MOLECULAR GENETICS OF AUTOSOMAL RECESSIVE RETINITIS PIGMENTOSA IN CONSANGUINEOUS PAKISTANI FAMILIES

Abstract

It is an established fact that genetic disorders are one of the most important threats to human health. Several genetic disorders have been described clinically but their etiology is still unidentified and mysterious. The molecular basis for most of them is also unknown. With the advancement in the field of molecular biology different powerful techniques have been developed to understand the molecular basis of hereditary disorders. This would help in the subsequent identification of causative genes and mutations. Blindness and visual impairment due to genetic disorders are more common in developing countries like Pakistan than in developed countries. Retinitis pigmentosa (RP) is a major form of incurable blindness affecting one out of 4000 people worldwide. This highly heterogeneous disease has numerous inheritance patterns with the end result of partial to complete irreversible blindness. Another ocular disorder called fundus albipunctatus (FAP) also has some symptoms similar to RP like night blindness. In FAP this night blindness occurs in childhood but it remains stationary and day vision is not affected as in the case of RP where constriction of day vision occurs gradually. The present study was aimed to analyze families with ocular disorder. Families with autosomal recessive hereditary retinitis pigmentosa were used for mapping the disease genes and mutations. Seven consanguineous unrelated families (RP8, RP9, RP11, RP12, RP13, RP14 and RP16) with inherited RP were ascertained from different regions of Pakistan. The mode of inheritance in all families was inferred as autosomal recessive. The strategy used for this study was candidate gene approach. Linkage analysis was performed by PCR using STR (short tandem repeats) microsatellite markers for the known loci/genes. Direct sequencing (next generation sequencing) of the PCR products was carried out for identification of pathogenic mutations. In the present study linkage to crumbs homolog 1 (CRB1) gene on chromosome 1q31.3 was confirmed in family RP12. A novel missense mutation in human CRB1 gene has been found after sequence analysis of exon 6 of the CRB1 gene at nucleotide position xx 1459 (c.1459T>C). At protein level this mutation resulted in a substitution of proline for serine at amino acid 487 (p.Ser487Pro). It was inferred that mutation in this gene is strong enough to cause autosomal recessive retinitis pigmentosa. After the initial screening of autosomal recessive retinitis pigmentosa loci for family RP13, it was evident that there was no involvement of retinitis pigmentosal loci in the disease phenotype and it was a rare case of fundus albipunctatus, with RDH5 gene defect as the underlying cause. The family RP13 showed linkage to retinol dehydrogenase 5 (11-cis/9-cis) RDH5 gene after homozygosity mapping. A novel missense mutation at nucleotide position 602 (c.602 C>T) was identified after next generation sequencing of exon 4 of the RDH5 gene .This mutation resulted in substitution of phenylealanine for serine at amino acid 201 (p.Ser201Phe) of the RDH5 gene. The mutations in RDH5 gene are related to fundus albipunctatus (FAP). This is an exceptional form of stationary night blindness, it was deduced that mutation in this gene was responsible for autosomal recessive FAP in this family. The family RP14 showed exclusion to all the known genes and loci of RP. It was inferred that a novel locus/gene is responsible for causing RP in this family. The strongest candidate gene was RY2R which was earlier involved in cardiac disorder. Fine mapping in future would confirm the involvement of this gene in RP. Four families (RP8, RP9, RP11 and RP16) with some of the common selected loci/gene showed heterozygosity for the different combinations of the parental alleles in both affected and normal individuals after the linitial linkage. This heterozygosity confirmed exclusion to five selected known loci or genes on different chromosomes associated with autosomal recessive RP. Since many genes and loci are involved in this disease and genotyping using vertical polyacrylamide gel electrophoresis (PAGE) is a time taking and laborious method so commonly found genes in RP were initially selected which showed exclusion.On the basis of these exclusions it was inferred that a novel locus/gene or mutation is involved in these families which could be identified by SNP affymetrix array technique and sequencing. Many loci/genes/mutations are yet to be identified for this phenotype. It would be helpful in future to understand the disease prognosis. This research will also provide a smooth way for carrier screening, genetic counseling and prenatal diagnosis. This study may help gaining insight into the genetic causes underlying these disorders, to improve the clinical management and prevention.