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Decrease of FZD4 exon 1 methylation in probands from FZD4-associated FEVR family of phenotypic heterogeneity

Familial exudative vitreoretinopathy (FEVR) is an important cause of childhood blindness and is clinically characterized by phenotypic heterogeneity. FEVR patients harboring the same genetic mutation vary widely in disease severity. The purpose of this study was to explore non-genetic factors that regulate FEVR phenotypic heterogeneity. We detected methylation levels of 21 CpG sites located at the FZD4 exon 1 region of 11 probands, 12 asymptomatic/paucisymptomatic carriers and 11 non-carriers from 10 unrelated FZD4-associated FEVR families using bisulfite amplicon sequencing (BSAS). Our results showed reduced methylation level of FZD4 exon 1 in probands, suggesting that FZD4 exon 1 methylation level may be negatively linked with FEVR disease severity. It provided a new research direction for follow-up research, helping us better understand the complexity of the FEVR-causing mechanism.


Introduction
Familial exudative vitreoretinopathy (FEVR) is a clinically and genetically heterogeneous ophthalmic disorder characterized by incomplete retinal vascular development. The disease was first described in 1969 by Criswick and Schepens (1), and it is a major cause of vision loss in juveniles. Several sight-threatening complications are considered secondary to retinal avascularity, including retinal neovascularization and exudates, retinal fold and detachments, vitreous hemorrhage, and macular ectopia, ultimately leading to total blindness. The clinical appearance of FEVR varies considerably. The mildly affected patients may have only a small avascular area at the peripheral retina, which is visible only by fundus fluorescein angiography (FFA), while severely affected patients often registered as blind during infancy. With extensive fundus screening, we now realize that the prevalence of FEVR is much higher than we thought before (2). Therefore, it is imperative to study the mechanism underlying FEVR in order to seek for advanced therapy.
Gene expression variability is one of the important causes for phenotypic variation. Epigenetic modification affects gene expression. One of the best understood epigenetic modification is DNA methylation. DNA methylation usually occurs in the cytosine guanine (CpG) dinucleotide. It affects gene expression by changing chromatin structure, DNA conformation, DNA stability and protein accessibility to DNA (25,26). CpG islands are regions in the genome where CpG levels are significantly higher than the average CpG levels of the whole gene. CpG islands usually are found in the promoter or exon 1 region. Although few studies addressed DNA methylation change in FEVR, methylation changes have been demonstrated in other ocular vitreoretinopathy diseases such as age-related macular degeneration (AMD), proliferative vitreoretinopathy (PVR), and diabetic retinopathy (DR) (27)(28)(29)(30). Therefore, we hypothesized that DNA methylation changes exist in FEVR causative genes, and such changes could affect gene expression and leading to FEVR phenotypic heterogeneity.
In this study, 10 separate FZD4-associated FEVR families were recruited to investigate the FZD4 methylation status of FEVR probands and their asymptomatic/paucisymptomatic family members carrying the same mutation. Our results showed reduced FZD4 exon 1 methylation in probands, suggesting that FZD4 exon 1 methylation level may be negatively linked with FEVR disease severity. To the best of our knowledge, this is the first paper that attempts to investigate the association between DNA methylation and heterogeneous presentation of FEVR.

Methods
Subjects 760 probands were recruited based on a clinical diagnosis of FEVR at the ophthalmology department of Xinhua hospital from May 2010 to August 2019. Next Generation Sequencing (NGS) revealed that 112 probands carried only FZD4 gene mutations, without other known FEVR-associated gene mutation. We further collected family history information of these 112 FZD4 Gene mutation probands. Finally, 10 FZD4-associated FEVR families met the following inclusion criteria were recruited. each enrolled family contained at least one FEVR proband (Staged 3-5), one asymptomatic/ paucisymptomatic carrier of the mutant allele (clinically healthy or staged 1) and one noncarrier. Stage 2 individuals were excluded to maximize the phenotypic heterogeneity feature in our study design. Clinical staging was based on Trese's 5-stage system (31) and the severity of patients was determined by the highest stage of FEVR in either eye. Subjects with histories of oxygen inhalation and preterm birth were also excluded. all enrolled participants were diagnosed by ophthalmologist with expertise in FEVR Based on clinical manifestations, genotyping, fundus examination and FFA. This study was approved by the Institutional Review Board of Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. All work was performed in accordance with the approved study protocol. written informed content was obtained from all the participants or their parents.

Analysis of FZD Rs single nucleotide polymorphisms
Cis-regulatory variants might also modify mutation penetrance and contribute to clinical heterogeneity (32). FZD4 rs10128621 SNP is the only known SNP which could affect FZD4 gene expression (33). We performed Sanger sequencing to analyze FZD4 rs10128621 SNP in our cohort. The primer sequences were as follows: 5'-ACCTTGGCTTCCTAAAGTGTTG-3' (forward primer) and 5'-CTGCCTGAGAGACAGAGTAAGA-3' (reverse primer). The expected amplicon is 297 bp in length.

Bisulfite amplicon sequencing for methylation analysis
DNA methylation was analyzed by Bisulfite amplicon sequencing (BSAS). About 5 mL of peripheral blood samples were collected from each participant in a standard EDTA tube. Genomic DNA was extracted and purified using QIAamp R DNA Mini kit. One hundred thirty microliter of CT Conversion Reagent was added to 300 ng purified DNA samples as per the manufacturer's instructions. This process converted unmethylated cytosines (C) in CpG sites to uracil (U), whereas methylated C remained C. The primer sequences were as follows: 5 ′ -TTTAGTTTGGGGTTGTTTTTGTAGT-3 ′ (forward primer) and 5 ′ -CTTTTCCAAAAAAACTATCTCCTTC-3 ′ (reverse primer). PCR library was constructed using VAHTS Turbo DNA Library Prep Kit for Illumina and sequenced on a Miseq system (Illumina). Quality control was performed using FastQC v 0.11.2. Mapping and calling of the methylation levels were performed using bismark software and methylKit (R package 4.1.0), respectively.

Statistical analysis
Independent Samples T-test was used to pairwise compare the methylation differences among probands, carriers and noncarriers. Significant differences were determined as p < 0.05. Data analysis was performed using Graphpad 9.0.

Characteristics of subjects
Thirty-four members of 10 FZD4-associated FEVR families were recruited in our study, including 11 FEVR probands, 12 asymptomatic/paucisymptomatic carriers of the same mutation, and 11 non-carriers of the mutant alleles. FEVR probands and family members were studied as a whole to best ensure similar genetic and environmental backgrounds. While FEVR probands had the most advanced ocular manifestations early in life, most of the family members carrying the same gene mutation were asymptomatic, only 4 carriers presented with Stage 1 FEVR. The mean age of probands, carriers and non-carriers were 35.1 ± 48.5 months (average ± standard deviation), 32.2 ± 12.2 years and 32.5 ± 14.8 years, respectively. Other than mutations in FZD4, no additional known FEVRassociated mutation was found in these subjects. Family No. 2, 5, 8 (chr11-86665923 c.205C>T) and family No. 7, 10 (chr11: 86665894-86665911 c.217_234del) shared the same mutation, respectively. The clinical details and genetic test results were summarized in Table 1. Fundus photographs and FFA of family No. 8 were presented in Figure 1.

Map of CpG island in the FZD exon region
We confirmed the methylation of CpG islands in FZD4 using a genomic map of FZD4 (NM_012193.4). FZD4 is located on the negative strand of chromosome 11. Most of the CpG island (CpG:69) exists in the exon 1 region. Twenty-one CpG sites were identified from a fragment in the exon 1 region at 86,665,722-86,666,073 ( Figure 2).

Methylation analysis of CpG sites
Before DNA methylation, we performed Sanger sequencing to analyze FZD4 rs10128621 SNP in our cohort. Sanger sequencing results showed a complete consistency of G base. Our results did not support the role of FZD4 rs10128621 SNP in the phenotypic heterogeneity of our FEVR patients. (Supplementary Figure S1) DNA methylation levels of 21 CpG sites identified above in probands and their family members were presented in Figure 3, except for the non-carrier of family No. 8, whose blood sample was not obtained due to data missing. At almost all tested CpG sites (19 out of 21), FEVR probands held the lowest average methylation level compared to carriers and non-carriers, except for CpG4 and CpG5, where probands had slightly higher methylation levels than carriers. At more than half of the CpG sites (12 out of 21), carriers held lower methylation levels than non-carriers. DNA methylation levels of probands were significantly lower than symptomatic/paucisymptomatic carriers in CpG14 (p = 0.0009), CpG15 (p = 0.0489) and CpG16 (p = 0.0320). DNA methylation levels of probands were significantly lower than non-carriers at CpG1 (p = 0.0009), CpG9 (p = 0.0371) and CpG13 (p = 0.0021). Overall, the results suggested probands had lower DNA methylation compared with carriers and non-carriers in FZD4 exon 1 region.
The methylation levels of these six significantly different CpG sites in each of the 10 FEVR families were shown in Figure 4. It showed that FEVR probands had lower methylation levels nearly in almost all cases. For example, at CpG14, probands in all 10 families had lower methylation levels. In addition, we noticed a negative correlation at CpG14 between methylation levels and disease severity within three families (No. 2, 5 and 8) that happened to carry the same mutation (chr11-86665923 c.205C>T). Proband from family No. 2 had the most advanced FEVR and the lowest methylation level at CpG14 compared to the rest. Similar negative correlations also existed for CpG15 and CpG16.   C>T mutation. (G,H) Fundus photographs of mother. The mother was a non-carrier without any fundus abnormality.

Discussion
FEVR is a rare disease featured by peripheral retinal vascular abnormalities (19, 34). It is clinically characterized by phenotypic heterogeneity, suggesting that non-genetic factors are involved in disease etiology (2,22,23). Comparing the epigenetic features among FEVR family members with or without mutation would provide important information regarding epigenetic factors which could contribute to phenotypic heterogeneity. DNA methylation is a widely studied epigenetic modification type. Increasing number of evidence supported the role of DNA methylation in modulating the phenotypic expression of diseases (35)(36)(37). For example, Coppede et al. found that the increase of global DNA methylation might contribute to disease manifestation in amyotrophic lateral sclerosis (ALS) carriers of not fully penetrant SOD1 mutations (38). DNA methylation at promoter CpG islands (CGI) of Wnt pathway genes showed a significant association with progression-free survival in ovarian tumor patients (39). Although little is known about FEVR disease, altered DNA methylation has been demonstrated in many other vitreoretinopathy diseases such as age-related macular degeneration (AMD), proliferative vitreoretinopathy (PVR), and diabetic retinopathy (DR). Therefore, we hypothesized that DNA methylation changes might play a role in FEVR phenotypic heterogeneity.
Up to date, at least 9 genes have been reported to cause FEVR. In previous studies, we and others showed that with FZD4 mutations accounting for the greatest proportion of FEVR in Chinese population (23, 40,41). The FZD4 gene encodes a 537-amino acid protein, which functions as a receptor of the Wnt signaling pathway (42). According to our previous work, more than half (51.43%) in the FZD4 group were identified asymmetry, suggesting FZD4 mutations might initiate the most diverse and asymmetric phenotypes (22). Previous study suggested epigenetic modifications might involve .

FIGURE
The graph showed methylation levels of tested CpG sites. The horizontal axis represented the position of CpG sites and the vertical axis represented percentage of methylation. Blue, orange and green points respectively indicated to FEVR probands, carriers and non-carriers. Box plot data represented range (error bars), median (horizontal line), quartile values (box) and outliers (dots). Median methylation levels of probands, carriers and non-carriers were compared in pair using student's t-test. DNA methylation levels of CpG , CpG and CpG were significantly di erent between probands and non-carriers. DNA methylation levels of CpG , CpG and CpG were significantly di erent between probands and carriers. *, p < . ; **, p < . ; ***, p < . . in the developing fetal retina which caused high asymmetric FZD4 gene expression (43). Therefore, it is important to study non-genetic factors underlying FZD4-associated FEVR phenotypic heterogeneity.
Bisulfite sequencing was first reported by Frommer et al. in (44). The bisulfite treated-DNA is amplified by PCR, and then the cloning and sequencing results of PCR products are used to determine whether the CpG site was methylated. This method has been recognized as one of the most powerful approaches to investigate DNA methylation patterns due to its reliable results, high accuracy and high sensitivity. It allows the determination of methylation at a single-cytosine resolution and has become the "gold standard" for DNA methylation detection (45,46).
The current study aimed to access evidence of a link between FEVR phenotypic heterogeneity and FZD4 methylation status. We focused on 10 unrelated clinically heterogeneous FEVR families carrying only FZD4 gene mutation. In this study, our goal was to investigate whether there was a difference in methylation levels between probands at severe FEVR stage and parents with mild phenotype. Stage 1 FEVR has only peripheral avascular zone seen by fluorescein fundus angiography. Patients with stage 1 FEVR may be stable for decades without any progression. Therefore, we grouped asymptomatic and stage 1 carriers together. Our data indicated a negative correlation between DNA methylation in FZD4 exon 1 region and FEVR severity, which requires further validation. Among the six significantly different CpG sites, four sites (CpG13, CpG14, CpG15 and CpG16) were adjacent, suggesting that this region might play an important and unique role in the regulation of FZD4 expression. Although the CpG9 of Family No. 9 and No.10 are obviously different, the probands still had the lowest average methylation level of the three groups and non-carriers had the highest average methylation level when these two families were eliminated. Our study suggests that changes in DNA methylation might contribute to the severity in FZD4-associated FEVR patients. The function of intragenic DNA methylation hasn't been well recognized. Brenet et al. found that DNA methylation at the first exon is tightly linked to transcriptional silencing (47), while others believed that the methylation in the body of genes can actually activate gene transcription (48,49). Although it remains controversial, given that altered methylation of a specific CpG site might be enough to change gene expression [50], FEVR probands with reduced DNA methylation are highly likely to undergo altered gene expression, thus causing different degrees of disease severity.
This study is limited by the number of FEVR families enrolled. For 5 mutations analyzed here, only one family was found for each mutation. Alternatively, we took the approach of comparing the average overall methylation level in probands, carriers and non-carriers, which could have masked the effect of specific mutation on DNA methylation. In addition, age may be an independent factor in the methylation level. However, in this study, after excluding the "grandmother" and "grandfather" subjects in family 6, the probands still had the lowest average methylation level among the three groups, which is in line with our conclusion. What's more, we do not have RNA samples from our patients to validate the expression of FZD4. Future study with larger sample size is needed to validate our findings. Nevertheless, our results provided a new mechanism potentially responsible for phenotypic heterogeneity amongst FEVR patients.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by the Institutional Review Board of Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

Author contributions
ML and JLu are responsible for conceptualization, methodology, software, investigation, and writing original draft. HF is responsible for clinical data collection and writing. JLi and XZ are responsible for software and writing review. PF and PZ are responsible for supervision, writing review and funding acquisition. All authors contributed to the article and approved the submitted version.