Mingrui Dong1, Satoru Noguchi2, Yukari Endo1, Yukiko K Hayashi1, Shinobu Yoshida1, Ikuya Nonaka1, Ichizo Nishino1. 1. From the Department of Neuromuscular Research, National Institute of Neuroscience (M.D., S.N., Y.E., Y.K.H., I. Nonaka, I. Nishino) and Department of Clinical Development, Translational Medical Center (S.N., Y.E., Y.K.H., I. Nishino), NCNP, Tokyo, Japan; Department of Neurology (M.D.), China-Japan Friendship Hospital, Beijing, China; Department of Neurophysiology (Y.K.H.), Tokyo Medical University; and Department of Pediatrics (S.Y.), Omihachiman Community Medical Center, Shiga, Japan. 2. From the Department of Neuromuscular Research, National Institute of Neuroscience (M.D., S.N., Y.E., Y.K.H., I. Nonaka, I. Nishino) and Department of Clinical Development, Translational Medical Center (S.N., Y.E., Y.K.H., I. Nishino), NCNP, Tokyo, Japan; Department of Neurology (M.D.), China-Japan Friendship Hospital, Beijing, China; Department of Neurophysiology (Y.K.H.), Tokyo Medical University; and Department of Pediatrics (S.Y.), Omihachiman Community Medical Center, Shiga, Japan. noguchi@ncnp.go.jp.
Abstract
OBJECTIVES: To identify gene mutations in patients with dystroglycanopathy and prove pathogenicity of those mutations using an in vitro cell assay. METHODS: We performed whole-exome sequencing on 20 patients, who were previously diagnosed with dystroglycanopathy by immunohistochemistry and/or Western blot analysis. We also evaluated pathogenicity of identified mutations for phenotypic recovery in a DAG1-knockout haploid human cell line transfected with mutated DAG1 complementary DNA. RESULTS: Using exome sequencing, we identified compound heterozygous missense mutations in DAG1 in a patient with asymptomatic hyperCKemia and pathologically mild muscular dystrophy. Both mutations were in the N-terminal region of α-dystroglycan and affected its glycosylation. Mutated DAG1 complementary DNAs failed to rescue the phenotype in DAG1-knockout cells, suggesting that these are pathogenic mutations. CONCLUSION: Novel mutations in DAG1 are associated with asymptomatic hyperCKemia with hypoglycosylation of α-dystroglycan. The combination of exome sequencing and a phenotype-rescue experiment on a gene-knockout haploid cell line represents a powerful tool for evaluation of these pathogenic mutations.
OBJECTIVES: To identify gene mutations in patients with dystroglycanopathy and prove pathogenicity of those mutations using an in vitro cell assay. METHODS: We performed whole-exome sequencing on 20 patients, who were previously diagnosed with dystroglycanopathy by immunohistochemistry and/or Western blot analysis. We also evaluated pathogenicity of identified mutations for phenotypic recovery in a DAG1-knockout haploid human cell line transfected with mutated DAG1 complementary DNA. RESULTS: Using exome sequencing, we identified compound heterozygous missense mutations in DAG1 in a patient with asymptomatic hyperCKemia and pathologically mild muscular dystrophy. Both mutations were in the N-terminal region of α-dystroglycan and affected its glycosylation. Mutated DAG1 complementary DNAs failed to rescue the phenotype in DAG1-knockout cells, suggesting that these are pathogenic mutations. CONCLUSION: Novel mutations in DAG1 are associated with asymptomatic hyperCKemia with hypoglycosylation of α-dystroglycan. The combination of exome sequencing and a phenotype-rescue experiment on a gene-knockout haploid cell line represents a powerful tool for evaluation of these pathogenic mutations.
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