Familial exudative vitreoretinopathy caused by CTNNB1 gene de novo mutation in a Chinese family: a case report

Abstract

Background

Familial exudative vitreoretinopathy (FEVR) is an inherited disorder of retinal vascularization insufficiency caused primarily by genetic mutations. So far, FEVR has been less reported in the Chinese population. This study will provide a case of FEVR due to CTNNB1 splice mutation in a Chinese family, which will be helpful for genetic counseling and clinical diagnosis.

Case presentation

We collected a Chinese Han child with clinical manifestations of FEVR, accompanied by neurodevelopmental disorders. Whole exome sequencing (WES) showed the proband's CTNNB1 gene had a c.1060 + 1G > A de novo splicing mutation. Our analysis indicates that this variant produces a truncated protein that contributes to the development of the disease. Genetic testing confirmed the diagnosis of FEVR in proband from the study lineage. In addition, the proband also carries three novel gene mutation sites: the NIPBL gene c.3130G > A (p.Asp1044Asn), the CNGA1 gene c.568G > T (p.Glu190X), and the FBN2 gene c.5370A > G (p.Ile1790Met).

Conclusions

In this study, the c.1060 + 1G > A heterozygous mutation of the CTNNB1 gene is the main cause of FEVR disease in proband, and this pathogenic mutation expands the spectrum of CTNNB1 gene functional loss mutations in the Chinese population.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12887-025-05508-9.

Background

Familial exudative vitreoretinopathy (FEVR) is a lifelong, blinding genetic disease of vitreoretinal vascular dysplasia. The genetic pattern of this disease is autosomal dominant inheritance, mainly affects infants and young children, and may also occur in older adults with a hypomorphic phenotype. The incidence rate of infants and young children in China can reach 0.63%—1.19% [1, 2]. In the early stage of the disease, it may be limited to peripheral retinal vascular dysplasia. The onset of the disease is hidden, and the onset is usually without warning [3]. With the progression of the disease, retinal ischemia can be accompanied by avascular areas and proliferative lesions in the temporal peripheral retina, accompanied by intraretinal or subretinal lipid exudation, as well as retinal folds and macular displacement, which will eventually lead to retinal detachment due to organization and traction, resulting in severe visual loss and even blindness. Some patients exhibit neurological developmental disorders [4, 5]. FEVR has diverse genetic modes and high genetic heterogeneity, mainly including autosomal dominant inheritance, autosomal recessive inheritance, X-linked recessive inheritance, and other scattered genetic modes [6, 7]. Different genetic patterns are related to their associated gene mutation.

As of now, the discovered FEVR pathogenic genes include Atonal bHLH transcription factor 7 (ATOH7), Catenin alpha-1 (CTNNA1), Catenin beta-1 (CTNNB1), Exudative vitreoretinopathy 3 (EVR3), Frizzled class receptor (FZD4), Jagged protein 1 (JAG1) Kinesin family member 11 (KIF11), Low-density lipoprotein receptor-related protein 5 (LRP5), Norrie disease protein (NDP), RCC1 and BTB domain-containing protein 1 (RCBTB1) Tetraspanin 12 (TSPAN12) and Zinc finger protein 408 (ZNF408) [8–11]. These genes play important roles in signaling pathways such as Wnt, Notch, and Wnt/β-catenin. In this study, we analyzed a case of CTNNB1 gene mutation in a Chinese family to clarify the etiology of the patient and to provide a reliable basis for clinical diagnosis and genetic counseling.

Subject and Methods

Research subject

The patient is a 1-year-old Han male who gave birth vaginal delivery at term and has a normal weight. With intermittent strabismus, decreased visual acuity, occipitofrontal circumference (OFC) is small, shorter than normal peers, hand–eye coordination disorders, inability to track objects, inability to sit alone, intellectual disability, and so on, which come to our hospital for examination. His parents did not exhibit similar symptoms. The patient underwent a complete physical examination, ocular examination (including fundus examination, ocular ultrasound, fluorescein fundus angiography (FFA)), laboratory examination, medical history, and family history inquiry. This study was approved by the Ethics Committee of Medical Genetics and Prenatal Diagnosis of Luoyang Maternal and Child Health Hospital (the ethics number is LYFY-YCCZ-2023008), and the patients' families signed informed consent.

Ocular examination

We used the Retcam3 Neonatal Digital Wide-Field Fundus Imaging System (Cree, USA) to examine the patient's fundus, including the optic disc and macula lutea at the posterior pole, the retina temporal, inferior, nasal, and superior to the fundus, and observe the abnormalities of the fundus structures in each layer. Then, using the SW-2100 ophthalmic A/B-ultrasound diagnostic instrument (Suoer, China), the probe was placed on the patient's closed eyelids to examine the patient's vitreous, retina, and optic nerve conditions. Finally, the TRC-50DX fluorescein fundus angiography instrument (TOPCON, Tokyo Optical) was used to examine the vascular status of the patient's fundus, including vascular course, changes in vascular morphology, and vascular filling and leakage.

Extraction of Genomic DNA and Whole exome sequencing (WES)

DNA extraction was performed on peripheral blood and amniotic fluid samples using the Qiagen Genome DNA Extraction Kit (QIAGEN, Germany). The concentration of DNA (0.2—0.4 ng/μL) was detected using a Nano-Drop measuring instrument (Thermofisher), and the purity (A260/A280 ratio within the range of 1.8—2.0), the qualified DNA samples are stored at −20℃ for future use.

We used Enzymatics' fragmented enzymes to break the genomic DNA of family members into fragments of 250—300 bp in length. We amplified the genome library using the KAPA HiFi Ready Mix enzyme from KAPABiosystems. Then, IDT xGen Exome Research Panel v1.0 capture kit was used for full exon capture (covering 19,396 gene coding region DNA sequences, target region range 39 M, to detect point mutation and small fragment deletion insertion mutation within 20 bp). Next, genomic library concentration (concentration ≥ 10 ng/uL) was detected using Qubit 4.0 equipment and Qubit dsDNA HS Assay Kit, and genomic library fragment length (300—550 bp) was measured using the QSeq400 fragment analyzer. After passing the library inspection, the enriched target fragments were sequenced using the NovaSeq 6000 equipment from Illumina, USA, and the PE150 (read length 150 bp) mode was selected. The data output was about 10G, and the average sequencing depth of the entire exon sequencing target region was greater than 100x. More than 95% of the target sequence had a sequencing depth of 20x.

Sanger sequencing validation

We compared the sequencing data obtained with the reference sequence of the human genome GRCh37/hg19. If a suspicious mutation was detected, we used Sanger sequencing to verify the site of genomic DNA of family members, and designed primers targeting the CTNNB1: chr3:41,268,844-F: 5´-TGGCTCTTCTCAGACATGTG-3´, chr3:41,268,844-R: 5´-GCTACAATCCAGATGACAGG-3´, and amplified them. The PCR products were purified by agarose gel electrophoresis and sequenced by ABI3730xl genetic analyzer. The sequencing results were analyzed by Chromas software.

Pathogenicity analysis of CTNNB1 (Mut. c.1060 + 1G > A)

Mutational pathogenicity was first assessed through the American College of Medical Genetics and Genomics (ACMG) guidelines, the Clinvar database, and the Mutation Taster software.

Minigene technology (also known as in vitro validation of mRNA splicing anomalies) was then utilized by cloning the target genome fragment with a mutation site (c.1060 + 1G > A) (PrimerSTAR MAX DNA Polymerase, TaKaRa), constructing a recombinant expression vector (Rapid Plasmid Mini Kit, SIMGEN), transfecting Hela and 293 T cell lines, RNA extraction and cDNA inversion [DNA Gel Exctration Kit, SIMGEN; Trizol (RNAiso PLUS, TaKaRa); HifairTM 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus), YEASEN], and then verifying the effect of the mutation on mRNA splicing using electrophoresis and Sanger sequencing. The primers used in the experiment are shown in Supplementary Materials Table 1.

Results

Clinical manifestations

The patient is a 1-year-old Han male who underwent full-term vaginal delivery, with normal weight, no history of hypoxia at birth, normal cardiopulmonary function, and no history of maternal medication or radiation exposure. At the age of 1, the patient was shorter than normal peers (−1.5SD) and was diagnosed with microcephaly (OFC: −2.5SD). Clinical manifestations were intermittent esotropia, with a left eye deviation angle of 16° and a right eye deviation angle of 5°, left eye visual acuity of 0.5 and right eye visual acuity of 0.6 (Punctate visual acuity chart), hand–eye coordination disorders, inability to track objects, inability to sit alone, and developmental delay and intellectual disability (DD/ID). The patient's facial features were thinning hair, a wide nasal tip, relatively large ears, and a long philtrum (Fig. 1a). The patient's family tree diagram is shown in Fig. 1b, and he is a proband in the family.

Fig. 1.

Clinical features and pedigree of the patient. a The patient's facial features. b Genetic family tree. The pedigree showed that II−1 in the family had a heterozygous variant (CTNNB1: c.1060 + 1G > A), marked in black, and the arrow indicated the proband. The variant was not found in other members of this family. c Fundus examination. R: right eye, L: left eye. d FFA examination. e. Ocular ultrasonography. OD: right eye, OS: left eye

Fundus examination showed that the proband's peripheral avascular retina. Thin areas with suspected full-thickness holes were visible in the temporal peripheral retina of both eyes (Fig. 1c). FFA examination confirmed the presence of avascular areas around the retina accompanied by an increase in vascular branches, with distal branches being rigid and claw-shaped (Fig. 1d). Based on Pendergast's classification (https://doi.org/10.1016/S0161-6420(98)96002-X), the clinical stage of the proband's eye is stage 2 (avascular retinal periphery with extrinsic vascularization). Ocular ultrasound shows a flocculent moderate echo in the vitreous body of both eyes of the proband. In addition, a short strip of moderate echo can be seen, connected to the temporal bulbar wall, and the temporal bulbar wall is locally less smooth suggesting bilateral vitreous opacity (Fig. 1e).

WES and Sanger sequencing results and analysis

The proband underwent WES testing, and both he and his family were subsequently verified by Sanger sequencing. The results showed that the proband had a c.1060 + 1G > A heterozygous mutation at the chr3: 41,268,844, the disease corresponding to the phenotype of the proband is exudative vitreoretinopathy type 7 (MIM: 617,572) (Supplementary Materials Table 2), and the rest of the family members are wild-type at this locus, confirming this to be a de novo mutation (Fig. 2a). The proband carried a heterozygous mutation c.1060 + 1G > A in CTNNB1 gene, located at the + 1 position of intron8. The c.1060 + 1 base mutated from guanine to adenine, which belongs to the "Class I mutation region" that affects splicing (Fig. 2b). In addition, we also found that in addition to the c.1060 + 1G > A heterozygous mutation, the proband carried three unreported novel gene mutation sites: c.3130G > A (p.Asp1044Asn) in the NIPBL gene (ACMG rating of Uncertain significance), c.568G > T (p.Glu190X) in the CNGA1 gene (ACMG rating of Likely pathogenic), and c.5370A > G (p.Ile1790Met) in the FBN2 gene (ACMG rating of Uncertain significance) (Supplementary Materials Table 2).

Fig. 2.

CTNNB1 gene sequencing analysis results. a Verification results of Sanger sequencing. b. Location of c.1060 + 1G > A mutation in the corresponding exon or intron

Pathogenicity analysis of CTNNB1 mutation

The Mutation Taster software scored the c.1060 + 1G > A splicing mutation locus as 1.0, with a prediction level of D (the prediction range is from 0 to 1, the higher the score, the greater the harm), indicating that the mutation locus is highly deleterious. The c.1060 + 1G > A mutation corresponds to the disease and pathogenicity in the Clinvar database as inborn_genetic_diseases (pathogenic). Referring to the interpretation guide of ACMG gene mutation, the mutation at this site is determined as PVS1 + PM6 + PM2_Supporting, graded as Pathogenic.

We used Minigene technology to detect the pathogenicity of c.1060 + 1G > A splicing mutation. Insert a portion of Intron 7 (474 bp) – Exon 8 (145 bp)—and a portion.

of Intron 8 (566 bp) into the pcMINI vector containing the universal ExonA-IntronA-MCS-IntronB-ExonB (Fig. 3a). Then we performed Sanger sequencing verification on the constructed vector. The results showed that both wild-type and mutant minigenes were successfully inserted into the corresponding vectors (Fig. 3b). The RT-PCR detection results showed that the wild-type was a single band in HeLa and 293 T cells, which was consistent with the expected size (534 bp) and named band “a”; The mutant type is also a single band, named band “b” (389 bp) (Fig. 3c). Perform Sanger sequencing on the wild-type band “a” and mutant band “b” produced in two cell lines. The results show that wild-type band “a” is a normal shear band, with ExonA (192 bp) – Exon 8 (145 bp)—ExonB (57 bp) as the shear mode; Mutant band “b” is an abnormal shear band with Exon 8 jumping, and the shear mode is ExonA (192 bp)—ExonB (57 bp) (Fig. 3d). Exon 8 jumps in the cDNA representation: c.916_1060del (p.Leu306Valfs*6), and the mutation creates a premature termination codon (PTC) in Exon 9, generating a truncated protein of length 310aa (Fig. 3e).

Fig. 3.

Results of pcMINI carrier detection. a Schematic diagram of pcMINI carrier construction; (b) Sanger sequencing validation for vector construction; (c&d) RT-PCR transcriptional analysis agarose gel electrophoresis map and the corresponding Sanger sequencing result map of the shear band. The wild-type and mutant-type bands in Hela and 293 T cells were labeled a and b respectively. e Effects of the c.1060 + 1G > A mutation on protein structure

Discussion

In this article, we reported a splicing mutation (CTNNB1: c.1060 + 1G > A) associated with familial exudative vitreoretinopathy in a Chinese Han family using WES technology. The c.1060 + 1G > A mutation carried by the proband is a de novo mutation in the family, and his parents and younger brother (or sister) of unknown gender have been tested as wild-type at this gene locus. We predicted through various prediction software that this mutation has high pathogenicity. Subsequently, Minigene in vitro experiments showed that the mutation c.1060 + 1G > A affected the normal splicing of gene mRNA. Upon mutation, Exon8 jumps and generates a PTC in Exon9, producing a truncated protein of length 310aa. This is the main cause of the proband's illness. The facial features of the proband include thinning hair, a wide nasal tip, relatively large ears, and a long philtrum. Ocular examination showed the presence of avascular areas around the retina accompanied by an increase in vascular branches, thin areas with suspected full-thickness holes visible in the temporal peripheral retina of both eyes bilateral vitreous opacity, etc. Clinical manifestations include intermittent strabismus, decreased visual acuity, microcephaly, hand–eye coordination disorders, inability to track objects, inability to sit alone, and DD/ID. So far, the CTNNB1: c.1060 + 1G > A mutation is the first discovery in the Chinese population.

The CTNNB1 gene is located on chromosome 3p22.1 and encodes a protein (β-catenin) with adhesive connectivity function β-catenin, which supports the integrity between epithelial layers and mediates intercellular signal transduction. The β-catenin protein plays a crucial role in the Wnt signaling pathway, and its activation or ablation is closely related to tumorigenesis or the development of the nervous system [12–14]. In recent years, various heterozygous variants of CTNNB1 have been associated with human diseases, including neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV) (MIM 615075), and FEVR (MIM 617572). NEDSDV is an autosome dominant genetic disease, characterized by DD/ID, language disorder, microcephaly, motor retardation, autism spectrum disorder (ASD), muscle hypotonia, progressive peripheral spasm, craniofacial malformation, and visual abnormalities of different degrees [15, 16]. In 2022, Yan et al. mentioned in a study that 78.4% of Chinese NEDSDV phenotype patients and 69.4% of non-Chinese NEDSDV phenotype patients had visual defects. The clinical characteristics and genetic results of 24 patients with CTNNB1 pathogenic variation in the Chinese Mainland were also reported. This is currently the largest case series of NEDSDV caused by CTNNB1 mutation in China. Among them, Mild visual impairment accounts for about 79%, and FEVR accounts for about 4% [17]. Among the 50 CTNNB1-related neurodevelopmental disorders or NEDSDV patients previously reported by Rossetti L Z et al., approximately 74% were found to have ocular abnormalities (including strabismus, hyperopia, and astigmatism), including approximately 24% with vitreoretinopathy [18]. From this, it can be seen that CTNNB1 heterozygous mutations may simultaneously lead to clinical symptoms associated with NEDSDV and FEVR diseases in patients.

We searched the Clinvar database and found that a splicing mutation in the CTNNB1 gene c.1081 + 1G > C was reported in the 2015 study by Kuechler et al. [19]. This is the same mutation as the c.1060 + 1G > A splicing mutation in the CTNNB1 gene we are currently studying, except that their transcripts differ. In the report by Kuechler et al., the patient also had no relevant family history, presented with microcephaly (−2.5SD), thinning hair, wide nasal tip, intermittent strabismus, delayed motor development, hand–eye coordination disorders, and inability to sit alone, which is almost consistent with our current study. But there are several differences: firstly, in their report, the patient was diagnosed with intrauterine growth retardation and was born by caesarian section with low birth weight. Secondly, they did not describe the patient's intelligence but mentioned that the patient had language barriers, which we have not yet found in our current report. The reason is that in our study, the proband was a 1-year-old child, and language abnormalities may not be easily detected, so it is necessary to conduct follow-up. Finally and most importantly, in their report, the patient may not have had clinical manifestations of FEVR, so there is no mention of relevant information about the patient's ocular disease, including the necessary ophthalmologic examination and description, and they have not assessed the pathogenicity of the mutation. Regarding this mutation, Kuechler et al. mainly described the clinical symptoms of NEDSDV exhibited by the patient, whereas we also provided the clinical manifestations of the proband's FEVR in addition to NEDSDV and clarified the pathogenicity of the mutation. The phenomenon of relatively large ears in the proband mentioned in this study has also been reported in Yan et al. and Ho's cohort. In Yan et al.'s study, approximately 45% of patients with CTNNB1-related diseases had large or protruding ears [17]. In Ho et al.'s study, approximately 80% of patients with CTNNB1-related neurodevelopmental disorders had relatively large ears [16]. This phenotype is only reported in the Chinese population at present, whether there is a racial difference in the clinical presentation of these larger ears is unknown. Furthermore, the contribution of the three additional mutations identified in this study to disease in proband is unknown, and they have only been reported as novel mutations for the first time in the world.

The limitation of this study is that firstly, it did not provide information on the treatment status and current situation of patients, and lacked follow-up with patients and their families. Secondly, there is no information on whether there are more generations of members in the family who suffer from FEVR disease (however, the patient's maternal grandparents and paternal grandparents were known to be in good health and had not shown any relevant clinical manifestations). Finally, no in vivo experiments, such as animal experiments were conducted to verify the role of the c.1060 + 1G > A mutation in the CTNNB1 gene in the progression of FEVR disease. In future research, we will closely monitor the patient's condition, strengthen follow-up work, track the genetic diseases of more than three generations of family members, and record in detail the various conditions of each family member to better perfect family history. In addition, for this mutation, we can use the Luciferase reporter gene system to determine the activity of related signaling pathways, use Co-Immunoprecipitation (CO-IP) technology to determine whether the mutation affects the expression of β–catenin-related proteins, and design gene knockout mice to observe the development of retinal blood vessels in mice and study the interaction of related signaling pathways, etc.

This study used methods such as WES and Minigene technology to clarify the etiology and genetic mode of a case of FEVR disease, providing guidance for the future eugenics and fertility of this family and providing a reliable basis for future clinical diagnosis and genetic counseling.

Conclusions

The CTNNB1: c.1060 + 1G > A mutation produces a truncated protein with a length of 310aa, and this study found that this pathogenic mutation is the main cause of FEVR disease in the proband. This mutation enriches the spectrum of CTNNB1 gene loss-of-function variants in the Chinese population and provides a reliable basis for genetic counseling in the clinic.

Abbreviations

FEVR

Familial exudative vitreoretinopathy

CTNNB1

Catenin beta 1

NEDSDV

Neurodevelopmental disorders with spastic diplegia and visual impairment

DD/ID

Developmental delay/Intellectual disability

FFA

Fluorescein fundus angiography

WES

Whole exon sequencing

Authors’ contributions

Y.W. brewed and designed experiments, critically reviewed the knowledge content of the article, and obtained research funding. Y.C. and H.L. performed sequencing and analysis, Y.C. and W.Y. prepared figures, W.Z. provided administrative, technical, or material support, and Y.W. and Y.C. wrote the main manuscript.

Funding

This work was supported by the Department of Genetics and Prenatal Diagnosis of the Luoyang Maternal and Child Health Hospital. The funding agency did not participate in the design or implementation of this study.

Data availability

The datasets generated and analyzed during the current study are available in the Genome Sequence Archive for Human repository, and the accession number to datasets is HRA005666. It can be accessed from the following link: https://bigd.big.ac.cn/gsa-human/browse/HRA005666.

Declarations

Ethics approval and consent to participate

I confirm that the relevant guidelines and regulations performed all methods. The parents of the proband signed an informed consent form. The study was approved by the Ethics Committee of Medical Genetics and Prenatal Diagnosis of Luoyang Maternal and Child Health Hospital. The ethics number is LYFY-YCCZ-2023008.

Consent for publication

Obtain the informed consent of the proband and his family, and publish information and images in an online open-access publication.

Competing interests

The authors declare no competing interests.