Linkage Analysis of Pakistani Families with Autosomal Recessive Retinitis Pigmentosa

Abstract

Over the past decades progress in the field of molecular genetics has had an immense contribution to the better understanding of hereditary diseases. Hereditary retinal disorders are a group of diseases that affect the normal function of retina leading to partial or complete loss of eye sight. Depending upon the type and severity of the disease, loss of vision may occur suddenly or gradually. Despite the age of onset and symptoms, eye diseases generally affect the overall quality of life in the affected individuals of all races, cultures and ethnicities and thus remained an active area of research in the past and will be explored in the future as well. The current study focused on the genetic analysis of eight consanguineous Pakistani families (A-H) with multiple members suffering from autosomal recessive RP or retinal dystrophies. These families were enrolled from different rural villages of Pakistan including Punjab, Khyber PakhtunKhwa and Sindh provinces. Clinical data of the affected members of the families were obtained and diagnosis of RP was made after ophthalmic assessment by local ophthalmologist. Physical evaluations ruled out presence of extraocular phenotypes. Blood samples were collected from available members of families and genomic DNA was isolated for use in genetic analysis. Initially all collected families were tested by STS based homozygosity mapping which result in the mapping of family B to chromosome 16. Remaining seven families were subjected to SNP based genome scan which revealed their mapping to different genomic regions. Further follow up of these seven families led to the identification of three novel muta-tions; (c.244- 2A>C) in C8ORF37 (Family C), (c.786delT) FAM161A (Family D) and (g.[152634_42094] delins A) LCA5 (Family F) genes. However mutation analysis of ZNF513, C2ORF71, FAM161A, VSNL1 genes in family A and CLN3 gene in family B did not identify any pathogenic variation. Two families (Family E and H) with multiple homozygous regions and a third family (i.e family A) underwent RD panel based next generation sequencing which only resulted in the identification of a known c.1600G>A in family E in TRPM1 gene. Although we identified two heterozygous variants (c.5653 A>G and c.14662 A>T) in USH2A gene in family A by RD panel sequencing but these variants did not segregate with the disease phenotype in this family. The splice site mutation (c.244 -2A>C) identified in family C was further analyzed with a minigene assay which confirmed the loss of splice acceptor site and the activation of Linkage Analysis of Pakistani Families with Autosomal Recessive Retinitis Pigmentosa xvi Abstract cryptic splice site in exon 3. Sanger sequencing of the cDNA also confirmed the activation of the cryptic splice site within exon 3 which result in the deletion of 22 nucleotides from the RNA. This 22 nucleotide deletion probably results in the frameshift and premature truncation of the protein. DNA walking was used to identify the large LCA5 deletion in family F. Sanger seuencing of PCR products obtained with DNA walking kit revealed a large homozygous deletion of 110540 bps (g.[152634_42094] delins A) in the LCA5 gene. This deletion is predicted to affect the binding site for the basal transcriptional apparatus therefore disrupts the transcriptional regulation and normal gene activation. Family G showed a recurrent mutation c.25G>A in the NMNAT1 gene. While RD panel NGS identified a recurrent missense mutation c.1208G>A, (p.Arg403Gln) in exon 11 of the CNGB3 in family H which did not segregate with the disease phenotype in the family. As this family has been clinically diagnosed with retinitis pigmentosa the CNGB3 variant does not segregate with the disease phenotype therefore negates the disease causative nature of the variant in this family. Families that did not link to any of the known genes/loci by conventional sequencing techniques may have the potential to link to novel genes involved in the pathogenesis of retinal dystrophies. Whole exome sequencing or whole genome sequencing may be implemented to determine the underlying genetic factors for families A, B and H. Linkage Analysis of Pakistani Families with Autosomal Recessive Retinitis Pigmentosa xvii Abstract This study resulted in one publication,  Ravesh and El Asrag et al., 2015. Novel C8orf37 mutations cause retinitis pigmentosa in consanguineous families of Pakistani origin  Two manuscripts submitted and currently under review  Ansar and Ravesh et al., 2015. Detection of Novel Mutations Causing Autosomal Recessive Retinitis Pigmentosa in Pakistan   Ravesh et al., 2015. DNA walking reveals a large deletion of LCA5 in a consanguineous family from Pakistan  Abstracts Presented in International Conferences  Ravesh Z, Weisschu N, Wissinger B, Ansar M. (2015): Molecular genetic analysis of Hereditary Retinal Dystrophies in Consanguineous Families from Pakistan. (Asia ARVO 2015, Feb 16 - 19, Yokohama, Japan).  Ravesh Z, Weisschu N, Reuter P, Bonin M, Ansar M, Wissinger B. (2015): Molecular genetic analysis of Autosomal Recessive Retinitis Pigmentosa & Leber congenital amaurosis in Pakistani Population (25th Annual Meeting of the German Society of Human genetics, ESSEN 2014).