This study was carried out to localize/ identify the loci and genes responsible for causing retinal dystrophies in the Pakistani population. For this, a number of large inbred families as well as a large number of patients suffering from Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD) and retinitis . pigmentosa (RP) were collected with informed consent and screened for disease causing mutations.
The families were initially screened for all the known retinal dystrophy loci. Following the exclusion of all the known loci, a genome wide search was carried out using polymorphic microsatellite markers. For gene identification, databases were searched for candidate genes within the critical disease interval. For mutation detection in the retinal dystrophy panel, single stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (DHPLC) were carried out. Direct sequencing was performed to find out the disease causing mutation in the genes. The results are as follows.
During screening for the known retinal dystrophy loci, three families (3330RP, 111RP and 010LCA) were found to be linked at the CRB1 gene locus on chromosome 1 q31-32.1. Among these, two families were suffering from arRP with para-arteriolar preservation of retinal pigment epithelium (PPRPE) and one family with LCA. Sequencing analysis of the CRB1 gene revealed three novel disease-causing mutations. In the 3330RP family a homozygous G to A substitution was found at codon 846 in exon 7. This mutation replaced glycine with an arginine residue. Another T to C substitution at codon 1116 in exon 9 changed leucine with a proline in the 111 RP family. Family 010LCA had a homozygous T to C substitution at codon 989 that changed isoleucine with a threonine residue.
Another LCA family (011 LCA) was found to be linked to chromosome 17p13.1. Mutation screening of the AIPL1 gene (present on 17p13.1) revealed a homozygous C to A transversion at codon 116 in exon 2. This mutation changed the amino acid threonine with asparagine.
Two other families (1 CRD and 4CRD) suffering from CRD were observed to be linked to chromosome 14q11. This region of the chromosome harbors the RPGRIP1 gene. The RPGRIP1 gene has previously been reported to cause LCA. Mutation screening revealed a homozygous G to T point mutation in exon 16 (Arg827leu) in the 1 CRD family. Sequencing of the 4CRD family showed a homozygous G to T substitution in exon 13 (Ala547Ser).
This is the first report showing the involvement of RPGRIP1 gene mutation in the pathogenesis of CRD.
Linkage analysis of three arRP families (442RP, 452RP and 336RP) showed significant linkage with markers D8S285 and D8S1815. This locus contains the RP1 gene that has previously been reported to be associated with autosomal dominant RP. Sequencing analysis of the RP1 gene for 442RP and 452RP revealed a homozygous C to T substitution at nucleotide 1118 (Thr373lle). Another homozygous 4bp insertion (1461-1465insTGAA) was found in exon 4 in the 336RP family. This insertion produced a stop codon (TGA) immediately after codon 1461, resulting in the synthesis of a truncated protein of 487 amino acids instead of 2156 amino acids.
A panel of 150 patients was also screened for mutations in the RP1 gene. A heterozygous G to A substitution was found in exon 4 in one patient. This mutation replaced alanine with a threonine at codon 699. These results showed the involvement of the RP1 gene in the pathology of autosomal recessive RP.
The Semaphorin (SEMA4A) gene has been reported to cause severe retinal degeneration in a mouse model. To find out the association of human SEMA4A gene with retinal dystrophies, SSCP analysis was carried out using a panel of 190 patients. Sequencing analysis of the variant bands from SSCP gels revealed two compound heterozygous mutations in two patients with RP and two patients with CRD. These mutations were Asp345His and Phe350Cys. Both the mutations were found to segregate together in all the patients. The parents were heterozygous for each of the two mutations. This suggests that in the autosomal recessive mode of inheritance a single mutation does not lead to the disease state.
Another disease causing mutation was identified in three RP and one LCA patient. This heterozygous G to A mutation was found to change the codon for arginine with that of glutamine (Arg713Gln) suggesting autosomal dominant mode of inheritance. This mutation was not found in the 100 normal control subjects. This is the first report of the involvement of the SEMA4A gene in the pathogenesis of retinal dystrophies in humans.