Genetic Exploration and Analysis of Autosomal Recessive Cataracts

Abstract

The present work was designed to identify the genetic causes of autosomal recessive cataracts in consanguineous families from Pakistan. For this purpose, twenty five families were identified from different areas of Pakistan mainly from south Punjab. Blood samples were collected and DNAs were extracted for all the samples. Exclusion analysis was performed on genomic DNAs to exclude known genes/loci for recessive cataracts using the traditional homozygosity mapping technique. Three families were found linked to previously reported regions on different chromosomes. Family PKCC185 was found linked to chromosome 22q11.23 harboring CRYBB3, CRYBB1, CRYBA4, and CRYBB2 genes. A maximum two point logarithm of odds score of 3.0 was calculated with markers D22S1174, D22S419 and D22S315 at θ = 0. No causative mutation was found in CRYBB1, CRYBB2 and CRYBA4 genes. Sanger sequencing of CRYBB3 gene in this family identified an already reported missense mutation. In family PKCC214 cataract phenotype was found linked to chromosome 19q13.41. A maximum two point logarithm of odds score of 3.25 was calculated with markers D19S572 and D19S589 at θ = 0. This region harbors LIM2, an already reported gene in cataracts. Sanger sequencing of the gene revealed a novel missense mutation. In another large consanguineous family PKCC215; linkage was found in a region on chromosome 6p24.3-24.2 harboring GCNT2 gene with a maximum two point LOD score of 5.78 with marker D6S470 at θ = 0. PCR amplification for the Sanger sequencing of GCNT2 gene coding exon failed in affected individuals of PKCC215 thus indicating a large DNA deletion. Chromosomal walking and exome sequencing data analysis led to the identification of approximately 190 kb deletion resulting in excision of all the coding exons of GCNT2 gene. Failure to amplify the deletion breakpoints indicated a complex chromosomal rearrangement at this region most probably presence of an insertion in addition to the large DNA deletion. In linkage analysis, PKCC206 was found linked to a region on chromosome 1p36.13. A maximum two point LOD score of 3.36 was calculated with the marker D1S2672 at θ = 0. This region on chromosome 1p harbors EPHA2 gene. Sanger sequencing of coding exons of the genes did not reveal any causative variation. Thus a novel locus of cataract was identified on chromosome 1p36.13. Another family PKCC212 was found linked to a region on chromosome 22q11.23 with a maximum two point LOD score of 2.51 with marker D22S315, harboring cluster of crystalline genes including CRYBB3, CRYBB1, CRYBA4, and CRYBB2. No causative variation was found in CRYBB3, CRYBB1 and CRYBA4. While Sanger sequencing of CRYBB2 gene resulted in identification of a large DNA deletion. Interestingly this gene has been previously reported in autosomal dominant cataracts where it was responsible for causing cataracts in heterozygotes. While in case of PKCC212 heterozygous carrier were normal completely. Genome wide scan with MD-10 panel was done on two cataract families: PKCC208 and PKCC216. In PKCC208 a novel locus on chromosome 17p12 was identified. A maximum two point LOD score of 6.01 was calculated at recombination fraction of zero with marker D17S938. This study reports two novel and a previously reported mutation in known cataract genes in three consanguineous families. Furthermore this study also identified two novel loci and a novel gene in three consanguineous families responsible for cataracts.