Deep Intronic Sequence Variants inCOL2A1Affect the Alternative Splicing Efficiency of Exon 2, and May Confer a Risk for Rhegmatogenous Retinal Detachment

Abstract

(2016). Deep intronic sequence variants in COL2A1 affect the alternative splicing efficiency of Exon 2, and may confer a risk for rhegmatogenous retinal detachment. Human Mutation, 37(10), 1085-1096. DOI: 10.1002/humu.23050 General rightsCopyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from Discovery Research Portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain.• You may freely distribute the URL identifying the publication in the public portal. Take down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Stickler syndrome which has a high risk of retinal detachment and failure of the vitreous to develop normally. Exon 2 of COL2A1 is alternatively spliced, expressed in the eye but not in mature cartilage and encodes a region that binds growth factors TGFβ1 and BMP-2. We investigated how both a mutation in intron 2 and a normal variant allele altered the efficiency of COL2A1 exon 2 splicing and how the latter may act as a predisposing risk factor for the occurrence of rhegmatogenous retinal detachment (RRD) in the general population. Using both amplification of illegitimate transcripts and allele specific minigenes expressed in cultured cells, we demonstrate variability in exon 2 inclusion not only between different control individuals, but also between different COL2A1 alleles. We also identify possible trans-acting factors that bind to allele specific RNA sequences, and investigate the effect of both knockdown and over expression of these factors on exon 2 splicing efficiency. Finally, using a specific cohort of patients with RRD and a control population, we demonstrate a significant difference in the frequency of the COL2A1 intronic variant rs1635532 between the two groups.

Before progressing from “exomes” to “genomes”… don’t forget splicing variants

“…Splice site prediction program analysis of each variant was performed using Alamut ( http://www.interactive-biosoftware.com/alamut-visual/ ), which incorporates five different programs all of which have been developed for U2 splice sites rather than U12 splice sites (see Table 1 ). Whilst the three variants were not flagged, we considered they could alter splice site function due to their proximity to known splice sites, and assessed each by a minigene splicing assay [ 5 ], as transcriptomic sequencing was not possible (see Supplement for methodology).…”

Mutation Spectrum and De Novo Mutation Analysis in Stickler Syndrome Patients with High Myopia or Retinal Detachment

“…De novo mutations in Stickler syndrome have only been reported in a few cases [29]. The proportion of cases caused by de novo pathogenic variants is unknown.…”

A novel deep intronic COL2A1 mutation in a family with early‐onset high myopia/ocular‐only Stickler syndrome

“…Moving from exome sequencing to whole genome sequencing may be of great help in solving some of such mystery in the era of genome medicine, if the cost of whole genome sequencing goes down continuously. However, the pathogenicity of deep intronic variants needs to be validated by RT‐PCR on illegitimate RNA transcription of COL2A1 in patients' lymphoblasts or fibroblasts 48 . Unfortunately, in the current study indirect mini gene construction was used to test the effect of the variant for splice because the patients' lymphoblasts or fibroblasts were not available.…”