All 5 families (PKRP063, PKRP084, PKRP111, PKRP122, and PKRP171) were recruited from the Punjab province of Pakistan. Reviewed medical records of previous ophthalmological examinations suggested that all affected individuals in these families had experienced night blindness since early childhood, with progressive loss of peripheral vision and decreased visual acuity with age (). Family PKRP063 was recruited from the southern part of the Punjab province and had 9 family members, including 4 affected members who agreed to participate in the study (). Fundus examination in PKRP063 showed attenuated retinal blood vessels and typical RP pigmentation in the midperipheral retina (). Electroretinography in affected individuals demonstrated diminished rod and cone responses under scotopic conditions, whereas isolated cone responses using a 30-Hz flicker were absent ().
Clinical Characteristics of Individuals in the 5 Pakistani Families With Pathogenic Mutations in TULP1a
Figure 1 In PKRP063, haplotypes of 6p markers and segregation of c.1138A>G variation with the disease phenotype. A square represents a male individual; a circle, a female individual; shading, an affected individual; a double line, consanguinity; and a (more ...)
Figure 2 Fundus photographs in PKRP063 showing right eyes (left) and left eyes (right). A, Individual 9. B, Individual 11. C, Individual 12. Shown are several features associated with retinitis pigmentosa, including waxy pallor of the optic disc, attenuated arterioles, (more ...)
Figure 3 Electroretinographic responses in PKRP063 showing combined rod and cone responses and isolated cone responses in right eyes (left panels) and left eyes (right panels). A and B, Individual 9. C and D, Individual 11. E and F, Individual 12. Electroretinographic (more ...)
A genome-wide linkage scan was completed for PKRP063, and evidence of significant linkage was only observed with markers on chromosome 6p. Two-point LOD scores of 3.17 and 3.10 (at θ = 0.00) were obtained with markers D6S276 and D6S1610, respectively (). Subsequently, additional short tandem repeat markers were selected from the Marshfield database, and genomic DNA from all family members was genotyped. Two-point LOD scores of 3.16, 3.15, and 3.01 (at θ = 0.00) were obtained with markers D6S439, D6S1611, and D6S1645, respectively.
Two-Point Logarithm of Odds (LOD) Scores of 6p21.3 Markersa
Visual inspection of the haplotypes of chromosome 6p markers supported results of the linkage analysis (). There is a proximal recombination event at D6S422 in affected individual 9 and a distal recombination event at D6S257 in affected individuals 11 and 13. This places the disease locus in a 44.26-cM (35.54 Mb [million bases]) region on chromosome 6p, flanked by D6S422 proximally and D6S257 distally. This region harbors the previously reported RP-causing gene TULP1
Sequencing of the coding exons of TULP1 identified a single base pair substitution (c.1138A>G), resulting in threonine to alanine substitution (p.T380A) (). This mutation segregated with the disease phenotype in the family. All affected individuals were homozygous for the mutation, whereas unaffected individuals were heterozygous carriers or were homozygous for the wild-type allele. This mutation was not present in 192 racially/ethnically matched control chromosomes.
Figure 4 Forward and reverse sequence chromatograms in PKRP063. A, Individual 12, harboring wild-type allele. B, Individual 7, a heterozygous carrier. C, Individual 9, homozygous for the single base pair substitution c.1138A>G, resulting in threonine to (more ...)
Identification of a causal mutation in TULP1 prompted us to investigate our cohort of nuclear families with closely spaced fluorescently labeled short tandem repeat markers spanning the TULP1 locus. We identified 4 families in whom results of linkage and haplotype analyses were suggestive of linkage to chromosome 6p (). Maximum 2-point LOD scores (at θ = 0.00) were 3.58 with marker D6S1645 for PKRP084, 2.07 with marker D6S1645 for PKRP111, 2.56 with marker D6S439 for PKRP122, and 3.24 with marker D6S439 for PKRP171. Subsequent sequencing of the coding exons of TULP1 identified a single change (c.1466A>G) in all 4 families, which results in lysine to arginine substitution (p.K489R). All affected individuals were homozygous, whereas unaffected individuals were heterozygous carriers or homozygous for the wild-type allele. This variation was not present in 192 racially/ethnically matched control chromosomes.
Haplotypes of 6p markers and segregation of c.1466A>G variation with the disease phenotype. A, In PKRP084. B, In PKRP111. C, In PKRP122. D, In PKRP171. Symbols are explained in the legend for .
Next, we investigated the evolutionary conservation of T380 and K489 in other TULP1 orthologs. Both T380 and K489 are conserved, along with amino acid residues residing in the immediate neighborhood of both T380 and K489 (). Position-specific independent count score differences obtained from PolyPhen suggested that T380A and K489R substitutions could potentially have a deleterious effect on TULP1 structure, with scores of 1.92 and 1.52, respectively (a position-specific independent count score difference >1.0 is probably damaging).
Figure 6 Sequence conservation of T380 and K489 residues in TULP1 orthologs (primates are green, placental mammals are blue, and vertebrates are purple). The arrows point to amino acid residues T380 and K489, which were mutated in individuals with autosomal recessive (more ...)
All 5 families described herein reside in the same geographic region of Pakistan, but racially/ethnically they belong to different social castes. We were unable to identify any family relationships among these clans. Hence, the presence of common causal mutations in 4 families prompted us to investigate the ancestral relationships, if any, among these 4 families. We used single-nucleotide polymorphisms flanking the causal mutation and constructed a haplotype (CC/TGTC), which was suggestive of a common founder for the c.1466A>G variation ().
Single-Nucleotide Polymorphism (SNP) Haplotypes of Affected Individuals in PKRP084, PKRP111, PKRP122, and PKRP171 Harboring the p.K489R Mutation in TULP1