Two apparently unrelated, ethnically diverse, Pakistani arRP families from different geographical regions of Pakistan, RP21 from the Punjab province and RP53 from the North West Frontier province, were identified and analyzed by homozygosity mapping using highly informative microsatellite markers flanking the known arRP genes and loci. Initially, one of the families (RP21) showed homozygosity for the RHO (RP4) locus at 3q22.1 in all affected individuals of the family.
Mutations in the rhodopsin
gene have previously been shown to result in RP. However, most of the RHO
mutations have been found in families with adRP, Human Genome Mutation Database (HGMD
, rhodopsin mutation
), whereas only two mutations have been shown to cause RP with an autosomal recessive mode of inheritance [14
In this study, we identified the c.448G>A mutation in the RHO gene in two different consanguineous arRP families from Pakistan and also determined the frequency of the pathogenic allele in the normal population. While determining the population frequency, we found the c.448G>A mutation in the heterozygous state in one out of 100 control samples, but we did not find it in any of the other 28 families from our RP panel that were screened for the c.448G>A mutation. Further investigations of the family of the heterozygous individual from the control samples resulted in the identification of an additional arRP family (RP53). DNA sequencing revealed the same causative mutation in one branch of the family. There seems to be genetic heterogeneity of the RP in this family as individual III-12 may either carry a second, yet unidentified, variant in the RHO intronic sequences or a mutation in a different gene. The statistical analysis for the association/correlation showed that the mutant allele A is significantly associated (p<0.05) with the disease phenotype because the AA genotype is always observed in known affected individuals and is absent from the normal population (, ).
This c.448G>A mutation has previously been reported to cause arRP in an Indian family from southern India [15
], which is ethnically different and geographically isolated from the present families. We therefore compared, in the two Pakistani families and the Indian family, some of the SNPs from the surrounding region of RHO
to determine if they share a haplotype, suggesting a founder mutation in these families. Indeed, our analysis revealed a shared haplotype in the patients of the three families, suggesting a common ancestry. This is remarkable as the three families belong to diverse ethnic groups and have lived at their present location for several generations with no relative having moved to any other part of the subcontinent, at least for the last 10 generations; thus any commonality in ancestry must be more than several hundred years old.
Genetic screening of RP families is not only helpful in understanding the molecular mechanism of the disease; it is also important for genetic counseling. Traditional consanguineous marriages are major risk factors for autosomal recessive diseases, including retinopathies. Families have little knowledge of the inheritance of the diseases, and through such studies as the present one, many families could be counseled appropriately, as was done for members of families RP21 and RP53.
The rod visual pigment rhodopsin consists of the apoprotein opsin and the covalently bound chromophore 11-cis
]. This protein is a heptahelical G protein-coupled receptor synthesized at a high level in the rod inner segment and transported subsequently to the rod outer segment [22
]. The seven transmembrane segments (H1–H7) are sequentially linked by extracellular (E1–E3) and cytoplasmic (C1–C3) loops [23
]. The first reported null mutation (p.Glu249Ter) associated with arRP was shown to abolish the function of the helices H6 and H7, which contains the retinal binding site. Remarkably, this mutation caused an abnormal ERG pattern even in heterozygotes [14
Residue Glu150, which is embedded at the edge of the phospholipid bilayer, is located near the C-terminal region of the C2 loop, which is important for proper folding of opsin and its binding to transducin and thus for the initiation of the phototransduction cascade [24
]. However, Zhu et al. [25
] have shown that the homozygous p.Glu150Lys change does not affect binding of opsin to transducin but instead affects the export of the protein opsin from the Golgi apparatus. This is due to improper glycosylation of the protein, which results in retention of the protein inside the Golgi, leading to an insufficient supply of opsin to the rod outer segments. The processing of rhodopsin protein has been shown to be normal in the p.Glu150Lys heterozygotes [25
We conclude that linkage and mutation analysis of known retinal disease genes in Pakistani patients with RP resulted in the identification of a RHO mutation in two out of 30 families that very likely have a common ancestor and are also distantly related with the Indian family previously found to harbor this mutation. We also observed genetic heterogeneity in one of these two families. These findings have facilitated accurate genetic counseling of patients and unaffected family members.