A Study of Molecular and Genetic Determinants of Primary Congenital Glaucoma

Abstract

The study reported in this thesis document was undertaken to characterize molecular and genetic basis of primary congenital glaucoma (PCG) in Pakistani population. For this purpose, traditional strategy of homozygosity mapping was used to identify disease causing mutations and to map novel loci/genes responsible for autosomal recessive primary congenital glaucoma (arPCG). Thirty consanguineous families with arPCG were enrolled from different parts of Pakistan. Genomic DNAs from these families were subjected to linkage analysis for the exclusion of previously reported genes/loci for autosomal recessive primary congenital glaucoma. The phenotypes of 17 PCG families (PKGL028, 032, 040, 047, 050, 051, 058, 060, 065, 066, 067, 068, 069, 070, 071, 072 & 073) were found linked to GLC3A locus harboring Cytochrome P450 Family 1 Subfamily B Member 1 (CYP1B1) gene. Sanger sequencing of CYP1B1 identified five missense mutations; p.Y81N, p.E229K, p.R368H, p.R444Q and p.R469W in families PKGL051, PKGL047, PKGL058, PKGL050 and PKGL028 respectively. Another homozygous missense mutation; p.R390H was identified in nine PCG families designated as; PKGL040, PKGL060, PKGL065, PKGL066, PKGL067, PKGL069, PKGL070, PKGL071 and PKGL073. In PKGL032, a 10bp homozygous duplication; c.1200_1209dupTCATGCCACC (p.T404Sfs*30) was identified. Two novel frameshift mutations; p.W246Lfs*81 and p.P442Qfs*15 were identified in PKGL047 and PKGL068 respectively. Furthermore, mutational analysis identified a known homozygous nonsense mutation; p.Q37X in family PKGL072. Three consanguineous families; PKGL042 PKGL015 and PKGL076 were found linked to another known PCG locus; GLC3D harboring Latent Transforming Growth Factor Beta Binding Protein 2 (LTBP2) gene. Sanger sequencing of LTBP2 identified two missense and a frameshift mutation; p.D1010N, p.Q1143Rfs*35 and p.C1757Y in PKGL076, PKGL015 and PKGL042 respectively, all of these were novel. These results show that pathogenic mutations in CYP1B1 and LTBP2 gene are responsible for PCG phenotype in these nineteen families. Three large consanguineous PCG families which remained unlinked in linkage studies were selected for genome wide scan. A novel locus for autosomal recessive primary congenital glaucoma was mapped to chromosome 1p33-32.3 in one family; PKGL061 with a maximum two point LOD score of 5.33 obtained with marker D1S386 at recombination fraction zero. This study reports identification of five novel and eight known pathogenic mutations in already reported genes; CYP1B1 and LTBP2. Furthermore, a novel disease locus at chromosome 1p33-32.3 in a large consanguineous PCG family was identified. These findings provide insight into genetic and molecular determinants responsible for autosomal recessive primary congenital glaucoma, thus providing a better understanding of mechanisms underlying the disease.