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.


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.


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.


These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.

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Author contributions:

K.H.: literature review, data collection, and drafting the manuscript; E.K., Y.T., S.Mit., K.I., A.N.A., A.F., E.I., Y.U., N.T., Y.A., Y.M., M.O., M.N., T.M., N.Mi., H.S., and A.I.: data collection and manuscript revision; S.Miy., A.T., I.N., and N.M.: supervision of all aspects, including study design, data interpretation, and manuscript preparation.

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  1. Department of Human Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan

    • Kohei Hamanaka MD, PhD
    • , Satoko Miyatake MD, PhD
    • , Eriko Koshimizu PhD
    • , Satomi Mitsuhashi MD, PhD
    • , Kazuhiro Iwama MD
    • , Ahmed N. Alkanaq MD
    • , Atsushi Fujita PhD
    • , Eri Imagawa PhD
    • , Yuri Uchiyama MD, PhD
    • , Takeshi Mizuguchi MD, PhD
    • , Atsushi Takata MD, PhD
    • , Noriko Miyake MD, PhD
    •  & Naomichi Matsumoto MD, PhD
  2. Clinical Genetics Department, Yokohama City University Hospital, Yokohama, Kanagawa, Japan

    • Satoko Miyatake MD, PhD
  3. Clinical Research Institute, Kanagawa Children’s Medical Center, Yokohama, Kanagawa, Japan

    • Yoshinori Tsurusaki PhD
  4. Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Kumamoto, Japan

    • Nozomu Tawara MD
    • , Yukio Ando MD, PhD
    •  & Yohei Misumi MD, PhD
  5. Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

    • Mariko Okubo MD
    •  & Ichizo Nishino MD, PhD
  6. Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan

    • Mitsuko Nakashima MD, PhD
    •  & Hirotomo Saitsu MD, PhD
  7. Department of Clinical Genome Analysis, Medical Genome Center, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

    • Aritoshi Iida PhD
    •  & Ichizo Nishino MD, PhD
  8. Department of Genome Medicine Development, Medical Genome Center, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

    • Ichizo Nishino MD, PhD


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