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Mutations in TSPAN12 gene causing familial exudative vitreoretinopathy
Human Genomics volume 18, Article number: 22 (2024)
Abstract
Background
To report newly found TSPAN12 mutations with a unique form of familial exudative vitreoretinopathy (FEVR) and find out the possible mechanism of a repeated novel intronic variant in TSPAN12 led to FEVR.
Results
Nine TSPAN12 mutations with a unique form of FEVR were detected by panel-based NGS. MINI-Gene assay showed two splicing modes of mRNA that process two different bands A and B, and mutant-type shows replacement with the splicing mode of Exon11 hopping. Construction of wild-type and mutant TSPAN12 vector showed the appearance of premature termination codons (PTC). In vitro expression detection showed significant down-regulated expression level of TSPAN12 mRNAs and proteins in cells transfected with mutant vectors compared with in wild-type group. On the contrary, translation inhibitor CHX and small interfering RNA of UPF1 (si-UPF1) significantly increased mRNA or protein expression of TSPAN12 in cells transfected with the mutant vectors.
Conclusions
Nine mutations in TSPAN12 gene are reported in 9 FEVR patients with a unique series of ocular abnormalities. The three novel TSPAN12 mutations trigger NMD would cause the decrease of TSPAN12 proteins that participate in biosynthesis and assembly of microfibers, which might lead to FEVR, and suggest that intronic sequence analysis might be a vital tool for genetic counseling and prenatal diagnoses.
Background
Familial exudative vitreoretinopathy (FEVR, OMIM 133780) is hereditary angiogenesis of the retina whose characteristic is peripheral avascularity in the retina, described by Schepens and Criswick firstly in 1969 [1]. The retinal vascular abnormalities in FEVR might lead to incomplete and aberrant vascularization resulting in retinal fold, detachments, retinal neovascularization, fibrosis, folds, subretinal exudation, vitreous hemorrhage, macular ectopia, and eventually causing to total blindness. In China, FEVR was found in 217 infants (1.19% of all abnormalities) among 199,851 newborns reported according to a multicenter research.
FEVR can be genetically heterogeneous and inherited autosomal dominantly, autosomal recessively, or X-linked. Ten genes, including TSPAN12, FZD4, LRP5, KIF11, have been identified to the cause of FEVR [2,3,4]. TSPAN12 mutations have been reported in FEVRs with an autosomal dominant inheritance, which belongs to the phylogenetically ancient family of tetraspanins. However, a thorough understanding of the molecular mechanisms underlying TSPAN12 activity remains unclear.
TSPAN12 functions as a signaling platform within the plasma membrane whose relations to the Norrin/Frizzled4 signalling pathway have been demonstrated [5]. Norrin/Frizzled4 signaling was identified as a classic passthway causing the retinal vascular disease FEVR. According to Poulter et al., it was examined that TSPAN12 is crucial for Norrin/Frizzled4 signaling pathway, which is responsible for the development of avascular peripheral retinas and various other vitreoretinopathies. Junge et al. found that TSPAN12 plays a specific role in controlling Norrin/b-catenin signaling, rather than Wnt/b-catenin signaling, through its influence on FZD4 multimerization [6].
Herein, nine pathogenic mutations were screened for on 117 unrelated FEVR patients and afterwards the ocular manifestations were examined and analyzed in accordance with the mutation spectrum.
Methods
Study subjects and clinical assessments
This study focused on nine different FEVR patients with TSPAN12 mutation and their relatives who attended clinics and were diagnosed at the Eye and ENT Hospital of Fudan University, Shanghai, China. Informed consent has been obtained from all participants and written consent has been provided to us. All experiments were conducted as per the Helsinki Declaration tenets. This study was conducted in accordance with the approval fron the Ethical Oversight Committee of Eye and ENT Hospital of Fudan University. FEVR was evaluated according to the previously described diagnostic criteria [7]. In brief, the diagnosis of FEVR can be established by using fundus photography, slit-lamp biomicroscopy, type-B ultrasonic imaging, or fundus fluorescein angiography (FFA) by ophthalmologists according to the following criteria: (1) full term birth and no history of oxygen inhalation; (2) retinal avascular areas; and (3) retinal neovascularization or vascular leakage.
Whole exome sequencing and data analysis
The procedure of whole exome sequencing was done in Shanghai We-Health Biomedical Technology Co., Ltd. Genomic DNA samples obtained from individuals with FEVR were isolated from peripheral leukocytes utilizing a commercially available extraction kit (TIANGEN, China). The quality/quantity of DNA was evaluated utilizing Onedrop OD1000 spectrophotometer and by agarose gel electrophoresis. Exome capture was conducted with xGen Exome Research Panel v1.0 (Integrated DNA Technologies, Inc., Iowa, United States) and 150 base pair paired end sequencing was executed using the Illumina HiSeq4000 platform (San Diego, CA).The unprocessed reads were aligned using the Burrows–Wheeler Aligner (BWA) and SAMtools by the sequencing company. Then after eliminating duplicates from sorted alignment using Picard, variants were called using the Genome Analysis Toolkit (GATK v3.70) pipeline.
Variant validation
The suspected causal gene variants thereby discovered were demonstrated through Sanger sequencing. Genomic DNA was isolated from peripheral leukocytes ultilizing TIANamp Blood DNA Kit (TiangenBiotech, Beijing, China). Primers were designed using the Primer3Plus software (http://www.primer3plus.com/cgibin/dev/primer3 plus.cgi). PCR amplification was peformed by pre-denaturation at 95 °C for 5 min, and subsequently 35 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, with an extension at 72 °C for 5 min in the end. The products of PCR were then examined by 2% agarose gelelectrophoresis. Then the PCR products were sequenced directionally on an ABI3730xl DNA Analyzer (AppliedBiosystems, FosterCity, California, USA).
Construction of MINI-gene and transcription analysis
TSPAN12: c.612+1G>A
Based on the mutation information, mutant and wild-type plasmids were constructed. The cloning vehicle was pMini-CopGFP, with a cloning site of BamHI/XhoI. Three pairs of nested primers TSPAN12-612-AF/TSPAN12-612-AR, TSPAN12-612-BF/TSPAN12-612-BR and TSPAN12-612-CF/TSPAN12-612-CR were designed to amplify the genomic DNA of FEVR 48 (TSPAN12: c.612+1G>A) (intron 6 and 7 were too large and the target fragment was amplified in three segments, and the products were named TSPAN12-612A, TSPAN12-612B and TSPAN12-612C, respectively), and mutant and wild-type target fragments were acquired and by digestion and recombination reactions inserted into the cloning vector. The primer information is shown in Additional file 1.
TSPAN12: c.149+2T>C
Two pairs of nested primers TSPAN12-149-AF/TSPAN12-149-AR and TSPAN12-149-BF/TSPAN12-149-BR were designed to amplify the genomic DNA of FEVR 91 (intron 2 were too large and the target fragment was amplified in two segments, and the products were named TSPAN12-149A and TSPAN12-149B). The rest of the steps are as described previously. The primer information is shown in Additional file 1.
TSPAN12: c.360G>Ac
Two pairs of nested primers TSPAN12-360-AF/TSPAN12-360-AR, TSPAN12-360-BF/TSPAN12-360-BR and TSPAN12-360-CF/TSPAN12-360-CR were designed to amplify the genomic DNA of FEVR 104 (intron 4 and 5 were too large and the target fragment was amplified in two segments, and the products were named TSPAN12-360A, TSPAN12-360B and TSPAN12-360C). The rest of the steps are as described previously. The primer information is shown in Additional file 1.
Followed by incubation at 4 °C overnight, the recombinant vector was transformed into E. coli competent cells E.coli DH5ɑ. Culture 5–15 ml of bacterial broth, after 12 h of incubation, single clones were picked by PCR amplification and Sanger sequencing to identify if the target fragments of TSPAN12 gene was accurately inserted into the vectors. The correctly recombinant mutant minigene plasmids and wild-type minigene plasmids were picked out. TIANprep Mini Plasmid Kit (DP103-03) was used for vectors extraction according to the guidence of instruction book. The identified primers were shown in Additional file 1.
Transfection of recombinant vector with TSPAN12
293T cells were cultured using DMEM + 10% FBS. Wt and mut recombinant vectors were transiently transfected into 293T by Liposomal Transfection Reagent (40802ES03) according to the guidance of the instruction. Samples were collected after 48 h of incubation.
RT-PCR and Sanger sequence
The transfected cells were collected to extract RNA reverse transcribed cDNA. For RT-PCR amplification, primers were designed, while gel electrophoresis was performed. PCR amplification products were subjected to Sanger sequencing. The RT-PCR primer sequences are shown in Additional file 1.
Results
Clinical data
Next-generation sequencing (NGS) of the DNA samples from each patient can determine the genetic cause of FEVR. A total of 117 subjects received a panel-based NGS test in Fudan University Eye and ENT Hospital between 2015 Jan and 2022 Dec. The demographic characteristics of the overall patients are compiled below within Table 1. Among the variants recognized in these study participants, there were approximately 47.87% (56 of 117) of variants were found in 7 genes (FZD4, LRP5, TSPAN12, NDP, CTNNB1, ZNF408, and KIF11) (Table 2). 9 probands were identified as carrying TPSAN12 mutation, with a mutation detection rate of 15.25%. The proportion of missense mutations in positive mutated genes is shown in Table 2 and Fig. 1B. The highest non-missense mutation proportion was TSPAN12, reaching 44.44%.
The baseline characteristics of the 9 probands with TPSAN12 mutation are summarized in Table 3. In this cohort, 6 were female and 3 were male. The age of these patients ranging from 1 to 54 years. Overall, 7 patients were found to have the positive family history. Fathers of five of the 9 patients had FEVR histories and the mothers of two had histories of FEVR patients. The fathers of FEVR 50 and FEVR 104 carried the same genetic variant. Cosegregation analysis of FEVR50 and FEVR104 demonstrated a trans pattern and confirmed the autosomal dominant inheritance (Fig. 2). All 9 patients underwent graded assessment for FEVR stage, measurement of BCVA, and examination of associated pathology. FEVR 29 showed FEVR stage 4 in his left eye, with his BCVA not being detected, and the FEVR stage of the remaining 17 eyes were normal (stage 1). FEVR 29 had retinal detachment in her left eye and accepted the scleral bucking surgery. A total of 8 eyes in 4 patients developed drag-disc.
Patients with TSPAN12 variants
The genetic spectrum are as follows (Table 4). A total of 9 gene variants were found in the TPSAN12, of which 6 variants, c.402G>T, p.R134S; c.287A>G, p.Y96C; c.232G>A, p.G78R; c.149+2T>C, -; c.360G>A, p.M120I; c.449G>A, p.W150X, were novel, and 3 variants, c.352G>T, p.E118X; c.612+1G>A, -; c.146C>T, p.T49M, has been previously reported. Overall, the majority of probands carried missense mutations (55.56%). 5 were missense mutations, 2 were nonsense and 2 were splice site mutations. Four of the 9 TSPAN12 mutations were categorized as “pathogenic” or “likely pathogenic”, while the remaining missense mutations were considered variants of uncertain significance (VUS) in accordance with the guidelines of ACMG. The scores of Mutation Taster, Splice AI and SPIDEX predicted that the variants can negatively impact the protein's functionality. FEVR 13 was detected to have missense mutation c.402G>T (p.R134S), while Sample 40 had missense mutation c.287A>G (p.Y96C). Sample 45 was examined to have a missense variant c.232G>A (p.G78R). Sample 104 was identified to have a missense mutation c.360G>A (c.286_360del), which has been proved to induce exon 5 skipping or not. Sample 113 was also examined to have a missense variant c.146C>T (p.T49M) of TPSAN12. In addition, Sample 29 and Sample 106 had nonsense mutations c.352G>T (p.E118X) and c.449G>A (p.W150X), respectively. Sample 48 and Sample 50 had splice site mutations c.612+11G>A (c.469_612del in exon 8) and c.149+2T>C (c.67_149del in exon 3), respectively.
In order to explore the possibility of a novel splicing mode during gene transcription resulting from the TPSPAN12 mutation, transfection experiments were conducted using wild-type and mutant pcMINI-TPSPAN12 vectors respectively in 293T and HeLa cells. PCR and sequencing of recombinant plasmids showed the successful incorporation of both wild-type and mutant minigenes into their respective vectors (Figs. 3, 4 and 5). The extraction of total RNA was performed for RT-PCR. Results shows that in 293T cells and Hela cells. The splicing mode of FEVR 48 mutant-type band were reduced from 540 to 396 bp, causing exon 7 skipping (Fig. 3). The splicing mode of FEVR 50 mutant-type band were reduced from 426 to 343 bp, causing to exon 3 skipping (Fig. 4). There were one bands of the c.360G>A of FEVR 104, of which band was 390 bp in wild-type. The mutant-type exhibited a dual band pattern, designated as A and B correspondingly, of which band A was 390 bp in expected size and band B was smaller (Fig. 5B). These two bands were sent for sequencing and results revealed that band A of wild-type was the normal splicing band, whose splicing mode was exon 4—exon 5—exon 6.
The predicted 3D protein structures of TPSAN12 variants
The protein TPSAN12 was subjected to 3-Dimensional structure prediction using Swiss-Pdb Viewer 4.1, revealing that the variants could potentially affect the functionality of the proteins by disrupting hydrogen bonding, or making skipping. The absence of an appropriate model in the SWISS-MODEL database hindered the construction of TPSAN12 protein structure, considering its splice variation.
The predicted protein structures of TSPAN12 are used to better explain the potential pathogenic effects and protein function of TSPAN12 mutation variants. Mutations of c.612+1G>A, c.149+2T>C and c.360G>A could affect mRNA splicing which cause exon skipping. Nonsense mutations c.352G>T and c.449G>A could cause premature termination of transcription. Mutation of c.232G>A: p.G78R could cause non-polar amino acids becoming polar amino acids, which affects the protein structure: wild-type GKY-78 formed hydrogen bonding with TYR-82 at a spacing of 2.4 Å and with LEU-74 at a spacing of 1.8 Å; mutant ARG-78 formed hydrogen bonds with several surrounding amino acids including TYR-82, LEU-74, TRY-96, and GLY-81. Mutation c.360G>A could produce two forms of mRNA splicing, of which one mutation affecting mRNA splicing shown above, another mutation p.M120I could cause polar amino acid becoming non-polar amino acid, which affects the TSPAN12 protein structure: wild-type MET-120 forms hydrogen bonds with spacing of 2.3 Å and 3.2 Å with GLN-124 and 2.4 Å with GLN-117; mutant ILE-120 forms hydrogen bonds with spacing of 2.4 Å with GLN-124 and 2.6 Å with GLN-117. Mutation of c.402G>T: p.R134S could cause basic amino acid changed to neutral amino acid, which affects the TSPAN12 protein structure: wild-type ARG-134 formed hydrogen bonding with a spacing of 2.1 Å with THR-130; mutant SER-134 formed hydrogen bonding with a spacing of 2.1 Å and 3.5 Å with THR-130, 2.7 Å with LEU-131, 2.4 Å with MET-135, and TYR-138 to form hydrogen bonds with a spacing of 2.0 Å. Mutation of c.146C>T: p.T49M could cause the amino acid threonine to transfer to hydrophobic ones from hydrophilic ones. Mutation of c.287A>G: p.Y96C could cause aromatic-like amino acid changed to sulfur-containing amino acid, which affects the TSPAN12 protein structure: wild-type TYR-96 formed hydrogen bonding with LEU-92 at a spacing of 2.5 Å, LEU-100 at a spacing of 2.2 Å, SER-99 at a spacing of 2.8 Å, and ASN-18 at a spacing of 2.7 Å. The mutant CYS-96 formed hydrogen bonding with LEU-92 at a spacing of 2.2 Å and LEU-100 at a spacing of 2.4 Å. The mutant CYS-96 formed hydrogen bonding with LEU-92 at a spacing of 2.4 Å. The mutant CYS-96 forms a hydrogen bond with LEU-92 at a spacing of 2.2 Å and a hydrogen bond with LEU-100 at a spacing of 2.4 Å (Fig. 6).
Discussion
Familial exudative vitreoretinopathy (FEVR), as described for the first time in 1969 by Schepens and Criswick, is a hereditary condition which is characterized by peripheral retinal abnormalities and incomplete vascularization [8]. The presence of incomplete and aberrant vascularization is associated with the development of a variety of eye complications, which includes retinal exudates and neovascularization, retinal folds and detachments, vitreous hemorrhage, macular ectopia, and eventually resulting in total blindness.
FEVR has great phenotypic and genetic heterogeneity. Inheritance of FEVR, which is genetically determined, can be dominant, recessive or X-linked, with dominant inheritance being the most common manner. It has been currently found that at least nine genes are identified as responsible for the development of FEVR, which includes NDP, FZD4, LRP5, TSPAN12, ZNF408, KIF11, RCBTB1, CTNNB1, and JAG1 [9]. The proteins from the initial four genes work together in the Norrin/β-catenin signaling pathway, known as the Norrin/Frizzled-4 signaling pathway as well, and exhibit strong mutual interaction [10].
In previous study, we characterized the subtypes of FEVR by genetic analyses combined with clinical symptoms. Significant phenotypic difference was shown in TSPAN12 mutations with ocular developmental disorder affecting retinal exudates and neovascularization, retinal folds and detachments, vitreous hemorrhage, and macular ectopia [11]. In this study, we investigated a cohort of FEVR patients with TSPAN12 mutations. Compound heterozygous or homozygous TSPAN12 variants were found in 6 affected patients with a unique combination of lens and iris abnormalities.
Approximately 47.87% (56 of 117) of the variants were found in 7 genes (FZD4, LRP5, TSPAN12, NDP, CTNNB1, ZNF408, and KIF11) among the variants recognized in these study participants. 9 probands were identified as carrying TPSAN12 mutation, with a mutation detection rate of 15.25%. The highest non-missense mutation proportion was TSPAN12, reaching 44.44%.
In this study, these TSPAN12 variants were identified as being deleterious by virtue of the results of bioinformatic analyses and the rareness in general population, but further functional experimental research were needed to formally confirm their deleterious properties. To understand the clinical significance of TPSAN12 variants, functional study of the 3 mutations (c.612+1G>A, c.149+2 T>C and c.360G>A) on TPSAN12 expression was made in this study. Minigene analysis confirmed the 3 mutation on TPSAN12 found in FEVR patients caused alters splicing resulting in loss of TPSAN12 expression.
The novel intronic variants in TPSAN12 was shown a deleterious effect on silico algorithms. An analysis of mini-gene splicing assay in vitro revealed that these variants alter splicing, causing frameshifts and predicted premature termination codons, resulting in protein dysfunction from this allele via nonsense-mediated mRNA decay (NMD).
TSPAN12 is a tetraspanin which is encoded by chromosome 7q31 and composed of 305 amino acids. An intracellular loop and two extracellular loops (ECL-1 and ECL-2) comprise four transmembrane domains. TSPAN12 was revealed to associate selectively with Norrin/β-catenin pathway as the significant auxiliary constituent part of Norrin/FZD4/LRP5 complex [12]. Through interacting between the FZD4 and LEL, TPSAN12 is anchored into the Norrin receptor complex. It improves its signal transduction and enhances the selectivity of ligand-receptor binding by promoting the polymerization of Norrin/FZD4/LRP5 complex, and ultimately assists in reducing the phosphorylation and degradation of β-catenin, making more β-catenin into the nucleus to act as transcription factors. TSPAN12 participates in retinal angiogenesis significantly under physiological conditions through Norrin/β-catenin signaling pathway. In contrast, TSPAN12 mutations have been reported to be associated with vascular injury, nerve cell damage, microaneurysms and other retinal vascular diseases [13].
In patients who have complex heterozygous mutations, inactivation of the PTC-containing allele by NMD results in a halving of the total amount of effective TSPAN12 proteins, thus possibly becoming the reason of diseases due to haploinsufficiency. The dominant system between Norrin/β-catenin pathway and Wnt/β-catenin pathway systems presents a difference between numerous regions of the central nervous system, among which the Norrin/β-catenin pathway system makes the major contribution to the retina [14]. Recessive loss-of-function mutations in TPSAN12 results in aberrant Norrin/β-catenin pathway, rather than affecting Wnt/β-catenin pathway. Therefore, the imbalanced signaling and relative enhancement of the latter become potential risk factors of diseases associated with TPSAN12 and FEVR. The specific promotion of the Norrin/β-catenin pathway owing to TSPAN12 mutations will cause spectrum of retinal vascular diseases (Fig. 7).
The detailed molecular mechanism by TSPAN12 mutations is still lacking. Further integrative functional studies in the comprehension to TSPAN12 sequence variants is needed. For gaining a deeper understanding of the influence of TSPAN12 in ocular system, animal model is needed to build in our further study.
Conclusions
In conclusion, this study expanded the variants spectrum and phenotype of TSPAN12 gene. Besides, function validation assay showed that the expression of mRNAs and proteins of TSPAN12 were decreased in transfected cells with mutant TSPAN12 plasmids. Intronic sequence analysis, increasing the interpretation of molecular mechanism of TSPAN12 pathogenesis at the transcription and translation levels, might be a vital tool for genetic counseling and prenatal diagnoses.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- FEVR:
-
Familial exudative vitreoretinopathy
- NGS:
-
Next-generation sequencing
- PTC:
-
Premature termination codons
- NMD:
-
Nonsense-mediated mRNA decay
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Acknowledgements
The authors would like to thank all the members of this project supported by the National Natural Science Foundation of China (Grant Nos. 82070975, 81770908, 82070943), as well as all the volunteers who participated in the project.
Funding
This study was supported by the National Natural Science Foundation of China (Grant Nos. 82070975, 81770908, 82070943), the Shanghai Science and Technology Commission (Scientific Innovation Action Plan, Grant No. 18411965200) and Shanghai Science and Technology Commission (Grant No. 21ZR1411400).
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Conceptualization: YJ, TC, QC, XH. Methodology: YJ, TC, LR, YZ, QC, XH. Investigation: YJ, TC, LR, YZ. Visualization: YJ, TC, LR, YZ. Funding acquisition: QC, XH. Supervision: QC, XH. Writing—original draft: YJ, TC. Writing—review and editing: YJ, TC, QC, XH. YJ, TC, QC, XH had directly accessed and verified the underlying data. All authors have read and approved the final version of the manuscript. All authors had final responsibility for the decision to submit the paper.
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All experiments were conducted as per the Helsinki Declaration tenets. This study was conducted in accordance with the approval fron the Ethical Oversight Committee of Eye and ENT Hospital of Fudan University.
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Additional file 1
. Table S1: The primers uesd in pMINI-gene construction. Table S2: The primers uesd in Amplification product identification. Table S3: The primers uesd in RT-PCR.
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Ju, Y., Chen, T., Ruan, L. et al. Mutations in TSPAN12 gene causing familial exudative vitreoretinopathy. Hum Genomics 18, 22 (2024). https://doi.org/10.1186/s40246-024-00589-6
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DOI: https://doi.org/10.1186/s40246-024-00589-6