PMID- 36579833
OWN - NLM
STAT- MEDLINE
DCOM- 20230426
LR  - 20230426
IS  - 1460-2083 (Electronic)
IS  - 0964-6906 (Print)
IS  - 0964-6906 (Linking)
VI  - 32
IP  - 9
DP  - 2023 Apr 20
TI  - A mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured 
      cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived 
      iPS cells.
PG  - 1511-1523
LID - 10.1093/hmg/ddac306 [doi]
AB  - At the neuromuscular junction, the downstream of tyrosine kinase 7 (DOK7) 
      enhances the phosphorylation of muscle-specific kinase (MuSK) and induces 
      clustering of acetylcholine receptors (AChRs). We identified a patient with 
      congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, 
      c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology 
      domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C 
      caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a 
      cryptic splice site deleting seven nucleotides in exon 6. p.G64R reduced the 
      expression of DOK7 to 10% of wild-type DOK7, and markedly compromised AChR 
      clustering in transfected C2C12 myotubes. p.G64R-DOK7 made insoluble aggresomes 
      at the juxtanuclear region in transfected C2C12 myoblasts and COS7 cells, which 
      were co-localized with molecules in the autophagosome system. A protease 
      inhibitor MG132 reduced the soluble fraction of p.G64R-DOK7 and enhanced the 
      aggresome formation of p.G64R-DOK7. To match the differentiation levels between 
      patient-derived and control induced pluripotent stem cells (iPSCs), we corrected 
      c.190G>A (p.G64R) by CRISPR/Cas9 to make isogenic iPSCs while retaining 
      c.653-1G>C (CMS-iPSCsCas9). Myogenically differentiated CMS-iPSCs showed 
      juxtanuclear aggregates of DOK7, reduced expression of endogenous DOK7 and 
      reduced phosphorylation of endogenous MuSK. Another mutation, p.T77M, also made 
      aggresome to a less extent compared with p.G64R in transfected COS7 cells. These 
      results suggest that p.G64R-DOK7 makes aggresomes in cultured cells and is likely 
      to compromise MuSK phosphorylation for AChR clustering.
CI  - (c) The Author(s) 2022. Published by Oxford University Press.
FAU - Zhang, Shaochuan
AU  - Zhang S
AUID- ORCID: 0000-0002-4853-4525
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Ohkawara, Bisei
AU  - Ohkawara B
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Ito, Mikako
AU  - Ito M
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Huang, Zhizhou
AU  - Huang Z
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Zhao, Fei
AU  - Zhao F
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Nakata, Tomohiko
AU  - Nakata T
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Takeuchi, Tomoya
AU  - Takeuchi T
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
FAU - Sakurai, Hidetoshi
AU  - Sakurai H
AD  - Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 
      606-8507, Japan.
FAU - Komaki, Hirofumi
AU  - Komaki H
AD  - Department of Pediatrics, National Institute of Neuroscience, National Center of 
      Neurology and Psychiatry, Kodaira, Tokyo 187-8551, Japan.
FAU - Kamon, Masayoshi
AU  - Kamon M
AD  - Department of Peripheral Nervous System Research, National Institute of 
      Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 
      187-8551, Japan.
FAU - Araki, Toshiyuki
AU  - Araki T
AUID- ORCID: 0000-0003-3625-2042
AD  - Department of Peripheral Nervous System Research, National Institute of 
      Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 
      187-8551, Japan.
FAU - Ohno, Kinji
AU  - Ohno K
AUID- ORCID: 0000-0002-1529-2750
AD  - Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya 
      University Graduate School of Medicine, Nagoya 466-8550, Japan.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Hum Mol Genet
JT  - Human molecular genetics
JID - 9208958
RN  - 0 (DOK7 protein, human)
RN  - 0 (Muscle Proteins)
RN  - EC 2.7.10.1 (MUSK protein, human)
RN  - 0 (Receptors, Cholinergic)
SB  - IM
MH  - Humans
MH  - Cells, Cultured
MH  - *Induced Pluripotent Stem Cells/metabolism
MH  - Muscle Proteins/genetics
MH  - Mutation
MH  - *Myasthenic Syndromes, Congenital/genetics/metabolism
MH  - Phosphorylation
MH  - Receptors, Cholinergic/genetics/metabolism
PMC - PMC10117378
EDAT- 2022/12/30 06:00
MHDA- 2023/04/24 06:41
PMCR- 2022/12/29
CRDT- 2022/12/29 08:02
PHST- 2022/07/22 00:00 [received]
PHST- 2022/12/12 00:00 [revised]
PHST- 2022/12/21 00:00 [accepted]
PHST- 2023/04/24 06:41 [medline]
PHST- 2022/12/30 06:00 [pubmed]
PHST- 2022/12/29 08:02 [entrez]
PHST- 2022/12/29 00:00 [pmc-release]
AID - 6964998 [pii]
AID - ddac306 [pii]
AID - 10.1093/hmg/ddac306 [doi]
PST - ppublish
SO  - Hum Mol Genet. 2023 Apr 20;32(9):1511-1523. doi: 10.1093/hmg/ddac306.