Table3_Case report: Incomplete penetrance of autosomal dominant myotonia congenita caused by a rare CLCN1 variant c.1667T>A (p.I556N) in a Malaysian family.DOCX

Myotonia congenita (MC) is a rare neuromuscular disease caused by mutations within the CLCN1 gene encoding skeletal muscle chloride channels. MC is characterized by delayed muscle relaxation during contraction, resulting in muscle stiffness. There is a lack of MC case reports and data on the prevalence among Malaysians. We report a clinical case of a 50-year-old woman presents with muscle stiffness and cramp episodes that started in early childhood. She had difficulty initiating muscle movement and presented with transient muscle weakness after rest, which usually improved after repeated contraction (warm-up phenomenon). She was diagnosed with MC after myotonic discharge on electromyography (EMG). Her brother had similar symptoms; however, no additional family members showed MC symptoms. Serum creatine kinase levels were elevated in both the proband and her brother with 447 U/L and 228 U/L recorded, respectively. Genetic analysis by whole-exome sequencing (WES) revealed a previously reported pathogenic CLCN1 gene variant c.1667T>A (p.I556N). Genetic screening of all family members revealed that the same variant was observed in the children of both the proband and her brother; however, the children did not present with either clinical or electrophysiological MC symptoms. The multiplex ligation-dependent probe amplification (MLPA) analysis conducted identified neither exon deletion nor duplication in CLCN1. In conclusion, this report describes the first case of MC in Malaysia in which incomplete penetrance observed in this family is caused by a known pathogenic CLCN1 variant.</p

Table2_Case report: Incomplete penetrance of autosomal dominant myotonia congenita caused by a rare CLCN1 variant c.1667T>A (p.I556N) in a Malaysian family.DOCX

Myotonia congenita (MC) is a rare neuromuscular disease caused by mutations within the CLCN1 gene encoding skeletal muscle chloride channels. MC is characterized by delayed muscle relaxation during contraction, resulting in muscle stiffness. There is a lack of MC case reports and data on the prevalence among Malaysians. We report a clinical case of a 50-year-old woman presents with muscle stiffness and cramp episodes that started in early childhood. She had difficulty initiating muscle movement and presented with transient muscle weakness after rest, which usually improved after repeated contraction (warm-up phenomenon). She was diagnosed with MC after myotonic discharge on electromyography (EMG). Her brother had similar symptoms; however, no additional family members showed MC symptoms. Serum creatine kinase levels were elevated in both the proband and her brother with 447 U/L and 228 U/L recorded, respectively. Genetic analysis by whole-exome sequencing (WES) revealed a previously reported pathogenic CLCN1 gene variant c.1667T>A (p.I556N). Genetic screening of all family members revealed that the same variant was observed in the children of both the proband and her brother; however, the children did not present with either clinical or electrophysiological MC symptoms. The multiplex ligation-dependent probe amplification (MLPA) analysis conducted identified neither exon deletion nor duplication in CLCN1. In conclusion, this report describes the first case of MC in Malaysia in which incomplete penetrance observed in this family is caused by a known pathogenic CLCN1 variant.</p

Table1_Case report: Incomplete penetrance of autosomal dominant myotonia congenita caused by a rare CLCN1 variant c.1667T>A (p.I556N) in a Malaysian family.XLSX

Myotonia congenita (MC) is a rare neuromuscular disease caused by mutations within the CLCN1 gene encoding skeletal muscle chloride channels. MC is characterized by delayed muscle relaxation during contraction, resulting in muscle stiffness. There is a lack of MC case reports and data on the prevalence among Malaysians. We report a clinical case of a 50-year-old woman presents with muscle stiffness and cramp episodes that started in early childhood. She had difficulty initiating muscle movement and presented with transient muscle weakness after rest, which usually improved after repeated contraction (warm-up phenomenon). She was diagnosed with MC after myotonic discharge on electromyography (EMG). Her brother had similar symptoms; however, no additional family members showed MC symptoms. Serum creatine kinase levels were elevated in both the proband and her brother with 447 U/L and 228 U/L recorded, respectively. Genetic analysis by whole-exome sequencing (WES) revealed a previously reported pathogenic CLCN1 gene variant c.1667T>A (p.I556N). Genetic screening of all family members revealed that the same variant was observed in the children of both the proband and her brother; however, the children did not present with either clinical or electrophysiological MC symptoms. The multiplex ligation-dependent probe amplification (MLPA) analysis conducted identified neither exon deletion nor duplication in CLCN1. In conclusion, this report describes the first case of MC in Malaysia in which incomplete penetrance observed in this family is caused by a known pathogenic CLCN1 variant.</p

The breakpoints of disease-associated interchromosomal insertions at Xq27.1 localize near the center of 180 bp palindrome sequence.

<p>Cartoon depicts a portion of the palindrome sequence (chrX:139,502,939–139,502,970) with the positive strand folded upon itself in a hairpin loop (black). The four non-palindromic bases in the middle of the 180 bp sequence (TATC, bolded black) are predicted to form the head of the hairpin loop. The locations of the breakpoints on chromosome Xq27.1 for CMTX3 (orange); hypertrichosis¹ (red, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref037" target="_blank">37</a>]); hypertrichosis² (blue, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref037" target="_blank">37</a>]); hypertrichosis³ (green,[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref038" target="_blank">38</a>]); ptosis (pink; Bunyan [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref039" target="_blank">39</a>]); and XX sex reversal (purple, Haines [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006177#pgen.1006177.ref040" target="_blank">40</a>]) are marked out on the hairpin structure. Single breakpoints are depicted by a solid line. Multiple breakpoints are indicated by broken lines.</p

Average depth ± SD of sequence coverage across 8q24.3.

<p>Average depth ± SD of sequence coverage across 8q24.3.</p

Characterization of the CMTX3 insertion.

<p>Sequence analysis of the proximal (A) and distal (B) breakpoints. Reference sequence for chromosome X and chromosome 8 are indicated in blue and orange, respectively. The distal breakpoint includes additional sequence from chromosome 12 (in green) and small rearrangements of the chromosome X sequence including an inversion of 12 bp, and a base pair substitution and a base pair deletion. (C) The 78 kb 8q24.3 sequence (in orange) contains the partial 5’<i>ARHGAP39</i> transcript which has been inserted 330 kb downstream and 84 kb upstream of the genes <i>LOC389895</i> and <i>SOX3</i>, respectively (in blue). The direction of transcripts are indicated by the arrow. (D) Location of the 78kb 8q24.3 insertion sequence (in orange) relative to the whole of the 5.7 Mb CMTX3 locus (in blue).</p

Identification and confirmation of a 78 kb chromosome 8q24.3 insertion in patients with CMTX3.

<p>(A) Whole genome sequencing depth of coverage for affected (red) and normal (black) males across the 8q24.3 insertion and flanking sequence. (B) Depiction of wild type chromosome X (top) and mutant chromosome X (bottom). The location of primers and amplicon sizes for the multiplex PCR genotyping assay are shown. Dotted red lines represent insertion breakpoints. (C) Size fractionation of multiplex PCR genotyping assay for a subset of family members from CMT623. Individual genotypes are depicted above the gel lane. Expected band sizes for the various primer combinations are listed to the right. Unaffected hemizygous males and homozygous females generate a single 340 bp amplicon; affected hemizygous males generate 595 bp and 235 bp amplicons crossing the proximal and distal breakpoints, respectively; carrier females amplify all three amplicons.</p

Split-reads and discordant paired ends mapping to Xq27.1 and 8q24.3 in whole genome sequencing data from affected males.

<p>Split-reads and discordant paired ends mapping to Xq27.1 and 8q24.3 in whole genome sequencing data from affected males.</p