A homozygous p.Glu150Lys mutation in the opsin gene of two Pakistani families with autosomal recessive retinitis pigmentosa

Abstract

Purpose: To identify the gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in Pakistani families.

Methods: A cohort of consanguineous families with typical RP phenotype in patients was screened by homozygosity mapping using microsatellite markers that mapped close to 21 known arRP genes and five arRP loci. Mutation analysis was performed by direct sequencing of the candidate gene.

Results: In two families, RP21 and RP53, homozygosity mapping suggested RHO, the gene encoding rhodopsin, as a candidate disease gene on chromosome 3q21. In six out of seven affected members from the two families, direct sequencing of RHO identified a homozygous c.448G>A mutation resulting in the p.Glu150Lys amino acid change. This variant was first reported in PMK197, an Indian arRP family. Single nucleotide polymorphism analysis in RP21, RP53, and PMK197 showed a common disease-associated haplotype in the three families.

Conclusions: In two consanguineous Pakistani families with typical arRP phenotype in the patients, we identified a disease-causing mutation (p.Glu150Lys) in the RHO gene. Single nucleotide polymorphism analysis suggests that the previously reported Indian family (PMK197) and the two Pakistani families studied here share the RHO p.Glu150Lys mutation due to a common ancestry.

Figure 1

Pedigrees and 3q21 marker haplotypes spanning the RHO gene in autosomal recessive retinitis pigmentosa families RP21 (A) and RP53 (B). A: Family RP21 consists of four generations. White circles represent unaffected females, filled circles affected females, white squares unaffected males, and filled squares affected males. Deceased individuals are shown with a slanting line across the symbol. Also shown are the marker loci and their positions in an 18.5-Mb interval, which were used for fine mapping of the disease locus. Marker allele sizes were not accurately determined and therefore we only give two-allele designations. Arrowheads designate crossovers that define the 6.8-Mb minimal critical region between D3S1589 and D3S2322. B: Family RP53 consists of five generations. Patient V-3 in one branch of the family is homozygous for marker alleles flanking RHO. The disease in affected individual III-12 could be RHO unrelated, suggesting locus heterogeneity in the different branches of the family. Haplotypes were established for all individuals for which marker alleles are depicted, except for RP21-III-6, the haplotypes of whom were deduced (indicated with brackets around the haplotypes).

Figure 2

Haplotype analysis for single nucleotide polymorphisms (SNPs) in close proximity to the RHO mutation. Frequencies [26] of the combined homozygous variants based on individual allele frequencies in the studied population are: rs789231 (CC), 0.25; RHO (AA), 0.000025; rs2855557 (AA), 0.178084; rs2625961 (GG), 0.207025.

Figure 3

Fundus photograph of an individual affected with retinitis pigmentosa. The fundus appearance of individual RP21-IV-1 is characteristic of advanced RP, with optic disc pallor, attenuation of the retinal vessels, peripheral pigment epithelial atrophy, and bone spicule pigmentation. The posterior pole shows macular edema, and small white dots are present in the mid-periphery at the level of the retinal pigment epithelium

Figure 4

Sequence analysis of RHO in family RP21. A: Sequence trace of part of exon 2 of an affected individual (RP21-IV-1) showing the homozygous mutant sequence c.448G>A. B: Sequencing results of RP21-IV-7 showing the heterozygous c.448G>A sequence. C: Sequence trace of an unaffected individual with the homozygous wild-type c.448G. d