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. 2017 Feb;25(2):192-199.
doi: 10.1038/ejhg.2016.162. Epub 2016 Dec 14.

Limb Girdle Myasthenia With Digenic RAPSN and a Novel Disease Gene AK9 Mutations

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Free PMC article

Limb Girdle Myasthenia With Digenic RAPSN and a Novel Disease Gene AK9 Mutations

Ching-Wan Lam et al. Eur J Hum Genet. .
Free PMC article

Abstract

Though dysfunction of neuromuscular junction (NMJ) is associated with congenital myasthenic syndrome (CMS), the proteins involved in neuromuscular transmission have not been completely identified. In this study, we aimed to identify a novel CMS gene in a consanguineous family with limb-girdle type CMS. Homozygosity mapping of the novel CMS gene was performed using high-density single-nucleotide polymorphism microarrays. The variants in CMS gene were identified by whole-exome sequencing (WES) and Sanger sequencing. A 20 MB-region of homozygosity (ROH) was mapped on chromosome 6q15-21. This was the only ROH that present in all clinically affected siblings and absent in all clinically unaffected siblings. WES showed a novel variant of AK9 gene located in this ROH. This variant was a start-gain mutation and introduced a cryptic 5'-UTR signal in intron 5 of the AK9 gene. The normal splicing signal would be interfered by the cryptic translation signal leading to defective splicing. Another 25 MB-ROH was found on chromosome 11p13-q12 in all siblings. WES showed a homozygous RAPSN pathogenic variant in this ROH. Since RAPSN-associated limb-girdle type CMS was only manifested in AK9 homozygous variant carriers, the disease phenotype was of digenic inheritance, and was determined by the novel disease modifier AK9 which provides NTPs for N-glycosylation. This is the first time that this specific genotype-phenotype correlation is reported. Importantly, the AK9-associated nucleotide deficiency may replete by dietary supplements. Since AK9 is a disease modifier, enhancing N-glycosylation by increasing dietary nucleotides may be a new therapeutic option for CMS patients.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Affymetrix SNP 6.0 microarray analysis. The samples of the father (F), mother (M), clinically affected sibling 1 (P1), clinically affected sibling 2 (P2), clinically unaffected sibling 1 (S1), clinically unaffected sibling 2 (S2) were analyzed. Upper panel: The only ROH (region of homozygosity) shared by P1 and P2, colored in green and brown, respectively, was located on chromosome 6q15–21. This is a new CMS locus since no known CMS genes are located in this region: ALG2 on chr. 9q, ALG14 on chr. 1p, AGRN on chr. 1p, CHAT on chr. 10q, CHRNA1 on chr. 2q, CHRNB1 on chr. 17p, CHRND on chr. 2q, CHRNE on chr. 17p, COLQ on chr. 3p, DOK7 on chr. 4p, DPAGT1 on chr. 11q, GFPT1 on chr. 2p, LAMB2 on chr. 3p, LRP4 on chr. 11p, MUSK on chr. 9q, PREPL on chr. 2p, RAPSN on chr. 11p, SCN4A on chr. 17q, SNAP25 on chr. 20p, SYT2 on chr. 1q and GMPPB on chr. 3p. Lower panel: Another ROH region located on chromosome 11p13–q12 was shared among P1, P2, S1 and S2, colored in green, brown, blue, and violet, respectively. Among all known CMS genes, RAPSN gene is located in this region.
Figure 2
Figure 2
Results of ROH on chromosome 6 shared by clinically affected siblings P1 and P2 and ROH on chromosome 11 shared by all siblings (P1, P2, S1, S2). (a) The number of filtered variants remained after each filtering step. (b) WES mutant depth of the filtered SNVs in the family. Sanger sequencing confirmed the WES genotypes of the AK9 (NM_001145128.2) and RAPSN (NM_005055.4, NG_ 008312.1) genes of the family members. The start-gain mutation ATG is underlined in P1 and P2. The parents were doubly heterozygous for AK9 and RAPSN variants. Chr, Chromosome; hom, homozygous; het, heterozygous.
Figure 3
Figure 3
The NM_001145128.2(AK9):c.332-14A>G creates a new start codon ATG in intron 5 (shown as ata>G). The promoter sequence was underlined and shown in black and the cryptic transcription start site is shown in red. The uORFs were underlined and shown in red. The cryptic 5′-UTR is of 327 bp in size. This may result in transcriptional interference.
Figure 4
Figure 4
Potential N-glycosylation sites of the RAPSN protein predicted by NetNGlyc.
Figure 5
Figure 5
The new link between AK9, N-glycosylation and RAPSN in NMJ. In normal state, AK9 involves in phosphorylation NDP to NTPs. NTPs are substrates for synthesis of NDP-sugars and are the substrates for N-glycosylation of AChR and RAPSN proteins (step 1). A high-surface density of AChR in the NMJ is mediated by RAPSN which is an essential protein for AChR co-clustering (step 2) (upper). A defective AK9 reduces N-glycosylation of AChR and RAPSN proteins while a defective RAPSN impairs the co-clustering of AChR in the post-synaptic membrane (lower). Taken together, this two-hit model explains the myasthenic symptoms developed in P1 and P2. ER, endoplasmic reticulum.

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