Molecular Genetic Analysis of Pakistani Families With Autosomal Recessive Congenital Cataracts by Homozygosity Screening

Abstract

Purpose: To identify the genetic origins of autosomal recessive congenital cataracts (arCC) in the Pakistani population.

Methods: Based on the hypothesis that most arCC patients in consanguineous families in the Punjab areas of Pakistan should be homozygous for causative mutations, affected individuals were screened for homozygosity of nearby highly informative microsatellite markers and then screened for pathogenic mutations by DNA sequencing. A total of 83 unmapped consanguineous families were screened for mutations in 33 known candidate genes.

Results: Patients in 32 arCC families were homozygous for markers near at least 1 of the 33 known CC genes. Sequencing the included genes revealed homozygous cosegregating sequence changes in 10 families, 2 of which had the same variation. These included five missense, one nonsense, two frame shift, and one splice site mutations, eight of which were novel, in EPHA2, FOXE3, FYCO1, TDRD7, MIP, GALK1, and CRYBA4.

Conclusions: The above results confirm the usefulness of homozygosity mapping for identifying genetic defects underlying autosomal recessive disorders in consanguineous families. In our ongoing study of arCC in Pakistan, including 83 arCC families that underwent homozygosity mapping, 3 mapped using genome-wide linkage analysis in unpublished data, and 30 previously reported families, mutations were detected in approximately 37.1% (43/116) of all families studied, suggesting that additional genes might be responsible in the remaining families. The most commonly mutated gene was FYCO1 (14%), followed by CRYBB3 (5.2%), GALK1 (3.5%), and EPHA2 (2.6%). This provides the first comprehensive description of the genetic architecture of arCC in the Pakistani population.

Figure 1

Work flow of the present study.

Figure 2

The 13 arCC pedigrees collected from Pakistan including 10 families that were mapped through homozygosity mapping and 3 families that were mapped by genome-wide linkage analysis (denoted by asterisks). Filled symbols denote affected individuals. Pedigrees include haplotypes for two microsatellite and gene mutations. The blackened bars correspond to affected haplotypes with alleles that cosegregate with the disease and that are homozygous in affected individuals.

Figure 3

Domain structure and evolutionary conservation of proteins with missense mutations. Graphic overview of the proteins encoded by genes in which mutations were identified in EPHA2, FOXE3, FYCO1, TDRD7, AQP0, HSF4, GALK1, and CRYBA4. Structural or functional domains are depicted, as well as the position of the mutation. Amino acid sequence conservation around residues affected by missense mutations are shown for the five known cataract genes identified in this study. The red bar indicates the position of the mutation. The sequences of proteins or predicted translation products from nine species from humans to zebrafish have been compared and aligned.

Figure 4

Frequency of cataract gene mutations in the Pakistani population. Pie chart showing the frequencies of cataract gene mutations in the Pakistani population as seen in this study and our previous studies.