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Hereditary neuromuscular disorders are a clinically and genetically heterogeneous group of genetic conditions that affect about 1 in 1000 individuals worldwide. These diseases affect the muscles and their direct nervous system control. The conditions are characterized by progressive muscle degeneration and weakness and constitute a great disease burden. Genetic defects in the proteins that maintain the motor demand and neural inputs lead to neuromuscular disorders. This study was aimed to explore the genetic cause of four neuromuscular disorders identified in five Pakistani families using whole exome and Sanger sequencing. Genomic DNA was extracted from peripheral blood of the recruited families. Whole exome paired-end sequencing was performed by generating 51 Mb Sure Select V4 libraries. DNA shearing, hybridization using RNA-based library baits, target capture and bridged amplification were subsequently carried out. The imaging and extension was achieved in automated cycles by mounting the clusters-bearing flow cell onto the Illumina HiSeq 2000/2500 sequencer. The data was analysed using standard bioinformatic pipeline. Raw read sequences were proceeded for recalibration of the base quality and removal of duplicates. Genome Analysis Toolkit (GATK3.v4) was used to call variant quality score recalibration and short insertions and deletions. Picard-tools- 1.118 was used to mark the duplicates. The variants with minor allele frequency value less than 0.01 were considered rare pathogenic variants. Deleterious effects of the variants on the structure and function of the protein were predicted through various bioinformatics tools. The variants present in the healthy unrelated individuals were excluded. The genotype of candidate variants was confirmed in the family members through Sanger sequencing. The proband of the family A [lab ID: NP-08] affected with autosomal dominant familial hypokalemic periodic paralysis (hypoKPP) was subjected to whole exome sequencing. A heterozygous missense variant (c.919A>G; p.Met307Val) was found in KCNJ2. Sanger sequencing verified the variant segregation in the family with disease phenotype with incomplete penetrance. The bioinformatics tools ranked the p.Met307Val change in KCNJ2 as deleterious. The variant was conserved in the 100 vertebrate species and is the likely cause of the hypoKPP in the Pakistani family. This investigation expands the underlying genetic etiology of familial hypoKPP. The proband of the family B [lab ID: NP-05] affected with autosomal recessive Charcot- Marie-Tooth disease type II was subjected to whole exome sequencing. A homozygous missense variant (c.1591C>A; p.Pro531Thr) in IGHMBP2 was shortlisted. Sanger sequencing revealed full segregation of the variant with the disease phenotype in the family. The parents of the affected individuals were heterozygous for the position. In silico analysis of the c.1591C>A substitution predicted deleterious effect on the protein structure and function. The variant was conserved in the vertebrate species. We conclude that IGHMBP2 c.1591C>A variant is the likely cause of the CMT2 disease in the family. Whole exome sequencing of the proband of the family C [lab ID: NP-07] affected with autosomal recessive Charcot-Marie-Tooth disease type IV was performed. No variant was found in the genes previously linked to neuromuscular disorders. Following disease model and tissue and organ specific expression, three rare candidate variants were shortlisted including compound heterozygous variant (c.874_875insGA, p.Lys292fs; c.871_872delCG, p.Arg291fs) in HADHA, homozygous missense mutation (c.2062C>T; p.Arg688Cys) in SLC6A6 and homozygous missense variant (c.63917G>A; p.Arg21306His) in TTN. However, none of the variants cosegregated with the disease phenotype in the family. These results support the evidence of further genetic heterogeneity in CMT disease. We believe that a hitherto unidentified genetic or epigenetic factor is the cause of the disease in the family. The families D [lab ID: NP-12] and E [lab IDs: NP-13] affected with autosomal recessive infantile-onset Pompe disease (IOPD) were subjected to Sanger sequencing. Short oligonucleotide sequences were used to screen GAA in both the families. A rare novel homozygous missense c.2561G>A variant was found segregating in the family D. The parents were carriers for this variant. While a rare heterozygous variant (c.2773 A>C) in GAA was identified in the family E. The genotype of the mother was heterozygous for the variant but no GAA variant identified in the father. The other unidentified variant in the family may be present in the promoter or regulatory sequence. The variants were not present in our in-house database of the local unrelated healthy population. Different bioinformatic tools predicted the identified variants to have deleterious effects on GAA protein structure and function. The variants are also conserved in the vertebrate species. We suggest that these GAA variants are the likely cause of IOPD in these families. In conclusion, the study highlights the clinical significance of whole exome sequencing in the molecular diagnosis of heterogeneous hereditary neuromuscular diseases. The data should also help in the prenatal diagnosis and improved genetic counselling of the families.
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