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.

Figure 1

Fundus Photograph Showing Part of the Avascular Periphery of Patient IV:2 from Family A Temporal periphery of the left fundus in patient IV:2 from family A (numbering according to the pedigree presented in Figure 2A). The retinal vasculature (at the left side) ends in small aberrant ramifications in the equatorial area of the fundus. The peripheral retina (at the right side) is avascular; only choroidal vessels are present in this area.

Figure 2

Pedigree Structure and Genome-wide Linkage Analysis for FEVR Families A and B (A) Pedigree structure of those parts of families A and B that were selected for Illumina 6K SNP genotyping analysis. Individuals of whom the clinical status is uncertain are depicted in gray. Probands are indicated with an arrow. Eight members of family A and 15 individuals of family B, indicated with asterisks, were genotyped. (B) Genome-wide LOD score calculations, with the use of the 6K SNP array genotyping data. In family A, two peaks are visible, on chromosomes 7 and 8, respectively. In family B, only one clear peak is visible, on chromosome 7, with a genome-wide-significant LOD score of 3.31. Interestingly, the two peaks on chromosome 7 in families A and B, indicated by arrows, represent the same genomic region.

Figure 3

Genomic Structure, Next-Generation Sequencing, and Mutation Analysis of TSPAN12 (A) Upper panel, part of chromosome 7 showing the linkage intervals and the corresponding flanking SNPs for FEVR families A and B. The linkage interval from family A was enriched for targeted next-generation sequencing technology. The TSPAN12 gene is located in the shared interval between families A and B. Lower panel, overview of the genomic structure of the TSPAN12 gene, showing all introns and exons. For all exons, as well as a highly conserved region between exons 7 and 8, the sequence coverage histograms are presented. (B) Overview of all patient reads for exon 8 of TSPAN12. In total, more than 30 reads were representing parts of this exon. (C) Sequence traces for the twenty unique reads covering the TSPAN12 heterozygous G-to-C mutation. For these 20 reads, ten were representing the reference allele and ten were showing the mutant allele. At the protein level, the G-to-C transition results in the substitution of a proline for an alanine residue (c.709G>C [p.Ala237Pro]). (D) Topology of TSPAN12 and the positions of the missense mutions identified in the FEVR families. TSPAN12 shows the characteristic domain structure of tetraspanins, including four transmembrane (TM) domains (in green). A cysteine residue preceding TM domain 1 (C9) and another following TM domain 2 (C83) are probably palmitoylated (bright green wavy lines). The second extracellular loop (EC2) contains three α helices (boxes), the typical C-C-G motif characteristic for all tetraspanin proteins, followed by one pair and two single cysteine residues. These six cysteines are predicted to form disulphide bonds (stippled lines). The glycine residue that is mutated into an arginine (G188) precedes one of these cysteines. The alanine residue that is mutated into a proline (A237) resides within an α-helical structure within the fourth TM region. (E) Sequence comparison of a number of vertebrate TSPAN12 proteins. Depicted are parts of TSPAN12 that harbor the amino acid residues that are mutated. Both the glycine residue at position 188 (upper panel) and the alanine residue at position 237 (lower panel) are completely conserved throughout vertebrate evolution. Residues identical in all sequences are black on a white background, whereas similar amino acids are black on a light gray background. Nonconservative changes are indicated in white on a black background. UniProt accession number of the protein sequences used for sequence comparison are as follows: human TSPAN12, O95859; bovine TSPAN12, Q29RH7; mouse TSPAN12, Q8BKT6; chicken TSPAN12, Q5ZIF5; zebrafish TSPAN12, Q7T2G0.