Abstract
Purpose :
Primary open angle glaucoma (POAG) is a genetic disease with known variants in many different genes, some causal and some conferring risk only. Traditional annotation methodology prioritises non-synonymous over synonymous variants, potentially overlooking the influence variants may have upon splicing. This study interrogates the human myocilin (MYOC) gene for splice altering variants, in search of missing POAG heritability.
Methods :
358 POAG patients were selected from a previously described UK cohort by disease phenotype (intraocular pressure = 21-40 mm Hg, cup:disc ratio ≥ 0.6 and visual field mean deviation ≤ -3) and Caucasian ethnicity.
Targeted sequencing of the entire myocilin (MYOC) gene was performed using Nextera rapid capture custom design service (Illumina) on a NextSeq 500. Raw NGS data were processed through a bioinformatics pipeline, producing a list of variants in a variant call format (VCF) file. Splice altering variants (SAVs) were further annotated using publicly available online tools MutPred Splice and Human Splicing Finder (HSF).
Results :
In silico analysis of the MYOC gene revealed 12 exonic variants including one potential splice altering variant (SAV) with a significant MutPred Splice score of 0.605. This SAV was observed in a single POAG patient of the 358 cohort tested. Of 161 intronic variants in MYOC, 30 were predicted to have potential to alter splicing, 26 within intron 1, and 4 within intron 2. Only one of these variants has previously been proposed to alter splicing in POAG [c.604+667C>T (rs537883267) in MYOC intron 1].
Conclusions :
The HSF tool predicts a mechanism whereby exonic splice altering variant c.376C>T could introduce a new 5’ donor splice site within MYOC exon 1 at base 372, truncating the protein coding sequence by at least 232bp (~77 AA residues). Conventional annotation software would usually report this variant as a rare non-synonymous variant (R126W), yet aberrant splicing would skip this part of MYOC exon 1 entirely.
HSF predicts intronic MYOC variants in this cohort are capable of creating up to 29 new acceptor and donor splice sites and may also break a wild-type branch point in intron 2. This project thus informs future studies, with the potential to investigate new treatment possibilities for POAG such as the aminoglycosides and ataluren drugs – currently used to correct exon skipping aberrations in degenerative disease.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.