Mutation of RCBTB1 in a severe syndromic retinal ciliopathy

Abstract

Purpose: To identify and functionally study a novel disease gene mutated in a Turkish consanguineous family with a severe retinal ciliopathy, characterized by retinitis pigmentosa, hypothyroidism, hypogonadism (amenorrhea), short stature, intellectual disability, facial dysmorphism.

Methods: Genome-wide SNP arrays were used for homozygosity mapping in three affected and one healthy sibling of one consanguineous family. Two affected individuals underwent whole exome sequencing (WES) (HiSeq2000, Illumina; CLC bio). Segregation analysis of variants was done using Sanger sequencing. RCBTB1 expression was tested using commercial human cDNAs. Localization studies were carried out in mouse retina using a commercial anti-RCBTB1 antibody (abcam). Rcbtb1 in situ hybridization was performed in zebrafish. Knockdown and RNA rescue experiments in zebrafish are ongoing.

Results: Homozygosity mapping revealed a single homozygous region on chromosome 13 (chr13: 41820714-53537171) shared by the three affected individuals of two branches of the family. WES identified a novel missense variant, c.973C>T p.(His325Tyr) (rs200826424, MAF 0.1%), in RCBTB1 (NM_018191.3). This variant was found to be homozygous in the three affected individuals and heterozygous in the healthy sibling and in the parents of the affected persons. Another family member with polydactyly but without retinal involvement was heterozygous for c.973C>T but no second RCBTB1 mutation was found, suggesting another condition. Strong conservation and different prediction tools point toward an effect of the variant on protein function. RCBTB1 expression was demonstrated in human retina, consistent with retinal expression in databases such as BioGPS - also showing thyroid expression - and with preliminary in situ hybridization findings in zebrafish. Immunostaining in mouse retina showed a ciliary staining in inner segments. Knockdown and RNA rescue experiments in zebrafish are ongoing.

Conclusions: By combining homozygosity mapping and WES, a missense variant was identified in the RCBTB1 gene encoding a regulator of chromosome condensation (RCC1) and BTB (POZ) domain containing protein 1, in a family with a retinal ciliopathy. Interestingly, RCC1-like domains are also present in NEK8 and RPGR, which are known ciliary proteins implicated in nephronophthisis and XLRP, respectively. Further functional characterization of RCBTB1 will provide more insights in its role in the pathogenesis of ciliopathies.