A Survey of Multigenic Protein-Altering Variant Frequency in Familial Exudative Vitreo-Retinopathy (FEVR) Patients by Targeted Sequencing of Seven FEVR-Linked Genes

Abstract

While Inherited Retinal Diseases (IRDs) are typically considered rare diseases, Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie Disease (ND) are more rare than retinitis pigmentosa. We wanted to determine if multigenic protein-altering variants are common in FEVR subjects within a set of FEVR-related genes. The potential occurrence of protein-altering variants in two different genes has been documented in a very small percentage of patients, but potential multigenic contributions to FEVR remain unclear. Genes involved in these orphan pediatric retinal diseases are not universally included in available IRD targeted-sequencing panels, and cost is also a factor limiting multigenic-sequence-based testing for these rare conditions. To provide an accurate solution at lower cost, we developed a targeted-sequencing protocol that includes seven genes involved in Familial Exudative Vitreo-Retinopathy (FEVR) and Norrie disease. Seventy-six DNA samples from persons refered to clinic with possible FEVR and some close relatives were sequenced using a novel Oakland-ERI orphan pediatric retinal disease panel (version 2) providing 900 times average read coverage. The seven genes involved in FEVR/ND were: NDP (ChrX), CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11). A total of 33 variants were found that alter protein sequence, with the following relative distribution: LRP5 13/33 (40%), FZD4 9/33 (27%), ZNF408 6/33 (18%), (KIF11 3/33 (9%), NDP 1/33 (3%), CTNNB1 1/33 (3%). Most protein-altering variants, 85%, were found in three genes: FZD4, LRP5, and ZNF408. Four previously known pathogenic variants were detected in five families and two unrelated individuals. Two novel, likely pathogenic variants were detected in one family (FZD4: Cys450ter), and a likely pathogenic frame shift termination variant was detected in one unrelated individual (LRP5: Ala919CysfsTer67). The average number of genes with protein-altering variants was greater in subjects with confirmed FEVR (1.46, n = 30) compared to subjects confirmed unaffected by FEVR (0.95, n = 20), (p = 0.009). Thirty-four percent of persons sequenced had digenic and trigenic protein-altering variants within this set of FEVR genes, which was much greater than expected in the general population (3.6%), as derived from GnomAD data. While the potential contributions to FEVR are not known for most of the variants in a multigenic context, the high multigenic frequency suggests that potential multigenic contributions to FEVR severity warrant future investigation. The targeted-sequencing format developed will support such exploration by reducing the testing cost to $250 (US) for seven genes and facilitating greater access to genetic testing for families with this very rare inherited retinal disease.

Kinesin-5 Eg5 is essential for spindle assembly, chromosome stability and organogenesis in development

“…The Eg5-associated disease represents a potential system ciliopathy with defects in photoreceptor development and retinal angiogenesis [ 22 , 46 ]. The Eg5 vascular endothelial cell-specific knockout mouse models suggest that the Eg5 -deficient retinal vascular phenotype is independent of the established Norrin Wnt β-catenin signaling pathway [ 54 , 55 ]. In this study, we have demonstrated that Eg5 inhibition results in cell apoptosis in the eye, which further contributes to the disorganization of retinal cells and cell layers.…”

Mechanisms Underlying Rare Inherited Pediatric Retinal Vascular Diseases: FEVR, Norrie Disease, Persistent Fetal Vascular Syndrome

“…One such recent example was reported by our group recently, Cys450Ter, a nonsense variant that generates an early termination codon within TM helix-6. This was predicted to result in the loss of TM helix-7 and the C-terminal PDZ-binding domain of the FZD4 protein [17].…”

“…Norrie disease, Coats disease, retinopathy of prematurity (ROP), and familial exudative vitreoretinopathy (FEVR) belong to a family of rare retinopathies that are characterized by irregular vascularization or even lack of vascularization of the retina [16]. Our group has contributed to continuing efforts to identify variants in several genes that play a role in the pathogenesis of these diseases, which include NDP, FZD4, TSPAN12, and LRP5, members of the canonical Norrin Wnt-signaling pathway [17]. Recent studies have also uncovered additional genes, some that have no direct participation in the Norrin-signaling pathway.…”

“…The multiple allele knockout studies by Junge et al (2009) in mice suggested the possibility that more severe FEVR-like phenotypes could result from combinations of two or more different alleles that have a minimal impact alone [66]. Our group recently surveyed a cohort of FEVR patients and immediate relatives to confirm that the incidence of protein-altering variants in two or three different FEVR-linked genes was substantially greater than in the general population [17]. Thus, it is possible that combined mild-alleles might result in more severe phenotypes, but we have not yet described clear examples of this in FEVR.…”

Mechanisms Underlying Rare Inherited Pediatric Retinal Vascular Diseases: FEVR, Norrie Disease, Persistent Fetal Vascular Syndrome

“…Norrie disease, Coats disease, retinopathy of prematurity (ROP) and familial exudative vitreoretinopathy (FEVR) belong to a family of rare retinopathies that are characterized by irregular vascularization or even lack of vascularization of the retina [11]. Our group has contributed to continuing efforts to identify variants in several genes that play a role in the pathogenesis of these diseases, which include NDP, FZD4, TSPAN12, and LRP5, members of the canonical Norrin Wnt-signaling pathway [12]. Recent studies have also uncovered additional genes, some that have no direct participation in the Norrin-signaling pathway.…”