Whole Exome Sequencing Revealed Three Novel Variants in TSPAN12 and LRP5 Genes for Two Families with Familial Exudative Vitreoretinopathy

Background: Familial exudative vitreoretinopathy (FEVR) is a complex form of blindness-causing retinal degeneration. This study investigated the potential disease-causing variant and molecular basis in two Chinese families with FEVR. Methods: All family members underwent detailed ophthalmological examinations, including best-corrected visual acuity, fundus examination, and uorescein fundus angiography. Whole exome sequencing, bioinformatics analysis, and Sanger sequencing of available family members were used to conrm the disease-causing gene variant. Results: The proband F1-II:1, his mother, and proband F2-II:1 were diagnosed with FEVR based on clinical symptoms, fundus manifestation, and fundus uorescein angiography. Whole exome sequencing showed that the proband F1-II:1 had a novel heterozygous variant in the TSPAN12 gene, c.236T>G p. (Met79Arg), which may disturb the transmembrane domain of the TSPAN12 protein. The variant was cosegregated with the phenotypes of FEVR in the family. The proband F2-II:1 carried novel compound heterozygous variants, c.1210G>A p. (Gly404Arg) and c.1612C>T p. (Arg538Trp), in the LRP5 gene, which may disturb the second β-propeller motif of the LRP5 protein. These three variants were classied as likely pathogenic variants. The variants were predicted to be damaging or deleterious using multiple lines of prediction algorithms; they were not frequently found in multiple population databases. The residues affected by these variants are highly conserved among different species. The three novel variants may be potential disease-causing variants in the two families. Conclusions: These results expand the spectrum of variants in TSPAN12 and LRP5 genes and enrich the understanding of the molecular etiology of FEVR. In conclusion, through whole exome sequencing and bioinformatics analysis, we identied a variant in the TSPAN12 gene and compound variants in the LRP5 gene in two families with FEVR. To our knowledge, this is the rst report regarding 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, as potential disease-causing variants for FEVR. These results expand the spectra of variants in the TSPAN12 and LRP5 genes; they also enrich the understanding of the molecular etiology of FEVR. We presume that these ndings will provide insights regarding accurate diagnosis, family genetic counseling, and future gene therapy for FEVR.


Background
Familial exudative vitreoretinopathy (FEVR, OMIM: 133780) is a clinically and genetically heterogeneous inherited ophthalmic disorder (1,2). It is characterized by incomplete retinal vascular development and pathological neovascularization (3). Patients usually complain of reduced visual acuity or blindness in early childhood. The fundus can exhibit peripheral retinal avascularization, falciform retinal folds, macular ectopia, retinal exudate, retinal neovascularization, and retinal detachment (4). However, some patients may not complain of any visual impairment; they may only exhibit peripheral avascularization (5). The reported prevalence is approximately 0.11% in newborns (6), but, the actual prevalence may be underestimated because some patients are asymptomatic and demonstrate peripheral retinal involvement only (7).
FEVR can be inherited in autosomal dominant, autosomal recessive, or X-linked manners; the most common mode of inheritance is autosomal dominant (8).
Moreover, one locus, EVR3, which maps to 11p13-p12, can also lead to FEVR; its causative gene has not been fully identi ed (20). Among these pathogenic genes, FZD4, LRP5, and TSPAN12 are the most common disease-causing genes related to FEVR (21). The rst ve genes are involved in the Norrin or Wnt/βcatenin signaling pathway and have functions in cell adhesion, migration, and signaling (22).
Although increasing numbers of gene variants have been identi ed using next generation sequencing technology, these reported gene variants are responsible for only 50-60% of FEVR cases. Moreover, some patients may exhibit rapid progression without correct diagnosis and intervention. Thus, it is imperative to ascertain genetic etiology and achieve accurate diagnosis for affected patients, especially patients who are asymptomatic and exhibit peripheral retinal involvement alone. In this study, two families were diagnosed with FEVR based on clinical manifestations. We performed whole exome sequencing of probands and Sanger sequencing of available family members to elaborate the underling disease-causing gene variant.

Participants
The study was authorized by the medical ethics committee of Henan Provincial People's Hospital and conformed to the tenets of the Declaration of Helsinki. All participants were consecutively recruited in our hospital. Before all examinations, informed consent was obtained from each participant (or parents/guardians of participants ≤ 18 years of age).

Clinical examinations
Detailed premature delivery history, oxygen uptake history, family history, and birth weight information were acquired for the probands. Exhaustive ophthalmological examinations were completed, including best-corrected visual acuity, intraocular pressure, slit-lamp microscopy, ophthalmoscopy, fundus photography, and fundus uorescein angiography (FFA). All participants underwent pupillary dilation with a mixture of 0.5% phenylephrine hydrochloride and 0.5% tropicamide eye drops (Santen Pharmaceutical, Osaka, Japan Samples were sequenced on a HiSeq platform (Illumina, San Diego, California, United States) using a whole exome sequencing protocol, in accordance with the manufacturer's instructions. Sequence data were analyzed for corresponding ophthalmologic inherited genes, especially inherited retinal disease genes; sequences were aligned using Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/   Figure 1E). Both of the proband's parents did not complain of any symptoms, and they exhibited normal FFA appearance.
Both probands had no history of premature delivery, problems with oxygen uptake, or low birth weight; neither proband had clinically signi cant family medical history. All participants had no systematic complaints or extraophthalmic abnormalities that could be identi ed through conventional examinations (e.g., short stature or microcephaly). The ophthalmic features of all participants are summarized in Table 1. Based on clinical manifestations, proband F1-II:1, his mother, and proband F2-II:1 were diagnosed with FEVR.  Figure 2A. The variant cosegregated with the FEVR phenotypes in the family. The pedigree of the family is presented in Figure 2B. We concluded that the variant was inherited in an autosomal dominant manner. The variant information is summarized in Table 3. In particular, the variant was located in exon 4 of the TSPAN12 gene; this was predicted to affect the transmembrane domain of the TSPAN12 protein. Schematic representations of the genomic and protein structures are shown in Figure 2C and 2D.
The mean sequencing depth of proband F2-II:1 was 155.29, with 30X coverage over 96.77% of the target region. In total, 82.98 million reads mapped to the human reference genome and 86,987 variants were called. Sequencing information regarding the proband is summarized in Table 2. Whole exome sequencing and Sanger sequencing showed that the proband carried novel compound heterozygous variants, c.1210G>A p. (Gly404Arg) and c.1612C>T p.
(Arg538Trp), in the LRP5 gene. Sanger sequencing chromatographs of the proband are shown in Figure 2A. The pedigree of the family is presented in Figure  2B. Information regarding the variants is summarized in Table 3. In particular, the variants were located in exons 6 and 8 of the LRP5 gene, respectively; both residues affected by the variants were located in the second β-propeller motif of the LRP5 protein. Schematic representations of the genomic and protein structures are shown in Figure 2C and 2D.  Table 3 Information regarding variants in TSPAN12 and LRP5 genes detected in this study These three variants were predicted to be damaging or deleterious, using multiple lines of prediction algorithms; they were not frequently found in ethnically matched populations in multiple population databases. Predictive functional effects and population distribution frequencies are summarized in Table 4. (The table is too wide for A4 or Letter landscape page, it was uploaded as an additional le). The variants were classi ed as likely pathogenic, based on ACMG guidelines. Evolutionary conservation alignment showed that the variants were highly conserved among different species ( Figure 2E). Based on Sanger sequencing and bioinformatics analysis, we inferred that c.236T>G p. (Met79Arg) in the TSPAN12 gene was the potential disease-causing variant in family 1; compound variants, c.1210G>A p. (Gly404Arg) and c.1612C>T p. (Arg538Trp), in the LRP5 gene were potential disease-causing variants in family 2. 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 ndings 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.

Gene
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 rst, 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 rst 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 rst 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 rst 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.

Conclusion
In conclusion, through whole exome sequencing and bioinformatics analysis, we identi ed a variant in the TSPAN12 gene and compound variants in the LRP5 gene in two families with FEVR. To our knowledge, this is the rst report regarding 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, as potential disease-causing variants for FEVR. These results expand the spectra of variants in the TSPAN12 and LRP5 genes; they also enrich the understanding of the molecular etiology of FEVR. We presume that these ndings will provide insights regarding accurate diagnosis, family genetic counseling, and future gene therapy for FEVR.

Supplementary Files
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