Kohei Hamanaka1, Satoko Miyatake1,2, Eriko Koshimizu1, Yoshinori Tsurusaki3, Satomi Mitsuhashi1, Kazuhiro Iwama1, Ahmed N Alkanaq1, Atsushi Fujita1, Eri Imagawa1, Yuri Uchiyama1, Nozomu Tawara4, Yukio Ando4, Yohei Misumi4, Mariko Okubo5, Mitsuko Nakashima6, Takeshi Mizuguchi1, Atsushi Takata1, Noriko Miyake1, Hirotomo Saitsu6, Aritoshi Iida7, Ichizo Nishino5,7,8, Naomichi Matsumoto9. 1. Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan. 2. Clinical Genetics Department, Yokohama City University Hospital, Yokohama, Kanagawa, Japan. 3. Clinical Research Institute, Kanagawa Children's Medical Center, Yokohama, Kanagawa, Japan. 4. Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Kumamoto, Japan. 5. Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. 6. Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. 7. Department of Clinical Genome Analysis, Medical Genome Center, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. 8. Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. 9. Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan. naomat@yokohama-cu.ac.jp.
Abstract
PURPOSE: The diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20-40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants. METHODS: Aberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES. RESULTS: We identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases. CONCLUSION: These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.
PURPOSE: The diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20-40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants. METHODS: Aberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES. RESULTS: We identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases. CONCLUSION: These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.
Entities:
Keywords:
NEB; RNA sequencing; deep intron; exome sequencing; nemaline myopathy
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