The families described in this study, PKRP161 and PKRP183, are from the Punjab province of Pakistan (). All affected individuals underwent a thorough ophthalmologic examination that concluded that all affected individuals in the two families fulfill the diagnostic criteria of RP. Affected patients presented symptoms of night blindness in early childhood. Fundus examination of affected patients illustrated cardinal features of RP, namely arteriolar constriction, waxy pallor optic disc, and pigmentation on the peripheral as well as central retina (). ERG recordings showed absence of both rod and cone responses; scotopic and photopic ERG responses were highly reduced or absent ().
Figure 1 Pedigree drawings of families PKRP161 and PKRP183 with haplotyes of five adjacent chromosomes 4p microsatellite markers and variations identified in PDE6β. Single base changes: c.1655G>A and c.1160C>T were identified in PKRP161 (more ...)
Figure 2 Fundus photographs of affected individuals of families PKFP161 and PKRP183. A: OD and OS of affected individual 17 of PKRP161 (age: 28 years). B: OD and OS of affected individual 7 of PKRP183 (age: 55 years). C: OD and OS of unaffected individual 19 of (more ...)
Figure 3 Electroretinography recordings of affected individuals of families PKFP161 and PKRP183. A: OD combined rod and cone response, and OD isolated cone response. B: OS combined rod and cone response; and OS isolated cone response of affected individual 17 (more ...)
Exclusion analyses with closely spaced STR markers spanning the known/reported loci for arRP were completed. The results of these analyses strongly suggested that the pathogenic mutation in PKRP161 and PKRP183 resides in a region on chromosome 4p. For PKRP161 maximum two-point LOD scores of 3.77, 3.75, 3.63, and 3.66 were obtained with D4S3360, D4S2936, D4S3038, and D4S1614, respectively at θ=0 (). Similarly for PKRP183, two-point LOD scores of 3.08, 3.12, and 3.16 were obtained for markers D4S2936, D4S3038, and D4S1614, respectively at θ=0 ().
Two-point LOD at different recombination fractions of families.
Haplotype analyses confirmed the linkage results, and visual inspection of these haplotypes localized the disease to chromosome 4p. We did not find any proximal recombination events in any affected individuals, whereas the distal boundary was defined by a recombination in affected individuals 17 and 22 of family PKRP161 and affected individual 12 of family PKRP183 at D4S3023. This places the disease interval in an 8.24 cM (4.3 Mb) region on chromosome 4pter-p16.2. Alleles for markers D4S3360, D4S2936, D4S3038, and D4S1614 are homozygous in affected individuals of the two families.
The linked region on chromosome 4pter-p16.2 harbors PDE6β, a gene that has previously been associated with the causality of arRP. We sequenced all coding exons, exon–intron boundaries, and the 5′ and 3′ untranslated regions of PDE6β. In PKRP161 we identified a missense mutation c.1655G>A (), which results in an arginine residue substituted by a glutamine residue at position 552: p.R552Q. Similarly, we identified the novel missense variation c.1160 C>T in PKRP183 (), which leads to a proline residue substituted by leucine at position 387: p.P387L. None of these two variations were present in 192 ethnically matched control chromosomes.
Figure 4 The sequence chromatograms of families PKRP161 and PKRP183. A: Unaffected individual 20 of PKRP161 heterozygous. B: Affected individual 17 homozygous for a single base change: c.1655G>A, leading to an Arginine to Glutamine substitution: p.R552Q. (more ...)
As shown in , both amino acid residues P387 and R552 are highly conserved among other PDE6β orthologs; therefore we next examined the possible impact of these two variations on PDE6β with SIFT
predictions suggested that both p.P387L and p.R552Q substitutions will not be tolerated by the native three dimensional structure of PDE6β. The affected protein function score for p.P387L and p.R552Q were 0.01 and 0.02, respectively (scores <0.05 are predicted to be damaging). Likewise, position-specific score differences obtained from Polyphen
suggested that both p.P387L and p.R552Q substitutions could potentially have deleterious effect on the PDE6β structure, with a position- specific independent count scores of 3.09 and 2.24, respectively (scores >1 are predicted to be damaging).
Figure 5 Amino Acid conservation of the P387 and R552 residues in other PDE6β orthologs. The arrows point to the amino acid residues P387 and R552, which are mutated in the affected individuals of families PKRP183 and PKRP161, respectively. Primates are (more ...)