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Calpain-3 deficiency: a mild muscular dystrophy in childhood: molecular and clinical findings
442
Abstracts
N. Terki ~, A. Masmoudi b, M. Chaouch¢, M. Tazir d, M. Ait-Kacid, D. Grid e
~Service d'Anatomie Pathologique, Hbpital Beni-Messous, Alger, Algeria, t'Service de Neurologie, Chu Bab-El-Oued, Alger, Algeria, "Sevice de Neurologie, Chu Ben-Aknoun, Alger, Algeria, dService de Neurologie, Htpital Ait-ldir, Alger, Algeria, ~Departement des affaires medicales, AFM, Evry, France
Eighty cases from 80 families with muscular dystrophies underwent clinical, biological, and electrophysiological evaluation. Cardiac and respiratory function were tested, and biologic and electromyographic examinations were carried out. Surgical biopsy was performed systematically. Histoenzymological and histochemical techniques were also used. Four antibodies (anti 50, 46, 35 dystrophin-associated glycoprotein antibodies, and anti-merosin) were used for immunochemistry. From this study, the prevalence of the various types of muscular dystrophies (excluding CMD) could be calculated. There were 32 cases of dystrophinopathies (27 DMD and 3 BMD), 30 cases of adhalinopathies (with different types of DAG deficits), and 18 cases of LGMDs with normal dystrophin and adhalin. Screening for mutations in the calpain gene has not yet been performed. The adhalinopathy cases are discussed; some correlations between morphological and clinical data have been established. These preliminary results make it possible to establish an accurate diagnosis and to envisage the possibility of prenatal diagnosis.
GP1B.14 Limb girdle muscular dystrophy with calpain deficiency in Guipuzcoa (Basque country, Spain) J.A. Urtizberea a, M. Urtasunc, I. Richard b, A. Saenz c, J.J. Poza c, A.M. Cobo c, A. Lopez de Munainc, J.S. Beckmannb
aAFM: 1, Rue de l'lnternationale, 91000 Evry, France, bGenethon: 1, Rue de l'lnternationale, 91000 Evry, France, CHospital N/S Aranzazu, 20080, San Sebastian, Basque country, Spain A survey of limb girdle muscular dystrophy has been carried out in 1995-1996 in the Basque province of Guipuzcoa (702@000 inhabitants). Patients were ascertained through medical records kept by local hospitals and through information held by patients support groups. The diagnostic criteria were those agreed at the ENMC workshop held in Naarden in 1995. All patients were re-examined by two neurologists and underwent electrophysiological and immunohistochemical studies whenever necessary. Serum CK, bidimensional echocardiography and respiratory evaluation were also performed in order to investigate more deeply phenotypic variations. Genetic studies were carried out at Genethon and were designed to demonstrate a possible linkage to chromosome 15 (LGMD2A locus) or to test known mutations. A primary screening enabled to select 62 patients out of which 8 turned out to have an alternative diagnosis (2 with Becker muscular dystrophy, 3 with SMA, 1 with myotonic dystrophy, 1 with ~-sarcoglycanopathy, 1 with an undetermined myopathy), and 4 were not available for the study. For the remaining 51 patients (29 men, 22 women, mean age 38.9 years + 14.24), the clinical presentation was consistent with previous descriptions of LGMD in patients reported by M. Fardeau in the Reunion. A molecular screening for CANP3 revealed that 29 families corresponding to 38 patients were harbouring a mutation in the calpain gene while the status of the 13 other patients is pending. Interestingly, 18 families were homozygous for a mutation in exon 22 whereas 8 families were heterozygous for exon 22 and 1 family was homozygous for exon 21. The prevalence rate of LGMD 2A found in this Basque population (68/100000) is one the highest so far reported world-wide. This cluster of patients, mostly of Basque origin, is presumably due to socioeconomical (inbreeding, no admixture with neighbouring Spanish people) and geographical (landlocked mountainous valleys) factors. The existence of one main mutation in exon 22 suggest a founder effect. Correlations between genotype and phenotype are still under way and demonstrates some similarities in the exon 22 mutation carriers and some discrepancies for the others.
GP1B.15 Calpain-3 deficiency: a mild muscular dystrophy in childhood: molecular and clinical findings P. Dinqer a, I. Richard b, M.Z. Ak~tren ~, J.A. Urtizberea c, J.S. Beckmannb, H. Topaloglu ~
aHacettepe University, Ankara, Turkey, bGenethon, Evry, France; %4FM, Evry, France Clinical and genetic presentations of limb-girdle muscular dystrophies are very heterogeneous. LGMD2A was the first to be defined by its linkage to chromosome 15q. Among our 20 families with LGMD2, 10 were documented to have muscle specific calpain-3 deficiency (LGMD2A). The age of onset was around 10 years of age. The current ages of the index cases were between 12 and 23 years. Clinically, the patients showed mild courses; none of the cases have lost autonomy so far. The dystrophy was mainly proximal and atrophic with calf enlargement and scapular wasting in some. In three cases walking was delayed. Creatine kinase levels were at least 10 times elevated. Cardiopathy was not a problem. All mutations in these families were eventually identified. Five families shared the same 55 ldelA frameshift mutation. Four of these five families had the same core-haplotype for the highly polymorphic markers for chromosome 15q15.l-q15.3. The family that demonstrates a totally different haplotype showed a different clinical phenotype, milder than the previous four. Other mutations were four missense (Y336N, R490Q, A702V and R748Q), one frameshift (19-23delGCATC) and one nonsense (Y537X). Calpain-3 deficiency constitutes 50% of our cohort with the LGMD2. Our results suggest that calpain-3 deficiency in general is a mild muscular dystrophy in childhood. Calpain-3 deficiency should be considered as the initial diagnosis in cases with preserved sarcoglycans.
Keywords: Calpain-3 deficiency; Molecular findings; Clinical findings Acknowledgement - This study has been supported by the Association Franqaise contre les Myopathies (AFM).
GP1B.16 Immunofluorescence and Western blot analysis on human skeletal muscle using antibodies directed against different regions of the muscle-specific calpain Norma Beatriz Romero a'd, Luc Camoin b, C. Federici b, I. Richard c, F. Fougeroussec, J.L. Guillaume b, M. Hattab b, Marie-Armelle Cheval d, F.M.S. Tom6 a, Y. Eshdat b, M. Fardeau ~, J. Beckmannc, A.D. Strosberg b
alNSERM U. 153, blCGM- CNRS UPR 415, CGenethon, dH. Robert Debrd, Paris, France Limb-girdle muscular dystrophies (LGMD) are a group of inherited neuromuscular diseases characterised by progressive weakness of the pelvic and shoulder girdle muscles. One of the best characterised LGMD (LGMD2A) is due to mutation of gene of the calpain 3 (CAPN3), a calcium activated neutral protease 3. The calpain 3 has four main protein domains with three muscle calpain-specific regions: NS in protein domain I, IS1 in domain II and IS2 in domain IIl. Furthermore, it was demonstrated that calpain 3 binds to titin through the IS2 region but the exact significance of this interaction is not yet understood. Polyclonal antibodies were raised in rabbits against synthetic peptides corresponding to the three different muscle calpain-specific regions. These antibodies were used for immunofluorescence and Western blot analysis. Only antibodies that recognise the IS2 region of muscle-specific calpain 3 showed immunoreactivity. In normal muscle, immunocytochemical studies showed a fine granular pattern in the cytoplasm which was more intense in type II than type I muscle fibres. A distinct band of 94 kDa was seen by immunoblotting. These preliminary studies are now continued in order to characterise the specificity of antibodies against the IS2 region of muscle-specific calpain 3. The peptide used for their production has a homology on six contiguous amino-acids with human 6-phosphofructokinase. This homology may suggest that part of the immunoreactivity could be due to the presence of this enzyme.
Abstracts
N. Terki ~, A. Masmoudi b, M. Chaouch¢, M. Tazir d, M. Ait-Kacid, D. Grid e
~Service d'Anatomie Pathologique, Hbpital Beni-Messous, Alger, Algeria, t'Service de Neurologie, Chu Bab-El-Oued, Alger, Algeria, "Sevice de Neurologie, Chu Ben-Aknoun, Alger, Algeria, dService de Neurologie, Htpital Ait-ldir, Alger, Algeria, ~Departement des affaires medicales, AFM, Evry, France
Eighty cases from 80 families with muscular dystrophies underwent clinical, biological, and electrophysiological evaluation. Cardiac and respiratory function were tested, and biologic and electromyographic examinations were carried out. Surgical biopsy was performed systematically. Histoenzymological and histochemical techniques were also used. Four antibodies (anti 50, 46, 35 dystrophin-associated glycoprotein antibodies, and anti-merosin) were used for immunochemistry. From this study, the prevalence of the various types of muscular dystrophies (excluding CMD) could be calculated. There were 32 cases of dystrophinopathies (27 DMD and 3 BMD), 30 cases of adhalinopathies (with different types of DAG deficits), and 18 cases of LGMDs with normal dystrophin and adhalin. Screening for mutations in the calpain gene has not yet been performed. The adhalinopathy cases are discussed; some correlations between morphological and clinical data have been established. These preliminary results make it possible to establish an accurate diagnosis and to envisage the possibility of prenatal diagnosis.
GP1B.14 Limb girdle muscular dystrophy with calpain deficiency in Guipuzcoa (Basque country, Spain) J.A. Urtizberea a, M. Urtasunc, I. Richard b, A. Saenz c, J.J. Poza c, A.M. Cobo c, A. Lopez de Munainc, J.S. Beckmannb
aAFM: 1, Rue de l'lnternationale, 91000 Evry, France, bGenethon: 1, Rue de l'lnternationale, 91000 Evry, France, CHospital N/S Aranzazu, 20080, San Sebastian, Basque country, Spain A survey of limb girdle muscular dystrophy has been carried out in 1995-1996 in the Basque province of Guipuzcoa (702@000 inhabitants). Patients were ascertained through medical records kept by local hospitals and through information held by patients support groups. The diagnostic criteria were those agreed at the ENMC workshop held in Naarden in 1995. All patients were re-examined by two neurologists and underwent electrophysiological and immunohistochemical studies whenever necessary. Serum CK, bidimensional echocardiography and respiratory evaluation were also performed in order to investigate more deeply phenotypic variations. Genetic studies were carried out at Genethon and were designed to demonstrate a possible linkage to chromosome 15 (LGMD2A locus) or to test known mutations. A primary screening enabled to select 62 patients out of which 8 turned out to have an alternative diagnosis (2 with Becker muscular dystrophy, 3 with SMA, 1 with myotonic dystrophy, 1 with ~-sarcoglycanopathy, 1 with an undetermined myopathy), and 4 were not available for the study. For the remaining 51 patients (29 men, 22 women, mean age 38.9 years + 14.24), the clinical presentation was consistent with previous descriptions of LGMD in patients reported by M. Fardeau in the Reunion. A molecular screening for CANP3 revealed that 29 families corresponding to 38 patients were harbouring a mutation in the calpain gene while the status of the 13 other patients is pending. Interestingly, 18 families were homozygous for a mutation in exon 22 whereas 8 families were heterozygous for exon 22 and 1 family was homozygous for exon 21. The prevalence rate of LGMD 2A found in this Basque population (68/100000) is one the highest so far reported world-wide. This cluster of patients, mostly of Basque origin, is presumably due to socioeconomical (inbreeding, no admixture with neighbouring Spanish people) and geographical (landlocked mountainous valleys) factors. The existence of one main mutation in exon 22 suggest a founder effect. Correlations between genotype and phenotype are still under way and demonstrates some similarities in the exon 22 mutation carriers and some discrepancies for the others.
GP1B.15 Calpain-3 deficiency: a mild muscular dystrophy in childhood: molecular and clinical findings P. Dinqer a, I. Richard b, M.Z. Ak~tren ~, J.A. Urtizberea c, J.S. Beckmannb, H. Topaloglu ~
aHacettepe University, Ankara, Turkey, bGenethon, Evry, France; %4FM, Evry, France Clinical and genetic presentations of limb-girdle muscular dystrophies are very heterogeneous. LGMD2A was the first to be defined by its linkage to chromosome 15q. Among our 20 families with LGMD2, 10 were documented to have muscle specific calpain-3 deficiency (LGMD2A). The age of onset was around 10 years of age. The current ages of the index cases were between 12 and 23 years. Clinically, the patients showed mild courses; none of the cases have lost autonomy so far. The dystrophy was mainly proximal and atrophic with calf enlargement and scapular wasting in some. In three cases walking was delayed. Creatine kinase levels were at least 10 times elevated. Cardiopathy was not a problem. All mutations in these families were eventually identified. Five families shared the same 55 ldelA frameshift mutation. Four of these five families had the same core-haplotype for the highly polymorphic markers for chromosome 15q15.l-q15.3. The family that demonstrates a totally different haplotype showed a different clinical phenotype, milder than the previous four. Other mutations were four missense (Y336N, R490Q, A702V and R748Q), one frameshift (19-23delGCATC) and one nonsense (Y537X). Calpain-3 deficiency constitutes 50% of our cohort with the LGMD2. Our results suggest that calpain-3 deficiency in general is a mild muscular dystrophy in childhood. Calpain-3 deficiency should be considered as the initial diagnosis in cases with preserved sarcoglycans.
Keywords: Calpain-3 deficiency; Molecular findings; Clinical findings Acknowledgement - This study has been supported by the Association Franqaise contre les Myopathies (AFM).
GP1B.16 Immunofluorescence and Western blot analysis on human skeletal muscle using antibodies directed against different regions of the muscle-specific calpain Norma Beatriz Romero a'd, Luc Camoin b, C. Federici b, I. Richard c, F. Fougeroussec, J.L. Guillaume b, M. Hattab b, Marie-Armelle Cheval d, F.M.S. Tom6 a, Y. Eshdat b, M. Fardeau ~, J. Beckmannc, A.D. Strosberg b
alNSERM U. 153, blCGM- CNRS UPR 415, CGenethon, dH. Robert Debrd, Paris, France Limb-girdle muscular dystrophies (LGMD) are a group of inherited neuromuscular diseases characterised by progressive weakness of the pelvic and shoulder girdle muscles. One of the best characterised LGMD (LGMD2A) is due to mutation of gene of the calpain 3 (CAPN3), a calcium activated neutral protease 3. The calpain 3 has four main protein domains with three muscle calpain-specific regions: NS in protein domain I, IS1 in domain II and IS2 in domain IIl. Furthermore, it was demonstrated that calpain 3 binds to titin through the IS2 region but the exact significance of this interaction is not yet understood. Polyclonal antibodies were raised in rabbits against synthetic peptides corresponding to the three different muscle calpain-specific regions. These antibodies were used for immunofluorescence and Western blot analysis. Only antibodies that recognise the IS2 region of muscle-specific calpain 3 showed immunoreactivity. In normal muscle, immunocytochemical studies showed a fine granular pattern in the cytoplasm which was more intense in type II than type I muscle fibres. A distinct band of 94 kDa was seen by immunoblotting. These preliminary studies are now continued in order to characterise the specificity of antibodies against the IS2 region of muscle-specific calpain 3. The peptide used for their production has a homology on six contiguous amino-acids with human 6-phosphofructokinase. This homology may suggest that part of the immunoreactivity could be due to the presence of this enzyme.