G.P.278

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Abstracts / Neuromuscular Disorders 24 (2014) 791–924

1 Hospital de Pediatria J.P. Garrahan, Buenos Aires, Argentina; 2 Hopital Bichat Claude Bernard, Paris, France; 3 Institut de Myologie, GHU Pitie-Salpetriere, Paris, France; 4 Hospital de Pediatria J.P. Garrahan, Institute for Neurological Research, Buenos Aires, Argentina

Mutations in the FKRP gene account for a broad spectrum of patients ranging from CMD to the much milder LGMD2I. We describe the first 3 children with FKRP muscular dystrophy from Argentina. In all of them muscular dystrophy with deficit of alpha-dystroglycan was observed on muscle biopsy. Case 1: An 18-month-old boy with a history of poor sucking at birth, respiratory difficulties, delayed milestones, microcephaly, proximal weakness, and calf hypertrophy. CPK: 5100 IU/ l. Normal brain MRI. Molecular studies: homozygous mutation c.946C>Gp.Pro318Ala. He was able to walk independently at 3 years of age. At 5 years old he was started on nocturnal non-invasive ventilation. Case 2: A 9-year-old girl with progressive weakness and hypotonia, generalized, mainly proximal and lower-limb weakness, a waddling, tiptoe gait, bilateral scapular winging, and difficulty getting up from the floor, running, and jumping. CPK: 15920 IU/l. Molecular studies: 3 heterozygous variants were identified: C.826C>A p.Leu276Ile, C.946C>G p.Pro316Ala, c.970G>C p.Glu324Gln. She is currently 10 years old and stable. Cardiological and respiratory studies are normal. Case 3: An 11-year-old boy with difficulty running since 5 years of age, calf pain, and fatigue. He had proximal muscle weakness, mainly in the lower limbs, marked calf hypertrophy, and a waddling gait. CPK: 8737 IU/l. Molecular studies: 3 heterozygous variants were identified: C.545A>G p.Tyr182Cys, C.946C>Gp.Pro316Ala, c.970G>C p.Glu324Gln. The mother showed the heterozygous variant C.545A>G p.Tyr182Cys. He is currently 13 years old, shows a waddling, tiptoe gait, difficulty climbing stairs, running, and jumping. There is no evidence of cardiomyopathy. Conclusion: Case 1 presented as a CMD while the other two mainly have a LGMD2I phenotype. The last two cases presented with two identical, not previously reported variants. http://dx.doi:10.1016/j.nmd.2014.06.351

G.P.276 Slowly progressive motor and respiratory dysfunction resulting from FKRP mutations: A natural history study C.D. Crockett, C.M. Stephan, S.R.H. Mockler, K.M. Laubscher, B.M. Zimmerman, K.D. Mathews University of Iowa, Iowa City, USA Mutations in FKRP, critical for normal glycosylation of alpha-dystroglycan, result in clinically heterogeneous muscular dystrophy. Purpose: Describe the clinical phenotype and progression in a cohort of patients with FKRP mutations who were followed with annual standardized assessments. Methods: IRB approval was obtained. Subjects are enrolled in an ongoing Dystroglycanopathy natural history study. The current report includes all patients with FKRP mutations. Data collection included MMT, myometry, timed function tests, mutation analysis and historical information. Forty-three participants, ages 1–51 years, have been followed for <9 years. Twenty-one are homozygous for the common c.C826A missense mutation, and none has known brain dysfunction or malformation. Disease progression was highly varied. Although there was a trend toward slower progression across outcome measures among c.C826A homozygotes, variability was not fully explained by genotype. Onset was significantly later for those homozygous for c.C826 compared to the rest of the population (Wilcoxon rank sum test p < 0.0001), median age at onset 11 (IQR: 10–25) years for homozygotes and 2 (1–3) years for all others. In this cohort, only 4 individuals are non-ambulatory (age at fulltime wheelchair use: 15, 18, 21 and 41 years) and an additional 5 have limited functional ambulation, as defined by 6 min walk distance

<250 m. Sitting FVC was <80% of predicted in 16 participants, and low FVC was weakly correlated with worse performance on timed function tests. Mutations in FKRP result in a range of phenotypes that are not fully explained by genotype, although those homozygous for the c.C826A mutation have later onset of symptoms. Progression of weakness is slow and most of our cohort is still ambulatory. Our data will be helpful in counseling individuals about progression, but the generally slow and variable progression creates challenges for design of clinical trials. http://dx.doi:10.1016/j.nmd.2014.06.352

G.P.277 LGMD2I: Is there a relationship between clinical phenotype, morphological alterations and level of alpha-dystroglycan glycosylation in patients with the same FKRP genotype? S. Lindal 1, E. Stensland 2, M. Rasmussen 3, C. Jonsrud 2, V. Brox 2, A. Maisoon 4, Ø. Nilssen 4 1 University Hospital of North Norway (UNN), Tromsoe, Norway; 2 University Hospital of North Norway, Tromsoe, Norway; 3 Oslo University Hospital, Oslo, Norway; 4 University of Tromsø, Tromsø, Norway To investigate if there is a correlation between clinical expression (duration of disease, age, walking function) and morphological alterations in affected muscle (structural changes, immunohistochemical and the level of alpha-DG glycosylation) among 25 patients with Limb Girdle Muscular Dystrophy type 2I (LGMD2I) all with the common C.826C>A mutation ). Muscle biopsies were obtained from 25 patients. Quantitative evaluation of morphological alterations in muscle sections, and immunohistochemistry (IHC) with antibodies directed against the alpha-dystroglycan glycan epitope, was performed by light microscopy. A semi-quantitative assessment of changes was recorded, point-graded and summarized as morphological sum-score for each biopsy.. Western blot (WB) analysis on muscle biopsy homogenates were carried with antibodies directed towards the core alpha-DG as well as the alpha-DG-glycan epitope. Laminin overlay was included. Muscle biopsies from 25 patients with LGMD2I presented large variation in morphological features. All biopsies presented reduced molecular weight of alpha-DG as detected with alpha-DG core antibody on WB analysis. Immunohistochemistry and WB analysis directed towards the alpha-DG-glycan epitope, as well as laminin overlay, demonstrated large variation in signal intensity among the LGMD2I patients. Results from IHC and WB/laminin overlay correlated poorly. There was no obvious correlation between ages at onset or duration of disease at biopsy versus the level of alpha-DG glycosylation. Likewise, there was no apparent association between severities of disease and the sum-scores of structural changes in the muscle biopsies. The clinical and morphological variability seen among LGMD2I patients with identical FKRP genotype must therefore be explained by other genetic or environmental mechanisms. http://dx.doi:10.1016/j.nmd.2014.06.353

G.P.278 Facial dysmorphism in FKRP limb-girdle muscular dystrophy: About two cases A. Magot 1, S. Mercier 2, C. Bouchet Seraphin 3, J.M. Mussini 1, Y. Peron 1 1 Centre de re´fe´rence neuromusculaire Nantes-Angers, Nantes, France; 2 Service de Ge´ne´tique Me´dicale, Nantes, France; 3 Service de Ge´ne´tique, Paris, France

Abstracts / Neuromuscular Disorders 24 (2014) 791–924 Mutations in the Fukutin-Related Protein gene (FKRP) account for a broad spectrum of phenotypes ranging from severe congenital muscular dystrophies to much milder limb-girdle muscular dystrophy (LGMD2I). In both, weakness and wasting of shoulder-girdle muscles, primary restrictive respiratory and cardiac involvement are classical features. To our knowledge, facial dysmorphic features have not been reported yet. The first woman developed, at age 12, a progressive pelvic weakness. Two biopsies were performed showing a dystrophic pattern with normal classical immunohistochemistry. At age 38, she was confined in a wheelchair and suffered from respiratory insufficiency. She also had a mild cardiomyopathy. As she presented macroglossia, analysis of the FKRP gene was performed and two compound mutations p.Leu276Ile and p.Glu343X were found. The second woman was born of a consanguineous union. From childhood, she had difficulties running and rising from the floor. At 12, she developed a limb-girdle weakness that confined her in a wheelchair at age 26. Vital capacity was normal, as well as US heart exam. Muscular biopsy showed classical dystrophic features with normal immunohistochemistry and Western blot. She had no macroglossia but her face looked alike our first FKRP patient. Indeed, both women presented hypertelorism, large palpebral fissures, depressed nasal bridge, prognathism and thick lips being responsible for a dramatic physical resemblance although they were unrelated. FKRP gene analysis was therefore performed and a homozygous mutation p.Leu276Ile was found. Limb-Girdle weakness associated with macroglossia and calf hypertrophy are classical clues for FKRP gene sequencing. Some mutations have more frequent macroglossia or calf hypertrophy than others. But facial dysmorphia has not been reported in this disease so far. This dysmorphia represent a real diagnosis help in our second patient and should be searched each time a LGMD patient is considered. http://dx.doi:10.1016/j.nmd.2014.06.354

G.P.279 Efficient AAV-mediated transfer of FKRP in a new mouse model of Limb Girdle Muscular Dystrophy 2I E. Gicquel, I. Richard Genethon CNRS, Evry, France Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha-dystroglycan (aDG), a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. The aDG glycosylation, a very complex process, requires many proteins whose functions are not fully elucidated. In particular, mutations in the FKRP gene encoding Fukutin related protein, lead to hypoglycosylation of aDG, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I). We generated a knock-in mouse model of LGMD2I, carrying the most frequent mutation (L276I) encountered in LGMD2I patients. Molecular characterization of this mouse model showed that the introduction of the mutation did not alter the expression of FKRP. However, the protein appears to have altered function since abnormal glycosylation of aDG and reduction of laminin binding was observed. Histologically, the muscles of this model show a dystrophic pattern starting from 6 months of age, consisting both in the presence of central nuclei and in fiber size variability. Interestingly, functional muscle impairment can be observed as early as 2 months of age by a decrease of the muscle resistance to eccentric mechanical stress. To evaluate gene transfer as a therapeutic approach, we cloned the FKRP cDNA in an AAV vector under the transcriptional control of the desmin promoter. The recombinant AAV2/9 vector was injected intramuscularly or intravenously in the mouse model. Expression of the FKRP transgene was obtained, both at RNA and protein levels. The glycosylation of aDG was restored as well as laminin binding. A histological rescue was observed by the decrease

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of fibers with centrally located nuclei. The AAV vector also improved the muscle function, since it conferred a better resistance to eccentric stress to the injected muscles. http://dx.doi:10.1016/j.nmd.2014.06.355

G.P.280 Preserved expression of truncated telethonin in a patient with LGMD2G V. Straub 1, N.P. Davies 2, R. Barresi 3, C. Morris 3, C. Pickthall 4, K. Bushby 5 1 Newcastle University, NE2 4AZ, UK; 2 Queen Elizabeth Hospital, Birmingham, UK; 3 Newcastle NHS Hospital, NE2 4AZ, UK; 4 Newcastle NHS Hospital, Newcastle upon Tyne, UK; 5 Newcastle University, Newcastle upon Tyne, UK Limb-girdle muscular dystrophy type 2G (LGMD2G) is a disorder caused by mutations in the TCAP gene that encodes for the striated muscle specific protein telethonin. LGMD2G is extremely rare and was initially identified in the Brazilian population. Recently, a small number of LGMD2G patients have been diagnosed in populations with diverse genetic backgrounds indicating a wider geographical distribution of this disorder. Here we describe a 49 year old male patient presenting a classical LGMD phenotype. He was born from non-consanguineous healthy parents of Indian descent. He had normal motor milestones but became noticeable slower in his early teens and presented scapular winging and Achilles tendon contractures. He eventually became wheelchair bound by age 47. There has been no cardiac involvement so far and the respiratory function is only mildly reduced with nocturnal hypoventilation. CK levels were 2541 iU/L. A muscle biopsy showed well preserved fascicular architecture with mild myopathic features. Immunohistochemistry with an antibody directed to the C-terminal portion of telethonin showed complete absence of labelling. However, an antibody directed against full-length telethonin showed some labelling on sections and a single band of 10 kDa on Western blot. Sequence analysis of the TCAP gene revealed a novel homozygous nonsense c.244C>T (p.Gln82X) mutation in exon 2, predicted to generate a truncated protein of molecular mass consistent with the Western blot findings. Double labelling for telethonin and filamin C showed overlap in a cross-striated pattern, consistent with the Z-disc localization of both proteins, indicating that the mutant telethonin is retained at the Zdisc. Our results suggest that the mutant protein is correctly incorporated in the sarcomere allowing sufficient binding to titin such that functional rather than structural defects may be the mechanism that leads to LGMD2G. http://dx.doi:10.1016/j.nmd.2014.06.356

G.P.281 Detection of homozygous and compound heterozygous deletions in TRIM32 in LGMD patients analyzed by a combined strategy of CGH-array and Massive Parallel Sequencing J. Nectoux 1, R. de Cid 2, S. Baulande 3, F. Leturcq 1, J.A. Urtizberea 4, I. Penisson-Besnier 5, A. Nadaj Pakleza 5, C. Roudaut 6, A. Criqui 3, L. Orhant 1, D. Peyroulan 7, R. Ben Yaou 8, I. Nelson 8, M.C. Arne´-Bes 9, P. Nitschke 10, M. Claustres 7, G. Bonne 8, N. Le´vy 11, J. Chelly 1, I. Richard 6, M. Cosse´e 7 1 Assistance Publique – Hoˆpitaux de Paris, Hoˆpital Cochin, Paris, France; 2 Genethon, UMR8587, Evry, France; 3 PartnerChip, Evry, France; 4 Centre de re´fe´rence neuromusculaire GNMH, Hendaye, Angers, Angers, France; 6 Genethon, Evry, France; 5 CHU 7 France; CHRU Montpellier, Montpellier, France; 8 GH Pitie´-Salpeˆtrie`re - Institut de Myologie, Paris, France; 9 CHU Toulouse, Toulouse, France; 10 Universite´ Paris Descartes, Paris, France; 11 Faculte´