Identification of novel disease-causing genes in inherited retinal dystrophy
Keywords: macular dystrophy
whole-exome sequencing
PARPBP
next generation sequencing
MTSS1
MYO5B
inherited retinal dystrophies
PIEZO1
disease-gene identification
retinitis pigmentosa
gene
gene expression

Over 270 genes underlying monogenic forms of inherited retinal dystrophies (IRDs) have been identified to date, enabling the development of new treatment regimens such as gene therapy. However, there are still IRD cases with underlying genetic causes yet to be identified, prompting the need for continued disease-gene identification studies. In this thesis, individuals from four families with dominant IRDs without a known genetic cause underwent whole-exome sequencing (WES). Rare pathogenic variants identified in each family were prioritised for segregation analysis using Sanger sequencing. Variants segregating with the condition in each family were considered as candidate genes and were further characterised for expression, localisation and potential function in the mammalian retina using techniques such as polymerase chain reaction (PCR), western immunoblotting (WB), immunohistochemistry (IHC), site-directed mutagenesis and gene transfection.
Together, four genes, PARPBP, MYO5B, PIEZO1 and MTSS1, with rare variants were identified in three families with dominant RP (adRP). The expression and function of these candidate genes in the mammalian retina have not been previously described. We provide experimental evidence of the expression of PARPBP, PIEZO1, MYO5B and MTSS1 in the human retina. We determined the sites of Parpbp and Mtss1 expression in the mouse retina using antibodies specific for human PARPBP and MTSS1. Parpbp localised to cells in the inner nuclear layer and ganglion cell layer of the mouse retina. Mtss1 localisation was identified in the inner and outer segment of rod photoreceptors, the synaptic layers and ganglion cells of the mouse retina. We also showed that MTSS1 colocalises with F-actin in the photoreceptor inner and outer segments and in the synaptic layers of the retina. By transfecting several in vitro models with wild-type and mutant constructs of MTSS1, we show that the MTSS1 variant identified in one of the families, alters the expression and function of MTSS1 at the cellular level including altered subcellular localisation and ability to induce extensive cellular processes. Retinal tissue from a previously generated Mtss1 knockout mouse model was examined for potential abnormalities in the eye that may be attributable to the lack of Mtss1 expression. However, findings were inconclusive due to extensive folding of the retinas examined and the identification of residual Mtss1 expression in the retina of these knockout mice.
Overall, the findings in this thesis demonstrate that combining modern gene sequencing technologies, such as next generation sequencing, with other molecular biology techniques enables the identification of candidate genes for IRDs. For the first time, this thesis describes the expression of MTSS1 in the retina and provides compelling evidence linking MTSS1 to IRD. This thesis also provides a list of other candidate genes for IRDs worth investigating further.