Identifying genetic defects associated with retinal dystrophies common in Pakistani population

Patients of unrelated families (A-C) with documented consanguineous marriages were recruited from the rural Pakistani populations. Blood samples were collected from available members of the families and a proband from each family was subjected to ophthalmic assessments. Families were assessed for involvement of retinal disease genes/loci reported in the Pakistani population and un-linked families were subjected to genome-wide genotyping applying the 250K NspI genotyping array (Affymetrix, USA). Homozygosity Mapper was applied to identify homozygous regions shared by affected family members. Candidate genes were further analyzed by Sanger sequencing, deletion mapping & minigene functional assay.

Genome-wide SNP analysis revealed large homozygous intervals 14Mb, 79Mb & 4Mb on chromosomes 2, 8 & 15 in families A, B & C respectively. The identified homozygous regions were analyzed to find known RD genes and Sanger sequencing of these genes led to the identification of a novel and a recurrent mutation. Affected members of family A with autosomal recessive retinitis pigmentosa (RP) shared a homozygous region on 2p24.3-p23.1 that contains ZNF513, C2ORF71 & VSNL1 genes. Yet sequencing all coding exons of these genes revealed no pathogenic mutation. Similarly, we identified a 79Mb region on chromosome 8p21.3-q22.3 in family B, which harbors the candidate gene, C8ORF37. Mutation screening revealed a novel splice acceptor site variation, which was further verified by the minigene splice assay. The splice variant results in the activation of a cryptic splice site resulting in the partial deletion of the adjacent exon 3 in transcripts and eventually leads to a translational frameshift followed by premature protein truncation. Sanger sequencing also confirmed the presence of a recurrent homozygous missense mutation in TRPM1 gene in the third family (C), which was mapped on chromosome 15q12-q13.3. The identified variants segregate in the respective families and were absent in 60 healthy controls from Pakistan.

Our research highlights the influence of consanguineous marriages and presence of genetic heterogeneity in arRDs in the Pakistani population. It is anticipated that these findings will broaden our perception of the disease pathogenesis.