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Retinitis pigmentosa (RP) is the most frequent genetically and clinically heterogeneous inherited retinal degeneration. To date, more than 80 genes have been identified that cause autosomal dominant, autosomal recessive and X linked RP. However, locus and allelic heterogeneity of RP has not been fully captured yet. This heterogeneity and lack of an accurate genotype phenotype correlation makes molecular dissection of the disease more difficult. The present study was designed to characterize the underlying pathogenic variants of RP in Pakistan. For this purpose, a large consanguineous family with RP phenotype showing autosomal recessive mode of inheritance was selected after a complete ophthalmological examination. Next generation sequencing was used for the identification of molecular determinant followed by Sanger-sequencing for confirmation. After sequence analysis a novel homozygous missense mutation, (c.602 C > T) in exon 4 of the RDH5 gene (MIM: 601617) was identified. This mutation resulted in substitution of phenyl alanine for serine at amino acid 201 (p.Ser201Phe) of the RDH5 gene. The same mutation was not detected in the 200 ethnically-matched control samples by Sanger sequencing. The identified mutant allele segregated in homozygous fashion in all the affected individuals of pedigree. Identification of this mutation reveals the allelic heterogeneity of RDH5 in patients with RP phenotype. The findings of this study demonstrate the clinical significance of next generation sequencing to understand the molecular basis of diseases and would help to reveal new proteins and their function in visual cycle will pave the way for early diagnosis, genetic counseling and better therapeutic inventions. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) causes severe visual impairment early in life. The purpose of this study was to identify families with autosomal recessive RP (arRP), their exclusion analysis and identification of novel loci/genes/mutations in affected individuals. Twenty five consanguineous Pakistani families suffering from non syndromic Retinitis pigmentosa (RP) were ascertained from different areas of Pakistan to participate in this study. 85 affected and 210 normal individuals of selected families and from the healthy population took part in this research work. The clinical records of affected family members were retrospectively analyzed and ophthalmological examinations were performed in selected families. Genomic DNA was extracted from peripheral blood samples of phenotypically healthy and affected individuals. Exclusion analysis was done initially for the screening of the causative gene using highly polymorphic microsatellite markers. Genetic investigations using polymorphic microsatellite markers revealed homozygosity with CRB1 gene on chromosome 1 in affected individuals of family RP1. All other families and 210 healthy or control samples showed exclusion with the selected loci/genes. Haplotypes were constructed to appraise the results statistically. For further confirmation of linkage, sequence analysis of the linked gene (CRB1) was done. Sequence analysis revealed a novel missense mutation (?.1459?>?) in exon 6 of the CRB1 gene segregating with disease phenotype. This mutation resulted in a substitution of proline for serine at amino acid 487 (p. Ser487Pro). These findings suggest a founder effect of the p. Ser487Pro mutation in the Pakistani population and extend the spectrum of known CRB1 mutations. It was therefore concluded that the missense mutation in CRB1, leading to premature termination of the protein, is responsible forRP phenotype in the affected individuals ofRPl. [ABSTRACT FROM AUTHOR]