In this study, we enrolled two probands with reduced vision who were diagnosed with FEVR based on clinical symptoms, as well as fundus and FFA examinations. We performed whole exome sequencing, Sanger sequencing validation, cosegregation analysis, functional prediction, population distribution analysis, and evolutionary conservation alignment. Our results suggested that novel variants—c.236T>G p. (Met79Arg) in the TSPAN12 gene, as well as c.1210G>A p. (Gly404Arg) and c.1612C>T p. (Arg538Trp) in the LRP5 gene—were potential disease-causing variants in two probands. Except the c.1210G>A p. (Gly404Arg) variant was reported to cause osteoporosis-pseudoglioma syndrome in another patient (27), the other two variants have not been associated with any disease.
Clinical symptoms and fundus appearances can vary distinctly among patients and genetic backgrounds in patients with FEVR; in some instances, disease presentation can vary between eyes in a single patient (28). The proband F1-II:1 exhibited different appearances between eyes, such that the right eye demonstrated normal vision and mild fundus abnormality, while the left eye demonstrated mild reduced vision and moderate fundus abnormality. Moreover, the progress of disease was asynchronous between eyes: the right eye showed minimal progression, while the left eye showed progression with falciform retinal folds and peripheral retinal exudates at the 2-year follow-up. Distinct fundus findings were also present in proband F2-II:1, such that the right eye showed severe retinal detachment, while the left eye showed mild abnormality. Although the two probands has similar disease courses and were of similar age, their symptom severities and fundus appearances were different.
NDP, FZD4, LRP5, and TSPAN12 gene variants can impair the Norrin or Wnt/β-catenin signaling pathways, which are responsible for angiopoiesis during retinal development (29). In the canonical Wnt/β-catenin pathway, FZD4 and LRP5 form a ternary complex as a coreceptor; Wnt binds to the coreceptor and activates downstream β-catenin signaling (30). In the Norrin/β-catenin pathway, NDP binds to the coreceptor and activates downstream β-catenin signaling with the TSPAN12 auxiliary component (31). When these signaling pathways are activated, β-catenin translocates to the nucleus and interacts with the T-cell factor/lymphoid enhancing factor family of transcription factors, thus initiating RNA transcription and elongation (32, 33).
The TSPAN12 gene encodes the TSPAN12 protein, which contains four-pass transmembrane domains and four cysteines in the second extracellular region, forming two extracellular loops and an intracellular loop. Xiao et al. reported that variants in the second extracellular loop comprised 38% of 40 causative variants (34). Variants in transmembrane domains and extracellular regions can severely impair function, variants in the C-terminal end can moderately impair function, and variants in the N-terminal end can slightly impair function. The transmembrane domains provide a scaffold for extracellular loops to change conformation and interact with FZD4 for allosteric modulation. The variant c.236T>G p. (Met79Arg) is located in the transmembrane domain and may disrupt the domain structure of the TSPAN12 protein, potentially preventing TSPAN12 incorporation into the receptor complex and destabilizing the NDP/FZD4/LRP5 interaction (35).
The LRP5 gene encodes the LRP5 protein, which contains a putative signal peptide, four β-propeller motifs at the amino terminal that alternate with four epidermal growth factor-like repeats, three low-density lipoprotein receptor-like repeats, a single transmembrane domain, and a cytoplasmic domain (36). Xiao et al. reported that variants in the first, second, and third β-propeller epidermal growth factor domains comprised 12%, 38%, and 17% of 58 causative variants, respectively (34). Although the exact functions of these domains are unknown thus far, studies of LRP6 (with strong homology and similar function to LRP5) showed that the first and second β-propeller motifs, the third and fourth β-propeller motifs formed tandems to function respectively (37). Variants located in the second β-propeller motif may destroy the stable structure of first two β-propellers and interrupt their interactions with NDP or FZD4. The c.1210G>A p. (Gly404Arg) and c.1612C>T p. (Arg538Trp) variants are located in the second β-propeller motif of the LRP5 protein; therefore, these two variants may impair the second motif and cause the first and second β-propeller motifs tandems to become inactive.
Although we found three novel disease-causing variants in two FEVR families, there were some limitations in this study. First, we only speculated that variants were potential disease-causing based on clinical manifestations, whole exome sequencing, and bioinformatics analysis. Second, the parents of proband F2-II:1 did not undergo complete Sanger sequencing because peripheral blood samples were unavailable. We plan to validate the pathogenicity of the three variants by in vivo and in vitro analyses, and we will attempt to complete Sanger sequencing of available family members in a future study.