Molecular genetic screening and association studies of adult-onset primary open angle and congenital glaucomas

Glaucoma is a group of disorders with a broad range of clinical and histopathological manifestations. This condition presents itself in many different ways and with distinct shared characteristics that include optic neuropathy due to optic nerve head damage (cupping) and visual field dysfunction with or without presence of increased intraocular pressure (lOP). Glaucoma is the leading cause of irreversible blindness, which afflicts nearly 67 million people worldwide. It is considered the second most frequent cause of bilateral blindness, affecting 6.7 million people worldwide. By 2020, glaucoma is anticipated to affect 79.6 million people, increasing the number of bilaterally blind individuals due to glaucoma to 11.1 million. This thesis aimed to provide new insights into the molecular genetics of adult-onset Primary Open Angle Glaucoma (POAG) and a paediatric form of Primary Congenital Glaucoma (PCG). In the first portion of this work, the GLC1B locus on chromosome 2pl1.2-qI2.2 was investigated using extensive linkage and saturation mapping in order to reduce the region and to select potential candidate genes for screening 9 previously linked families. Genomic Convergence and Proteomic Streamlining methods were used to select and prioritise the most likely candidate genes. The prioritisation was based on tissue expression, bioinformatics, microarray data, as well as assessment of their protein- encoded functions. This investigation leads to the hypothesis that there may actually be two areas of interest for the GLCl B locus, one on the p arm and the other on the q arm of chromosome 2. Since at least three other independent studies shared a common overlapping area on the q arm of chromosome 2, single nucleotide polymorphisms (SNPs) from each of the genes within this common region were selected for a regional gene-specific association study. After genotyping our linked-GLC1B POAG families, sporadic cases and other matched control subjects, the genes that showed strong association were selected and screened for mutations. After direct sequencing of these genes, 62 DNA alterations (known and novel intronic and exonic variants) were observed and their disease-causing nature was carefully investigated. In conclusion, the GLC1B locus has been significantly reduced to a region of approximately 6.66-Mb. A total of 10 candidate genes were selected, individually sequenced and ultimately excluded as being involved in the aetiology of a group of GLC1B-linked POAG families. Further work is necessary to identify the defective gene(s) at the GLC1B locus. In the second portion of this thesis, the GLC3C locus on chromosome 14q24.3- q31.1, the study aimed to prioritise genes using similar methods to the strategies previously used to identify candidate genes within the GLC1B locus. These included Genomic Convergence, Proteomic Streamlining and the information from other linked families within the region. A total of 7 regional candidate genes were selected, sequenced and excluded as being involved in the aetiology of our GLC3C-linked family. Altogether, 80 DNA alterations (known and novel intronic and exonic variants) were observed in the sequenced genes and their disease-causing nature was carefully investigated. In addition, to the screening of the above-mentioned GLC3C candidate genes, a newly described PCG-causing gene, LTBP2, was fully sequenced and excluded in our GLC3C-linked family. Additionally, a total of 94 familial and sporadic PCG and 96 matched-control subjects were fully sequenced for the entire 36-coding exons of the LTBP2 gene. Only two heterozygous DNA alterations were identified in 2 PCG individuals, which were absent in 96 normal control subjects. Since no homozygous mutations were observed, further investigation is required to determine the disease- causing nature of these two DNA alterations. Therefore, this study was unable to replicate previously described mutations in the PCG subjects from Pakistan and Iran. Likewise, at least one other study of American PCG cases did not find any gene mutations in the LTBP2 gene, thus further confirming the observation of this study. As this gene is implicated in diseases with multiple clinical presentations, further investigation is necessary to determine the overall role that LTBP2 plays in the isolated PCG phenotype as well as in identifying the causative gene(s) within the GLC3C locus.EThOS - Electronic Theses Online ServiceGBUnited Kingdo