed a simple, reliable, easily used tool to define the clinical expression of FSHD. Longitudinal studies will assess its sensitivity and utility in measuring changes for widespread use.

2010 - Comment on 'Huntington's disease presenting as ALS' [Articolo su rivista]
Mandrioli, Jessica; Bernabei, Chiara; Georgoulopoulou, Eleni; Nichelli, Paolo Frigio; Cortelli, Pietro; Tupler, Rossella; E., Signaroldi; P., Sola

This is a case report of patient with concurrent Huntington disease and Amyotrophic Lateral Sclerosis

2010 - The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis. [Articolo su rivista]
Forlani, G; Giarda, E; Ala, U; Di Cunto, F; Salani, M; Tupler, Rossella; Kilstrup Nielsen, C; Landsberger, N.

Rett syndrome is a severe neurodevelopmental disorder mainly caused by mutations in the transcriptional regulator MeCP2. Although there is no effective therapy for Rett syndrome, the recently discovered disease reversibility in mice suggests that there are therapeutic possibilities. Identification of MeCP2 targets or modifiers of the phenotype can facilitate the design of curative strategies. To identify possible novel MeCP2 interactors, we exploited a bioinformatic approach and selected Ying Yang 1 (YY1) as an interesting candidate. We demonstrate that MeCP2 interacts in vitro and in vivo with YY1, a ubiquitous zinc-finger epigenetic factor regulating the expression of several genes. We show that MeCP2 cooperates with YY1 in repressing the ANT1 gene encoding a mitochondrial adenine nucleotide translocase. Importantly, ANT1 mRNA levels are increased in human and mouse cell lines devoid of MeCP2, in Rett patient fibroblasts and in the brain of Mecp2-null mice. We further demonstrate that ANT1 protein levels are upregulated in Mecp2-null mice. Finally, the identified MeCP2-YY1 interaction, together with the well-known involvement of YY1 in the regulation of D4Z4-associated genes at 4q35, led us to discover the anomalous depression of FRG2, a subtelomeric gene of unknown function, in Rett fibroblasts. Collectively, our data indicate that mutations in MeCP2 might cause the aberrant overexpression of genes located at a specific locus, thus providing new candidates for the pathogenesis of Rett syndrome. As both ANT1 mutations and overexpression have been associated with human diseases, we consider it highly relevant to address the consequences of ANT1 deregulation in Rett syndrome.

2009 - Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy. [Articolo su rivista]
R., Darabi; J., Baik; M., Clee; M., Kyba; Tupler, Rossella; R. C., Perlingeiro

Muscular dystrophies (MDs) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon; however, to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell-derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD.

2009 - In vitro 1H and 31P NMR spectroscopy as a tool for investigating muscle energy state in facioscapulohumeral muscolar dystrophy (FSHD) mouse model [Abstract in Atti di Convegno]
Mucci, Adele; Ghiaroni, Valeria; Muscatello, Umberto; Tupler, Rossella; Schenetti, Luisa

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary neuromuscular disorder characterized by progressive weakness and atrophy of the facial, shoulder, abdominal and pelvic girdle muscles. We proposed that its pathogenesis could be associated with the over-expression of genes mapped at chromosome 4q35, ANT1, FRG1 and FRG2. Consistently, transgenic mice over-expressing FRG1 develop a progressive muscular dystrophy characterized by symptoms similar to those of human disease and thus it can be considered a reliable mice model to study FSHD. FSHD mouse model shows reduced tolerance to exercise and muscle weakness which can be related to disorders of muscle energy mechanisms.To investigate this hypothesis we have applied 1H and 31P NMR spectroscopy to wild-type and dystrophic vastus muscle PCA extracts to evaluate the muscle energy parameters by measuring the concentration of the major energy metabolites (ATP, ADP, AMP, Pi, Cr and PCr)

2008 - Facioscapulohumeral muscular dystrophy: a multicenter study on hearing function. [Articolo su rivista]
C. P., Trevisan; E., Pastorello; M., Ermani; C., Angelini; G., Tomelleri; P., Tonin; T., Mongini; L., Palmucci; G., Galluzzi; Tupler, Rossella; G., Marioni; A., Rimini

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant progressive myopathy, characteristically associated with a 4q35 deletion. In the unusual infantile-onset form of this degenerative disease, sensorineural hearing loss is a frequent clinical manifestation, whereas in patients with typical late-onset FSHD, investigations regarding hearing impairment yielded controversial results. We describe the findings of a multicenter investigation on possible auditory impairment in a series of 73 FSHD patients with a genetically confirmed diagnosis. Among them, 49 cases with no risk factors for deafness, aside from the disease, were identified by a clinical questionnaire and otoscopic examination (mean age 37.8 years, 31 males and 18 females). These subjects were evaluated by pure-tone audiometry. None were aware of hearing loss, while 4 had raised unilateral or bilateral pure-tone audiometric thresholds at 4000 and 8000 Hz, when evaluated by standardized tables. However, the mean raw pure-tone audiometric threshold values for these 49 cases were not significantly different from those of 55 controls (mean age 37.1 years, 32 males and 23 females). Moreover, by statistical analysis, age of onset, degree of muscular weakness and 4q35 EcoRI fragment size made no significant difference to auditory thresholds in our FSHD patients. Overall, the results of our multicenter study suggest that hearing loss in typical FSHD is not more prevalent than in the normal population.

2008 - Novel mitochondrial tRNA Leu(CUN) transition and D4Z4 partial deletion in a patient with a facioscapulohumeral phenotype. [Articolo su rivista]
Filosto, M; Tonin, P; Scarpelli, M; Savio, C; Greco, Francesca; Mancuso, M; Vattemi, G; Govoni, V; Rizzuto, N; Tupler, Rossella; Tomelleri, G.

Point mutations in mtDNA-encoded tRNA genes frequently cause isolated myopathies but rarely cause the facioscapulohumeral phenotype. We report on a patient affected with chronic progressive weakness of facioscapulohumeral/peroneal muscles whose muscle biopsy showed a mitochondrial myopathy. mtDNA direct sequencing and RFLP analysis revealed a heteroplasmic transition T12313C which disrupts a conserved site in the T Psi C stem of the tRNA(Leu(CUN)) gene and fulfills the accepted criteria of pathogenicity. A partial deletion of the nuclear DNA D4Z4 region with residual repeat sizes of 25 kb was also found in the patient and in her mother. This is the first reported case of mitochondrial myopathy/facioscapulohumeral muscular dystrophy (FSHD) "double trouble".

2007 - Structural and functional alterations of muscle fibres in the novel mouse model of facioscapulohumeral muscular dystrophy [Articolo su rivista]
D'Antona, G; Brocca, L; Pansarasa, O; Rinaldi, C; Tupler, Rossella; Bottinelli, R.

We recently generated a mouse model of facioscapulohumeral muscular dystrophy (FSHD) by selectively overexpressing FRG1, a candidate gene for FSHD, in skeletal muscle. The muscles of the FRG-1 mice did not show any plasmamembrane defect suggesting a novel pathogenetic mechanism for FSHD. Here, we study structure and function of muscle fibres from three lines of mice overexpressing FRG1 at different levels: FRG1-low, FRG1-med, FRG1-high. Cross-sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of identified types of muscle fibres from FRG1-low and FRG1-med mice were analysed and found to be lower than in WT mice. Fast fibres and especially type 2B fibres (the fastest type) were preferentially involved in the dystrophic process showing a much larger force deficit than type 1 (slow) fibres. Consistent with the latter observation, the MHC isoform distribution of several muscles of the three FRG1 lines showed a shift towards slower MHC isoforms in comparison to WT muscle. Moreover, fast muscles showed a more evident histological deterioration, a larger atrophy and a higher percentage of centrally nucleated fibres than the soleus, the slowest muscle in mice. Interestingly, loss in CSA, Po/CSA and Vo of single muscle fibres and MHC isoform shift towards a slower phenotype can be considered early signs of muscular dystrophy (MD). They were, in fact, found also in FRG1-low mice which did not show any impairment of function in vivo and of muscle size in vitro and in soleus muscles, which had a completely preserved morphology. This study provides a detailed characterization of structure and function of muscle fibres in a novel murine model of one of the main human MDs and suggests that fundamental features of the dystrophic process, common to most MDs, such as the intrinsic loss of contractile strength of muscle fibres, the preferential involvement of fast fibres and the shift towards a slow muscle phenotype can occur independently from obvious alterations of the plasma membrane.

2006 - Altered gene silencing and human diseases [Articolo su rivista]
G., Perini; Tupler, Rossella

Epigenetic regulation of gene expression is mediated through several mechanisms, including modifications in DNA methylation, covalent modifications of core nucleosomal histones, rearrangement of histones and RNA interference. It is now clear that deregulation of epigenetic mechanisms cooperates with genetic alterations in the development and progression of several Mendelian disorders. Here, we summarize the recent findings that highlight how certain inherited diseases, such as Rett syndrome, Immunodeficiency–centromeric instability–facial anomalies syndrome, and facioscapulohumeral muscular dystrophy, result from altered gene silencing.

2006 - Facioscapulohumeral muscular dystrophy and occurrence of heart arrhythmia [Articolo su rivista]
C. P., Trevisan; E., Pastorello; M., Armani; C., Angelini; G., Nante; G., Tomelleri; P., Tonin; T., Mongini; L., Palmucci; G., Galluzzi; Tupler, Rossella; A., Barchitta

Background: Subjects with facioscapulohumeral muscular dystrophy (FSHD) do not generally suffer from significant cardiac symptoms. Although with heterogeneous results, studies reported to date indicate that heart alterations unrelated to cardiornyopathy are possible in FSHD. Patients and Methods: We describe the findings of a multicenter investigation aimed at detecting cardiac abnormalities in 83 IFSHD patients, 44 males and 39 females with a mean age of 47 years. All patients underwent clinical heart examination, 12-lead electrocardiography and 24-hour Holter monitoring; echocardiography was also performed on most patients. Results:Among the 83 patients, 62 with no cardiovascular risk factors were identified. Ten of them manifested clinical or subdinical cardiac involvement: 5 reported symptoms represented mostly by frequent palpitations secondary to supraventricular arrhythmia and another 5 exhibited electrocardiographic signs of short runs of supraventricular paroxysmal tachycardia. In the absence of cardiovascular risk factors, we found symptoms or signs of heart involvement of mainly arrhythmic origin in 10 of our 83 IFSHD patients (12%). Conclusions: Considering our data and those available in the literature as a whole, arrhythmic alterations seernto bedetected more frequently than expected in FSHD patients. Copyright (c) 2006 S. Karger AG, Basel.

2006 - Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1 [Articolo su rivista]
D., Gabellini; G., D'Antona; M., Moggio; A., Prelle; C., Zecca; R., Adami; B., Angeletti; P., Ciscato; Ma, Pellegrino; R., Bottinelli; Mr, Green; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene(1,2). Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 ( ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing(5-7). We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

2005 - Facioscapulohumeral muscular dystrophy type 1A in northwestern Tuscany: A molecular genetics-based epidemiological and genotype-phenotype study [Articolo su rivista]
Sposito, R.; Pasquali, L.; Galluzzi, F.; Rocchi, A.; Solito, B.; Soragna, D.; Tupler, R.; Siciliano, G.

Facioscapulohumeral muscular dystrophy type 1A (FSHD1A) is an autosomal dominant inherited disorder characterized by early involvement of facial and scapular muscles with eventual spreading to pelvic and lower limb muscles. A high degree of clinical variability with respect to age at onset, severity, and pattern of muscle involvement, both between and within families, is present. For this reason, diagnosis of FSHD1A can be sometimes difficult and molecular diagnosis is then necessary. A clinical and molecular genetic-based epidemiological investigation has been carried out in the territory of northwestern Tuscany in central Italy to calculate the prevalence rate of FSHD1A as of March, 2004. The molecular diagnosis has been based on the detection of large deletions of variable size of kpnI repeat units on chromosome 4q35. Results have been compared to those of a previous study conducted in the same area in 1981 (in the premolecular diagnosis era). The minimum prevalence rate was 4.60 × 10-5 inhabitants, a value four times higher compared to our previous study. No significant correlation between fragment size and clinical severity has been observed. This study confirms in an Italian population a prevalence rate of FSHD1A similar to that observed in other populations. Furthermore, it underlines the usefulness of routine adoption of the genetic testing in confirming clinical suspicion of FSHD1A as well as in correctly diagnosing atypical and otherwise misclassified cases. © Mary Ann Liebert, Inc.

2004 - FSHD: a disorder of muscle gene derepression. [Capitolo/Saggio]
Tupler, Rossella; Davide, Gabellini

Facioscapulohumeral muscular dystrophy (FSHD), the third most common myopathy, is an autosomal dominant disease with an insidious onset and progression. Almost all FSHD patients carry deletions of an integral number of tandem 3.3 kb repeats, termed D4Z4, located on chromosome 4q35. In FSHD patients a deletion of the integral number of D4Z4 repeats generates a fragment that is usually smaller than 35 kb (fewer than 11 repeats), whereas in normal controls the size usually ranges from 50 to 300 kb (between 11 and 150 units). D4Z4 is a repetitive element with heterochromatic features. Recently, 4q35 genes located upstream of D4Z4 have been found to be inappropriately overexpressed specifically in FSHD muscle. An element within D4Z4 has been shown to behave as a silencer that provides a binding site for a transcriptional repressing complex. These results suggest a model in which deletion of D4Z4 leads to the inappropriate transcriptional derepression of 4q35 genes, resulting in disease.

2004 - Molecular basis of facioscapulohumeral muscular dystrophy. [Articolo su rivista]
Tupler, Rossella; Gabellini, D.

Facioscapulohumeral muscular dystrophy (FSHD), the third most common myopathy, is an autosomal dominant disease with an insidious onset and progression. Almost all FSHD patients carry deletions of an integral number of tandem 3.3 kb repeats, termed D4Z4, located on chromosome 4q35. In FSHD patients a deletion of the integral number of D4Z4 repeats generates a fragment that is usually smaller than 35 kb (fewer than 11 repeats), whereas in normal controls the size usually ranges from 50 to 300 kb (between 11 and 150 units). D4Z4 is a repetitive element with heterochromatic features. Recently, 4q35 genes located upstream of D4Z4 have been found to be inappropriately overexpressed specifically in FSHD muscle. An element within D4Z4 has been shown to behave as a silencer that provides a binding site for a transcriptional repressing complex. These results suggest a model in which deletion of D4Z4 leads to the inappropriate transcriptional derepression of 4q35 genes, resulting in disease.

2004 - When Enough is Enough: Genetic Diseases Associated with Transcriptional Derepression. [Articolo su rivista]
Gabellini, D.; M. R., Green; Tupler, Rossella

For many human genetic diseases, the underlying geneticdefect has been determined. Thus, although traditionally afield only for researchers in medicine or human genetics,human diseases are now opening up to molecular biologists,cell biologists and biochemists. Here we discuss four humangenetic disorders, Familial Alzheimer’s disease, Rett syndrome,Klippel—Trenaunay syndrome and Facioscapulohumeralmuscular dystrophy, and how investigations into these diseasesare providing important lessons about human biology.

2003 - A locus for migraine without aura maps on chromosome 14q21.2- q22.3. [Articolo su rivista]
D., Soragna; A., Vettori; G., Carraro; Marchioni, . E.; G., Vazza; S., Bellini; Tupler, Rossella; F. SAVOLDI, M. L. MOSTACCIUOLO

Migraine is a common and disabling neurological disease of unknown origin characterized by a remarkable clinical variability. It shows strong familial aggregation, suggesting that genetic factors are involved in its pathogenesis. Different approaches have been used to elucidate this hereditary component, but a unique transmission model and causative gene(s) have not yet been identified. We report clinical and molecular data from a large Italian pedigree in which migraine without aura (MO) segregates as an autosomal dominant trait. After exclusion of any association between MO and the known familial hemiplegic migraine and migraine with aura loci, we performed a genomewide linkage analysis using 482 polymorphic microsatellite markers. We obtained significant evidence of linkage between the MO phenotype and the marker D14S978 on 14q22.1 (maximum two-point LOD score of 3.70, at a recombination fraction of 0.01). Multipoint parametric analysis (maximum LOD score of 5.25 between markers D14S976 and D14S978) and haplotype construction showed strong evidence of linkage in a region of 10 cM flanked by markers D14S1027 and D14S980 on chromosome 14q21.2-q22.3. These results indicate the first evidence of a genetic locus associated with MO on chromosome 14.

2003 - An Italian family affected by Nasu-Hakola disease with a novel genetic mutation in the trem 2 gene [Articolo su rivista]
Soragna, D.; Tupler, Rossella; Ratti, M. T.; Montalbetti, L.; Papi, L.; SESTINI R. . J., NEUROSURG PSICHIATRY

Polycystic lipomembranous osteodysplasia with sclerosing leucoencephalopathy (PLOSL; MIM 221770), also known as Nasu-Hakola disease, is a recessively inherited disorder characterised by systemic bone cysts and progressive presenile dementia associated with sclerosing leucoencephalopathy. Some patients, however, do not have clinically manifest osseous problems despite the radiological demonstration of cystic bone lesions. The disease leads to death before the age of 50.1 The disease is characterised by genetic heterogeneity: mutations in two genes (TYROBP and TREM2) encoding different subunits of a membrane receptor complex in natural killer and myeloid cells have been associated with the disease.We report here the clinical and genetic analysis of an Italian family in which two siblings are affected by PLOSL. In the two affected sisters, sequencing analysis identified a homozygous C to T mutation at position 191 (191 C/T) in exon 2 of the TREM2 gene. The mutation changes glutamine 33 to a stop codon (Q33X). Our study shows that in Italy PLOSL is explained by two different mutations in TREM2 gene. Its prevalence is undetermined because the disease is likely to go unrecognised.

2003 - Transcriptional derepression as a cause of genetic diseases [Articolo su rivista]
Gabellini, D; Tupler, Rossella; Green, Mr

Transcription of DNA into mRNA is a highly regulated process directed by a complex molecular machine comprising more than 100 proteins. Regulation of transcription occurs by both positive (transcriptional activation) and negative (transcriptional repression) mechanisms. Recently, inappropriate transcriptional derepression has been found as the underlying basis of several human genetic diseases. The putative target genes, whose inappropriate expression is thought to cause disease, have remained elusive but are being searched for intensively.

2002 - A repressor complex binds a chromosomal repeat deleted in dystrophic muscle. [Articolo su rivista]
Gabellini, D; Green, Mr; Tupler, Rossella

Facioscapulohumeral muscular dystrophy (FSHD), a common myopathy, is an autosomal dominant disease of unknown molecular mechanism. Almost all FSHD patients carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Here, we find that in FSHD muscle 4q35 genes located upstream of D4Z4 are inappropriately over-expressed. We show that an element within D4Z4 specifically binds a multi-protein complex consisting of YY1, a known transcriptional repressor, HMGB2, an architectural protein, and nucleolin. We demonstrate that this multi-protein complex binds D4Z4 in vitro and in vivo and mediates transcriptional repression of 4q35 genes. Based upon these results we propose that deletion of D4Z4 leads to the inappropriate transcriptional derepression of 4q35 genes resulting in disease.

2001 - Expressing the human Genome [Articolo su rivista]
Tupler, Rossella; G. PERINI, G.; M. R., Green

We have searched the human genome for genes encoding new proteins that may be involved in three nuclear gene expression processes: transcription, pre-messenger RNA splicing and polyadenylation. A plethora of potential new factors are implicated by sequence in nuclear gene expression, revealing a substantial but selective increase in complexity compared with Drosophila melanogaster and Caenorhabditis elegans. Although the raw genomic information has limitations, its availability offers new experimental approaches for studying gene expression.

1999 - Cerebellar dysgenesis and mental ritardation associated with a complex chromosomal rearrangement. [Articolo su rivista]
Maserati, E; Verri, A; Seghezzi, L; Tupler, Rossella; Federico, A; Tiepolo, L; Maraschio, P.

Cerebellar hypoplasia, mild mental retardation, skeletal abnormalities, and ataxia were present in a 40 years old patient with a complex chromosome rearrangement (CCR). Chromosomes 2, 5, 16, and 17 were involved in the CCR. For the definition of the eight breakpoints leading to the rearrangement FISH with whole chromosomes paintings and specific telomeric probes was employed. Gene disruption, positional effect variegation, and sub-microscopic deletions are all possible causes for the abnormal phenotype observed in the patient.

1999 - Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy [Articolo su rivista]
Tupler, Rossella; G., Perini; M. A., Pellegrino; M. R., Green

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

1999 - Ring chromosome 9 with a 9p22.3-p24.3 duplication [Articolo su rivista]
L., Seghezzi; P., Maraschio; M., Bozzola; E., Maserati; Tupler, Rossella; A., Marchi; L., Tiepolo

A ring chromosome 9 containing an inverted 9p22.3-p24.3 duplication was found in a girl presenting with some of the phenotypic characteristics of ring 9 syndrome such as trigonocephaly, microcephaly, hypotelorism, micrognathia, single palmar crease, and bilateral clinodactyly. The typical facial dysmorphic features of 9p duplication, ascribed to trisomy of the band p22, were not present in this patient. Cytogenetic and molecular studies indicated that the duplicated region of band p22 in the ring is confined to the sub-band 22.3. CONCLUSION: The chromosome region responsible for the 9p duplication syndrome appears to be restricted to sub-bands p22. 1-22.2.

1998 - Identical de novo mutation at the D4F104S1 locus in monozygotic male twins affected by facioscapulohumeral muscular dystrophy (FSHD) with different clinical expression [Articolo su rivista]
Tupler, Rossella; L., Barbierato; M., Memmi; C. A., Sewry; D., DE GRANDIS; P., Maraschio; L., Tiepolo; A., Ferlini

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive hereditary neuromuscular disorder, transmitted in an autosomal dominant fashion. Its clinical expression is highly variable, ranging from almost asymptomatic subjects to wheelchair dependent patients. The molecular defect has been linked to chromosome 4q35 markers and has been related to deletions of tandemly repeated sequences located in the subtelomeric region detected by probe p13E-11 (D4F104S1). We describe a pair of monozygotic male twins affected by FSHD, carrying an identical de novo p13E-11 EcoRI fragment of paternal origin and showing great variability in the clinical expression of the disease, one being almost asymptomatic and the other severely affected. Their medical history was the same, with the exception of an anti-rabies vaccination performed at the age of 5 in the more severely affected twin. We hypothesise that the vaccination might have triggered an inflammatory immune reaction contributing to the more severe phenotype.

1998 - Involvement of 9q22.1-31.3 region in pyloric stenosis [Articolo su rivista]
P., Maraschio; E., Maserati; L., Seghezzi; Tupler, Rossella; M. P., Verri; A., MARCHI A; L., Tiepolo

A chromosome 9q22.1 -q3 1.3 direct duplication was found in a 40-year-old patient in a screening program for mentally retarded people. The duplicated region was confirmed by fluorescent in situhybridisation (FISH) showing a double fluorescent signal with 9561410 and 931D9 YACs, mapping respectively to q2 1.33-22.1 and 22.3 I -22.33. YAC 933C.5, mapping more distally in 31.3-32, resulted in a single signal.

1997 - A variant of the Nijmegen breakage syndrome with unusual cytogenetic features and intermediate cellular radiosensitivity [Articolo su rivista]
Tupler, Rossella; G. L., Marseglia; M., Stefanini; E., Prosperi; L. CHESSA, T. NARDO; A., Marchi; P., Maraschio

We report the first Italian case of Nijmegen breakage syndrome (NBS). The proband is an immunodeficient, microcephalic, 11 year old boy with a "bird-like" face. He developed a T cell rich B cell lymphoma. Spontaneous chromosomal instability was detected in T and B lymphocytes and fibroblasts; chromosomes 7 and 14 were only sporadically involved in the rearrangements and no clonal abnormality was present. The patient appeared to be sensitive both to ionising radiation and to bleomycin, although his sensitivity did not reach the level of AT reference cells. After bleomycin treatment, inhibition of DNA synthesis was low when compared with normal cells, but higher than observed in an AT reference strain. Moreover, cell cycle analysis, after drug exposure, showed a progressive reduction in the percentage of S phase cells, but the G1 arrest, found in normal cells, was not observed. On clinical evaluation our patient shares features with NBS subjects, but cytogenetic and cell biological data do not completely overlap with those reported in Nijmegen breakage syndrome. The ethnic origin of our patient might account for these differences, as expression of different allelic forms at the NBS locus.

1996 - A novel mechanism for the origin of supernumerary marker chromosomes [Articolo su rivista]
P., Maraschio; Tupler, Rossella; E., Rossi; L., Barbierato; F., Uccellatore; M., Rocchi; O., Zuffardi; M., Fraccaro

A ring chromosome 3 and a 47th chromosome formed by the portions of 3p and 3q distal to the r(3) breakpoints were found in a girl with mental retardation and minor facial anomalies. The supernumerary chromosome 3, rea(3), had a primary constriction inside its 3p portion (3p23) and was consistently stable both in lymphocytes and fibroblasts. In situ hybridization with alphoid probes revealed that the r(3) maintained its wild-type-centromere, whereas the rea(3) showed no alphoid-related signals. This case and a similar one recently reported demonstrate that acentric fragments can acquire a new centromere and become stable, and that supernumerary marker chromosomes can also originate by the junction of the acentric portions distal to the centric region forming a ring. The possibility of such a chromosome segregating will depend on its ability to (re)activate a new centromere.

1996 - An analysis of Xq deletions [Articolo su rivista]
P., Maraschio; Tupler, Rossella; L., Barbierato; E., Dainotti; D., Larizza; F., Bernardi; H., Hoeller; A., Garau; L., Tiepolo

We characterized by fluorescence in situ hybridization and Southern blotting 14 partial Xq monosomies, 11 due to terminal deletions and 3 secondary to X/autosome translocations. Three cases were mosaics with a XO cell line. In view of the possible role played by telomeres in chromosome segregation, we hypothesize a relationship between the loss of telomeric sequences in terminal deletions and the presence of 45,X cells. A correlation between phenotype and extent of deletion reveal that there is no correspondence between the size of the deletion and impairment of gonadal function. Turner stigmata are absent in patients without an XO cell line, when the breakpoint is distal to Xq24. A low birthweight is present whenever the breakpoint is at q22 or more proximal.

1996 - Mild phenotype associated with inv dup 8 (q21.2-q22.3) of maternal origin [Articolo su rivista]
Tupler, Rossella; E., Pagliano; L., Barbierato; G., Lanzi; P., Maraschio; E., Fazzi

We report on a girl with a de novo inverted duplication of chromosome 8 (q21.2-q22.3) associated with a mild phenotype. We were able to establish the maternal origin of the rearranged chromosome. We discuss the correlation between genotype and phenotype on the basis of a review of the findings from individuals with partial dup(8q).

1996 - Monosomy of distal 4q does not cause facioscapulohumeral muscular dystrophy [Articolo su rivista]
Tupler, Rossella; A., Berardinelli; L., Barbierato; R., Frants; J. E., Hewitt; G., Lanzi; P., Maraschio; L., Tiepolo

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary neuromuscular disorder transmitted in an autosomal dominant fashion. FSHD has been located by linkage analysis in the most distal part of chromosome 4q. The disease is associated with deletions within a 3.2 kb tandem repeat sequence, D4Z4. We have studied a family in which an abnormal chromosome 4 segregates through three generations in phenotypically normal subjects. This chromosome is the derivative of a (4;D or G) (q35;p12) translocation. Molecular analysis of the region 4q35 showed the absence of the segment ranging from the telomere to locus D4F104S1. Probe p13E-11 (D4F104S1), which detects polymorphic EcoRI fragments containing D4Z4, in Southern blot analysis showed only one allele in the carriers of the abnormal chromosome 4. Probe p13E-11 EcoRI fragments are contained in the subtelomeric region of 4q and their rearrangements associated with FSHD suggested that the gene responsible for the muscular dystrophy could be subject to a position effect variegation (PEV) because of its proximity to subtelomeric heterochromatin. The absence of the 4q telomeric region in our phenotypically normal cases indicates that haploinsufficiency of the region containing D4Z4 does not cause FSHD.

1994 - Balanced autosomal translocation and ovarian dysgenesis [Articolo su rivista]
Tupler, Rossella; L., Barbierato; D., Larizza; P., Sampaolo; F., Piovella; P., Maraschio

We report two unrelated women with gonadal dysgenesis, and a (6;15)(p21.3;q15) and a (8;9)(p11.2;q12) balanced translocation, respectively. The patients were of normal stature and showed no phenotypic abnormality or malformation other than ovarian failure. We are not aware of other reports of balanced autosomal translocations associated with gonadal dysgenesis in women. The occurrence of chromosome anomaly and sterility in the two females may be coincidental. However, studies on mouse gametic progression indicate that balanced autosomal translocations can cause oocyte degeneration and reduction of reproductive lifespan. On the basis of these observations, we cannot exclude that the ovarian failure in our patients is the result of oocyte degeneration because of as yet unidentified consequences of the balanced translocations.

1994 - Maternal derivation of inv dup (22) and clinical variation in cat-eye syndrome [Articolo su rivista]
Tupler, Rossella; H. HOELLER, A. P. E. Z. Z. O. L. O. P. MARASCHIO

Cytogenetic analysis in a male child with dismorphies and renal anomalies showed an extra bisatellited chromosome. In situ hybridization and an analysis of cytogenetic polymorphisms revealed that the abnormal chromosome derived from a single maternal chromosome 22.

1994 - Molecular analysis of a Y;1 translocation in an azoospermic male [Articolo su rivista]
P., Maraschio; Tupler, Rossella; E., Dainotti; M., Cortinovis; L., Tiepolo

Cytogenetic studies on an azoospermic male revealed a balanced Y;1 translocation: 46,X,t(Y;1)(q12;p34.3). In situ hybridization with the probe St35-239 (DXY64) and with a probe detecting telomeric sequences revealed that only the Y telomere is involved in the translocation. Fluorescence in situ hybridization with a chromosome 1 library on meiotic preparations revealed consistent contact of the painted chromosome 1 with the sex vesicle at pachytene, the most advanced stage of spermatogenesis observed. No deletions were observed after Southern blot analysis with probes p49f (DYS1), 50f2 (DYS7), and 52d (DYF27), which map in interval 6 of the Y chromosome, which includes the azoospermia factor (AZF) gene. The results indicate that the infertility of the translocation carrier could be due to an alteration of the sex vesicle structure or to a disturbance of X-chromosome inactivation as a result of the proximity to the autosomal portion.

1993 - A highly informative microsatellite repeat polymorphism in intron 1 of the human amyloid precursor protein (APP) gene [Articolo su rivista]
Tupler, Rossella; E., Rogaeva; G., Vaula; M., Mortilla; W., Leukiw; Y., Liang; R., Hancock; E., Rogaev; GEORGE HYSLOP, P. S. T.

A highly polymorphic microsatellite was found in the first intron of the beta Amyloyd Precursor Protein gene by screening a chromosome 21 cosmid genomic library.

1992 - A complex chromosome re arrangement with 10 breakpionts: tentative assignment of the locus for Williams syndrome to 4q33-q35.1 [Articolo su rivista]
Tupler, Rossella; P., Maraschio; A., Gerardo; R., Maineri; G., Lanzi; L., Tiepolo

An unbalanced complex chromosome rearrangement with 10 breakpoints resulting in four derivative chromosomes (1, 2, 4, and 11) was found in a girl with severe phenotypic abnormalities, many of which are characteristic of Williams syndrome. The patient was monosomic for the region 4q33----q35.1 and thus the mapping of the syndrome could tentatively be restricted to this region.

1992 - A novel but non-pathogenic mutation in exon 4 of the human amyloid precursor protein (APP) gene [Articolo su rivista]
G., Vaula; M., Mortilla; Tupler, Rossella; W., Lukiw; R., Tanzi; L., Nee; R., Polinsky; J. F., Foncin; A. C., Bruni; M. P., Montesi; S., Sorbi; P., St George Hyslop

Mutations in the beta-amyloid precursor protein (APP) gene have been associated with both familial Alzheimer disease (FAD) and with hereditary cerebral haemorrhage. The polymerase chain reaction was used to both amplify and sequence exon 4 of the APP gene from genomic DNA of subjects with FAD and normal control subjects. A novel, rare, conservative DNA sequence variant was discovered at nucleotide 459 of codon 153 (valine) in exon 4 of the APP gene in an affected member of a large FAD pedigree. Segregation studies indicate that this mutation is likely to be non-pathogenic, but must be recognized and discriminated from pathogenic mutations during sequencing studies of the APP gene in patients with FAD.

1992 - Assessment of amyloid b-protein precursor gene mutations in a large set of familiar and sporadic Alzheimer's disease cases [Articolo su rivista]
R. E., Tanzi; G., Vaula; D. M., Romano; M., Mortilla; T. L., Huang; Tupler, Rossella; W., Wasco; B. T., Hyman; J. L., Haines; B. J., Jenkins; M., Kalaitsidaki; A. C., Warren; M. C., Mcinnis; S. E., Antonarakis; H., Karlinsky; M. E., Percy; L., Connor; J., Growdon; D. R., Crapper McIachlan; J. F., Gusella; P. H., St George Hyslop

A genetic locus associated with familial Alzheimer disease (FAD) and a candidate gene, APP, encoding the amyloid protein precursor have both been assigned previously to chromosome 21, and, in a few FAD families, mutations of APP have been detected. However, obligate crossovers between APP and FAD have also been reported in several FAD pedigrees, including FAD4, a large kindred showing highly suggestive evidence for linkage of the disorder to chromosome 21. In case the apparent APP crossover in FAD4 actually represented an intragenic recombination event or segregation of different mutations in different family branches, we have performed a more detailed assessment of APP as a candidate gene in this family. The entire coding region of the APP gene was sequenced for FAD4 and for FAD1, a second large kindred. No mutations were found, indicating that, in at least one chromosome 21-linked FAD pedigree, the gene defect is not accounted for by a mutation in the known coding region of the APP gene. A total of 25 well-characterized early- and late-onset FAD pedigrees were typed for genetic linkage to APP, to assess the percentage of FAD families predicted to carry mutations in the APP gene. None of the FAD families yielded positive lod scores at a recombination fraction of 0.0. To estimate the overall prevalence of FAD-associated mutations in the beta A4 domain of APP, we sequenced exons 16 and 17 in 30 (20 early- and 10 late-onset) FAD kindreds and in 11 sporadic AD cases, and we screened 56 FAD kindreds and 81 cases of sporadic AD for the presence of the originally reported FAD-associated mutation, APP717 Val----Ile (by BclI digestion). No APP gene mutations were found in any of the FAD families or sporadic-AD samples examined in this study, suggesting that the mutations in exons 16 and 17 are a rare cause of FAD. Overall, these data suggest that APP gene mutations account for a very small portion of FAD.

1992 - Establishment and characterization of two cell lines derived from human glioblastoma multiforme [Articolo su rivista]
G., Bacciocchi; N., Gibelli; C., Zibera; P., Pedrazzoli; G., Bergamaschi; P., DE PICEIS POLVER; M., Danova; G., Mazzini; L., Palomba; Tupler, Rossella

We established and characterized two cell lines derived from glioblastoma multiforme. Both cell lines exhibited tumor cell morphology and growth kinetics and showed variable expression of glial fibrillary acidic protein (GFAP), S-100, fibronectin and vimentin. Cytofluorimetrical analysis of tumor samples showed a diploid DNA distribution, whereas permanent culture cells evolved to the hyperdiploid DNA content. Karyotype studies revealed cytogenetical abnormalities described in glial tumors including gain of chromosome 7, loss of chromosome 10 and presence of double minutes (DMs). Enhanced expression of Ha-ras and c-myc genes resulted from high p-21 and p-62 levels. The contemporary presence of TGF-alpha and EGF-Rc transcripts suggested an autocrine mechanism in the cell lines growth.

1992 - Interphase cytogenetics of the ICF syndrome [Articolo su rivista]
P., Maraschio; M., Cortinovis; E., Dainotti; Tupler, Rossella; L., Tiepolo

Interphase behaviour of centromeric heterochromatin of chromosomes 1 and 16 has been investigated in lymphocytes and fibroblasts of patients with ICF syndrome and of normal subjects with non-isotopic in situ hybridization, using the satellite II-related probe pHuR 195. We found evidence for interphase somatic pairing in ICF lymphocytes with a frequency higher than that found in normal cells. Lymphocytes of ICF patients showed nuclear protrusions and micronuclei and these nuclear abnormalities consistently involved a hybridization signal. Somatic pairing was also present in fibroblasts, but with frequencies similar in normal and ICF subjects. The fibroblasts do not have the major chromosomal abnormalities found in lymphocytes. The degree of heterochromatin condensation in fibroblasts was lower than that in lymphocytes and we postulate that the more decondensed state of chromocentres in the fibroblasts could be the reason for the absence of the major chromosomal abnormalities.

1992 - Molecular and prospective phenotypic characterization of a pedigree with familial Alzheimer's disease and a missense mutation in codon 717 of the beta-amyloid precursor protein gene [Articolo su rivista]
H., Karlinsky; G., Vaula; J. L., Haines; J., Ridgley; C., Bergeron; M., Mortilla; Tupler, Rossella; M. E., Percy; Y., Robitaille; N. E., Noldy; T. C. K., Yip; R. E., Tanzi; J. F., Gusella; R., Becker; J. M., Berg; D. R., Crapper McLachlan; George Hyslop, P. H. S. t.

We present prospective clinical and neuropathologic details of a pedigree segregating familial Alzheimer's disease (FAD) associated with a mutation (G->A substitution) at nucleotide 2149 in exon 17 of the amyloid precursor protein (APP) gene. This mutation, which is predicted to cause the missense substitution of isoleucine for valine at codon 717 of APP, cosegregated perfectly with the FAD trait (lod score = 3.49 at [Greek small letter theta with circumflex] = 0.00). The earliest clinical manifestations of the disease relate to deficits in memory function, cognitive processing speed, and attention to complex cognitive sets. These changes occurred in the absence of changes in nonmemory language and visuospatial functions. The neuropathologic features of FAD associated with the APP717 mutation in this family include severe neuronal loss, abundant neurofibrillary tangles, amyloid plaques, and amyloid angiopathy. These results provide independent confirmation that mutations in the APP gene are linked to the FAD trait in some families.

1992 - Molecular genetic evidence for etiologic heterogeneity of Alzheimer’s disease [Relazione in Atti di Convegno]
S. T. GEORGE HYSLOP P., H; MC LACHLAN C. D., R; HAINES J., L; BRUNI A., C; FONCIN J., F; LUKIV W. J., L; MONTESI M., P; Mortilla, M; Pinessi, L; POLINSKY L., J; Pollen, D; Rainero, I; Rogaev, E; Sorbi, S; Tanzi, R; Tupler, Rossella; Vaula, G.

n the last several years considerable evidence has accumulated which argues that Alzheimer’s disease (AD) is etiologically heterogeneous. In particular, molecular genetic studies of pedigrees with familial Alzheimer’s disease (FAD) have provided quite convincing evidence that different primary events (defects in different genes) are capable of causing the same general disease phenotype associated with AD (i.e., adult onset progressive dementia accompanied by the characteristic neuropathologic features of neurofibrillary degeneration and amyloid deposits, etc.). This review will examine some of the molecular genetic evidence for etiologic heterogeneity.

1992 - Paternal origin of the denovo deleted chromosome 4 in wolf-Hirschborn syndrome [Articolo su rivista]
Tupler, Rossella; L. B. o. r. t. o. t. t. o., .; E. M., Buhler; M., Alkan; N. J., Malik; N., Bosch Al Jadooa; L., Memo; P., Maraschio

The parental origin of the de novo deleted chromosome 4 was studied in five cases of Wolf-Hirschhorn syndrome using polymorphic probes mapping in the 4p16.3 region. In all the patients the deleted chromosome was found to be of paternal origin and these results, together with similar ones obtained by another group, make the preferential paternal origin of the de novo chromosome 4 deletion highly significant.

1990 - Deletion of specific sequences or modification of centromeric chromatin are responsible for Y chromosome centromere inactivation [Articolo su rivista]
P., Maraschio; O., Zuffardi; A., Caiulo; E., Dainotti; M., Piantanida; H., Rivera; Tupler, Rossella

Stable dicentric chromosomes behave as monocentrics because one of the centromeres is inactive. The cause of centromere inactivation is unknown; changes in centromere chromatin conformation and loss of centromeric DNA elements have been proposed as possible mechanisms. We studied the phenomenon of inactivation in two Y centromeres, having as a control genetically identical active Y centromeres. The two cases have the following karyotypes: 45, X/46,X,i(Y)(q12) and 46,XY/47,XY,+t(X;Y) (p22.3;p11.3). The analysis of the behavior of the active and inactive Y chromosome centromeres after Da-Dapi staining, CREST immunofluorescence, and in situ hybridization with centromeric probes leads us to conclude that, in the case of the isochromosome, a true deletion of centromeric chromatin is responsible for its stability, whereas in the second case, stability for its stability, whereas in the second case, stability of the dicentric (X;Y) is the result of centromere chromatin modification.

1990 - Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder [Articolo su rivista]
P. H., St George Hyslop; J. L., Haines; L. A., Farrer; R., Polinsky; C., Van Broeckhoven; A., Goate; D. R., Crapper McLachlan; H., Orr; A. C., Bruni; S., Sorbi††; I., Rainero; J. F., Foncin; D., Pollen; J. M., Cantu; Tupler, Rossella; N., Voskresenskaya; R., Mayeux; J., Growdon; V. A., Fried; R. H., Myers; L., Nee; H., Backhovens; J. J., Martin; M., Rossor; M. J., Owen; M., Mullan; M. E., Percy; H., Karlinsky; S., Rich; L., Heston; M., Montesi; M., Mortilla; N., Nacmias; J. F., Gusella; J. A., Hardy

ALZHEIMER'S disease, a fatal neurodegenerative disorder of unknown aetiology, is usually considered to be a single disorder because of tbe general uniformity of the disease phenotype. Two recent genetic linkage studies revealed co-segregation of familial Alzheimer disease with the D21S1/S11 and D21S16 loci on chromosome 21. But two other studies, one of pre-dominantly multiplex kindreds with a late age-of-onset, the other of a cadre of kindreds with a unique Volga German ethnic origin, found absence of linkage at least to D21S1/S11. So far it has not been possible to discern whether these conflicting reports reflect aetiological heterogeneity, differences in methods of pedigree selection, effects of confounding variables in the analysis (for example, diagnostic errors, assortative matings), or true non-replication. To resolve this issue, we have now examined the inheritance of five polymorphic DNA markers from the proximal long arm of chromosome 21 in a large unselected series of pedigrees with familial Alzheimer's disease. Our data suggest that Alzheimer's disease is not a single entity, but rather results from genetic defects on chromosome 21 and from other genetic or nongenetic factors.

1989 - Differential expression of the ICF (innunodefiency, centromeric heterochromatin, facial anomalies) mutation in lymphocytes and fibroblasts [Articolo su rivista]
P., Maraschio; Tupler, Rossella; E., Dainotti; M., Piantanida; G., Cazzola; L., Tiepolo

Fibroblasts from a patient with ICF syndrome were grown in the presence of excess of nucleotides, in media with different amounts of folic acid, and with caffeine in an attempt to induce the chromosomal anomalies observed in lymphocytes. We induced despiralisation and breakages in the centromeric heterochromatin of chromosomes 1 and 16 but not associations and multibranching. We suggest that the absence of the major chromosomal anomalies in fibroblasts from patients with ICF might be the result of both a longer G2 in these cells and differential patterns of interphase heterochromatin associations in the two tissues

1989 - Regional assignment of the loci for adenilate kinase to 9q32 and for a-acid glycoprotein to 9q31-q32. A locus for Goltz syndrome in region 9q32-qter? [Articolo su rivista]
O., Zuffardi; A., Caiulo; P., Maraschio; Tupler, Rossella; E., Bianchi; P., Amisano; G., Beluffi; G., Moratti; G., Liguri

Normal levels of adenylate kinase (AK-1) and of agr1-acid glycoprotein (ORM1) were found in a girl with a deletion 9q32-qter secondary to a maternal translocation (4q35; 9q32), thus excluding these loci from the deleted region. These results, and comparison with other informative data, map the locus for AK-1 to 9q32 and that for ORM1 to region 9q31-q32. The girl has several signs of the Goltz syndrome (focal dermal hypoplasia), which is listed in the McKusick catalog (no. 30560) as an X-linked dominant condition. Our finding indicates that the locus for Golz syndrome is autosomal and in region 9q32-qter or that there are two such conditions, one autosomal and one X-linked.