Family PKRP064 recruited from the Punjab province of Pakistan included six affected individuals in three consanguineous loops (). This family resides in remote villages of the Punjab province of Pakistan, and so the exact age of disease onset is not known. However, the medical records available to us along with communications with the family elders were suggestive of an early, most probably a congenital onset. The fundus photographs of affected individuals illustrate multiple round white deposits in the mid and peripheral regions of the retina (). The scotopic ERG responses were non-detectable, whereas the photopic response was severely reduced in affected individuals (). Taken together, the ophthalmological examination in PKRP064 identified typical features of FA with an early onset form of night blindness ().
Figure 1 Pedigree drawing of family PKRP064 with haplotypes of six adjacent 15q markers and segregation of c.466C/T variation with the disease phenotype. Squares are males, circles are females; filled symbols are affected individuals; a double line between individuals (more ...)
Figure 2 Fundus photographs of individuals of family PKRP064. (A) Individual 11: OD and OS; (B) unaffected individual 14: OD and OS. The fundus photographs show multiple round white deposits with relative absence in the central macula. OD, oculus dexter (right (more ...)
Figure 3 Electroretinographic (ECG) responses of family PKRP064. Individual 11: (A) OD combined rod and cone response, and cone flicker response; (B) OS combined rod and cone response, and cone flicker response. Unaffected individual 14: (C) OD combined rod and (more ...)
Clinical characteristics of affected individuals of family PKRP064
Genomic DNA of affected individuals was investigated with closely spaced STR markers that span all reported loci associated with FA (data not shown). Two-point linkage analysis with alleles of chromosome 15q STR markers was suggestive of linkage with significant LOD scores. Maximum two-point LOD scores of 4.77 and 3.19 were obtained with markers D15S1045, and D15S202, at θ=0, respectively ().
Two point limit of detection (LOD) scores of chromosome 15q markers of families PKRP064 and PKRP107
Haplotype analysis supported linkage to the chromosome 15q. There are recombination events in individuals 12 and 22 at D15S152 and D15S979, respectively, which defines the proximal boundary (). The distal boundary is defined by recombination in individual 19 at marker D15S207 (). In addition, lack of homozygosity at D15S116 in individual 22 suggests that the pathogenic mutation resides proximal to marker D15S116 in a 2.24 cM (1.18 Mb) region flanked by D15S979 proximally and D15S116 distally.
The critical interval harbours RLBP1, a gene previously associated with RPA and FA, and sequencing of the coding exons of RLPB1 identified a C to T transition c.466C→T (), which leads to premature termination of the protein R156X. This variation segregated with the disease phenotype in family PKRP064: all affected individuals were homozygous for the mutation, whereas unaffected individuals were either heterozygous carriers or were homozygous for the wild type allele (). The mutation was not present in 192 ethnically matched control chromosomes.
Figure 4 Forward and reverse sequence chromatograms of family PKRP064. (A) Individual 15 harbouring the wild type allele; (B) individual 14 heterozygous and (C) affected individual 12 homozygous for C to T transition c.466 C→T, resulting in a p.R156X. (more ...)
Subsequently, we identified a second family with typical clinical characteristics of FA (). Exclusion analyses localised the disease phenotype to chromosome 15q with suggestive LOD scores. Two-point scores of 3.04, 3.09, and 3.01 at θ=0 were obtained with markers D15S152, D15S979 and D15S1045, respectively (). Bi-directional sequencing identified a missense mutation c.346G/C, resulting in glycine to arginine substitution: p.G116R (). The mutation segregated with the disease phenotype in the family () and was not present in 192 ethnically matched control chromosomes. The mutation segregated with the disease phenotype in the family and was not present in 192 ethnically matched control chromosomes. We investigated the evolutionary conservation of glycine 116 in other RLBP1
orthologues using the UCSC genome browser (http://genome.ucsc.edu/cgi-bin/ hgGateway
) and as shown in , the amino acid glycine at position 116 is conserved in other RLBP1 orthologues.
Figure 5 Sequence chromatograms of family PKRP107 and alignment of G116 in RLBP1 orthologues. (A) Individual 15 harbouring the wild type allele; (B) individual 7 heterozygous and (C) affected individual 8 homozygous for G to C transition c.346G→C, resulting (more ...)
Figure 6 Pedigree drawing of family PKRP107 with haplotypes of six adjacent 15q markers and segregation of c.346G→C variation with the disease phenotype. Symbols are as described in . The alleles forming the risk haplotype are shaded black, and (more ...)