The molecular characterisation of foveal hypoplasia

Foveal hypoplasia (FH) is a phenotype observed in association with a series of rare and overlapping inherited retinal developmental diseases, which hinders diagnosis in patients. A recessive manifestation known as foveal hypoplasia 2 (FVH2),comprises of FH without pigmentary abnormalities, and is caused by pathogenic variants in SLC38A8.

This doctoral study began with genetic characterisation of SLC38A8 variants in FH, achieved by analysing an FH cohort from the European Retinal Disease Consortium, 100,000 genomes project (100KGP) and cases in the published literature. This expanded the known SLC38A8 mutation spectrum to 51 unique variants in 63 FVH2 affected probands. Haplotype analysis in an Indian, Pakistani and Bangladeshi patients harbouring the NP_001073911.1:p.(Tyr88*) variant confirms a founder mutation restricted to people of South Asian ancestry. Homology modelling and variant simulation suggest loss of function as the underlying disease mechanism in FVH2. Reviewing phenotypes in the 63 FVH2 affected probands confirms that the SLC38A8 phenotype constitutes FH and chiasmal misrouting, with limited evidence to support the presence of hypopigmentation in a few cases.

In a parallel study, whole genome sequencing in local patients and the 100KGP has identified 19 unique single nucleotide variants and 11 structural variants that cause FH. Amongst these are novel SVs affecting PAX6 and GPR143, which were redefined using Nanopore sequencing. The genomic diagnosis in 26 FH probands was attributed to PAX6 (5), GPR143 (5), OCA2 (4), SLC38A8 (4), TYR (2), CACNA1F (2), HPS5 (1), CNGA3 (1), CNGB3 (1) and ZNF408 (1). The diagnostic yield achieved from the analysis of patients in both the 100KGP and the local cohort with the clinical description of FH was 25% (14/56). The study also identified a founder SV in OCA2 that occurs at a high frequency in the African population. The haplotype in two African and an Afro-Caribbean patients with NC_000015.10: g.28017719_28020673del in OCA2 support the hypothesis.

Finally, autozygosity mapping and variant analysis in an unsolved FH patient highlighted LAMP1 as a candidate FH gene. Midigene splicing constructs were generated to assess the molecular consequence of LAMP1 variant NP_005552.3:p.(Gly370Alafs*14). These vectors were validated using a novel bioinformatic workflow developed for a cost effective, multiplexed and barcode free assembly of plasmids using Nanopore sequencing.