Abstract

Purpose: Identification and functional characterisation of a novel candidate gene for autosomal dominant retinitis pigmentosa (adRP).

Methods: Five affected and five unaffected individuals of a Belgian adRP family in which known adRP loci were excluded, were enrolled. They underwent genome-wide (GW) linkage analysis (BeadChip, Illumina). Whole exome sequencing was carried out in two affected individuals (HiSeq, Illumina; CLC bio). Segregation analysis of variants was done using Sanger sequencing and testing of 300 controls by HRM (LightScanner). Targeted resequencing of SF3B2 was performed (Miseq, Illumina) in 472 unrelated adRP patients. SF3B2 expression was tested using a commercial cDNA panel. Localization studies were carried out in 661W mouse cells using commercial anti-SF3B2 antibodies. Sf3b2 knockdown in Xenopus was done using targeted injection of a splicing blocking morpholino (MO) (GeneTools).

Results: GW linkage analysis revealed two novel candidate loci with a maximum LOD score of 1.7. In the 11q13 region, a missense variant c.2417A>G p.(Tyr806Cys) was found in the SF3B2 gene encoding the splicing factor 3b, subunit 2. The Tyr residue is highly conserved, the Grantham distance between Tyr and Cys is 194, predictions suggest an effect on protein function. The change is predicted to disrupt a phosphorylation site. The variant co-segregates with adRP and is absent in 300 controls. No additional SF3B2 mutations were found in a large adRP cohort.<br /> Ubiquitous expression of SF3B2 was demonstrated in human tissues, including retina and RPE, and localization in perinuclear and nuclear areas was shown in 661W mouse cells. Targeted MO knockdown in Xenopus showed gross developmental anomalies affecting the retina. Rescue experiments are ongoing.

Conclusions: SF3B2 was identified as a novel candidate gene for adRP. SF3B2 is required for binding of the U2 small nuclear ribonucleoprotein (snRNP) to the branchpoint and is involved in early spliceosome assembly. Interestingly, protein-protein interactions have been identified between SF3B2, SNRNP200 and PRPF8, the latter being two proteins implicated in adRP. So far, of the seven known adRP genes involved in splicing, six encode components of the U4/U6-U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. Our study potentially involves other components of the spliceosome apart from the tri-snRNP complex in adRP.