Silva, Raquel Sofia; (2020) Dissecting the molecular mechanism of developmental macular dystrophies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

North Carolina macular dystrophy (NCMD) and Progressive bifocal chorioretinal atrophy (PBCRA) are a rare set of dominantly inherited disorders that affect central vision from birth. Two linked loci had been previously identified at 5p21 and 6q16. Whole-genome sequencing (WGS) analysis of our cohort of 92 affected individuals has identified 3 novel causative structural variants (SVs) on 5p21 in 13 NCMD families, and 2 novel noncoding single nucleotide variants on 6q16 upstream of the promoter of PRDM13 in three PBCRA families. Combined, the rearrangements in 5p21 have a shared duplicated region of 39kb, located in a gene desert downstream of IRX1 and upstream of ADAMTS16. DNAse-seq data publicly available for human fetal retina identified active open chromatin sites at both loci at restricted times during retinal development. To dissect the molecular mechanism of the identified variants, skin-derived fibroblasts lines from selected patients were established; CRISPR-CAS9 technology was used to recreate the patient variants in a mouse model; and candidate genes expression profile was assessed in three stages of human fetal retina tissue. Additionally, chromosome conformation capture technology was performed in patient fibroblasts and in mouse developing tissue to characterise the genomic landscape of the locus and assess the effect of structural variants. Genome-wide analysis of chromosome occupancy was also performed for the architectural protein CTCF and for histone modifications relevant for identification of cis-acting elements. Expression studies showed altered expression of IRX genes and ADAMTS16 in mutant lines. Together with evidence from human retinal tissue immunohistochemistry and in situ expression data, these genes are suggested to be miss-expressed and/or ectopically expressed during macular development, with involvement of PRDM13, FGF8 and FGF10, which were also found miss-expressed in mutant lines . This work provides novel insight into the gene regulation landscape involved in human macular development and prognostic information for affected families.

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