Next-Generation Sequencing of a 40 Mb Linkage Interval Reveals TSPAN12 Mutations in Patients with Familial Exudative Vitreoretinopathy

Abstract

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

“…Analyses of the Tspan12-deficient mouse did not address a potential role in ADAM10 regulation but instead demonstrated a role for Tspan12 in the regulation of Frizzled-4-induced ␤-catenin signaling (16). Tspan12 deficiency leads to defective vascular development in the retina, and Tspan12 mutations cause the human blinding disease familial exudative vitreoretinopathy, thus phenocopying mutations in the Frizzled-4 signaling pathway (17,18). To address whether Tspan12 could regulate ADAM10 in a manner comparable with that of TspanC8 tetraspanins, Tspan12 was compared with Tspan14 for its capacity to interact with and promote ADAM10 maturation.…”

Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP

“…At least 40% of patients with FEVR do not carry mutations in FZD4 or LRP5 [Boonstra et al, 2009;Qin et al, 2005;Toomes et al, 2004a], so we are pursuing the identification of additional FEVR genes. Recently, in a cohort of eleven Dutch families that were tested negative for mutations in the ORF of FZD4, LRP5, and NDP, respectively; we identified TSPAN12 as a gene causative for autosomal dominant FEVR [Nikopoulos et al, 2010]. The gene was mutated in 5 out of the 11 Dutch families tested, rendering it a relative frequent cause of FEVR.…”

Genes, mutations, and human inherited disease at the dawn of the age of personalized genomics

“…Thus, any reliable estimate of the number of different functionally significant mutations yet to be identified in the extant human population is likely to remain a guessing game for the foreseeable future. What is clear, is that with the advent not only of massively parallel sequencing of the human exome [Choi et [Kryukov et al, 2009;Nikopoulos et al, 2010;Prabhu and Pe'er, 2009], but also of the successful application of direct RNA sequencing of the human transcriptome [Ozsolak et al, 2009;Wang et al, 2009b] and whole-genome sequencing Roach et al, 2010], the identification of inherited pathological mutations is entering a new era. This will be an era in which, for each patient, many genomic variants ''will be called but few will be chosen.''…”