Identification of mutations associated with congenital cataracts in nineteen Chinese families

Abstract

Background

Congenital cataracts (CC) are one of the leading causes of impaired vision or blindness in children, with approximately 8.3–25% being inherited. The aim of this study is to investigate the mutation spectrum and frequency of 9 cataract-associated genes in 19 Chinese families with congenital cataracts.

Purpose

To identify the gene variants associated with congenital cataracts.

Methods

This study included a total of 58 patients from 19 pedigrees with congenital cataracts. All probands were initially screened by whole-exome sequencing(WES), and then validated by co-segregation analysis using Sanger sequencing.

Results

Likely pathogenic variants were detected in 8 families, with a positivity rate of 42.1%. Variants in various genes were identified, including GJA3, CRYGD, CRYBA4, BFSP2, IARS2, CRYAA, CRYBA1, ARL2 and CRYBB3. Importantly, this study identified compound heterozygous variants of IARS2 in one family.

Conclusions

Our research findings have revealed multiple gene variants associated with cataracts, providing clinical guidance for improved molecular diagnosis of congenital cataracts in the era of precision medicine.

Background

Congenital cataracts (CC) are one of the leading causes of visual impairment or blindness in children [1]. Approximately 8.3–25% of congenital cataracts are hereditary [2], and there are around 314,000 new cases of pediatric cataracts (congenital and developmental) each year in the world. If left untreated, congenital cataracts might result in irreversible visual impairment [3]. At present, sequence variants in more than 50 genes are associated with inherited forms of isolated or primary cataracts-largely based on the current update of the Cat-Map database (https://cat-map.wustl.edu/, accessed on 12 June 2024) [4]. Variants associated with cataracts typically occur in genes that encode various crystallin proteins—including crystallins (CRYAA, CRYAB, CRYBA1, CRYBA2, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGA, CRYGB, CRYGC, CRYGD, and CRYGS), gap junction proteins (GJA3 and GJA8), intermediate filament proteins, membrane proteins (MIP and LIM2), cytoskeleton proteins (BFSP2 and VIM), developmental regulators, and transcription factors (MAF, PITX3, HSF4, PAX6, FOXE3, and EYA1) [2, 5,6,7]. Because of the extreme genetic heterogeneity of CC, molecular diagnosis is challenging for ophthalmologists and researchers.

Whole-exome sequencing (WES) is a powerful approach for detecting exome variants. In the past decade, WES has become an important method for identifying pathogenic genes in monogenic diseases [8, 9]. According to the National Disabled Persons’ Database of the China Disabled Persons’ Federation, there were approximately 1,109,000 visually impaired children aged 0–14 in China in 2021, with around 69,000 in Zhejiang Province. In this study, we aimed to perform WES to identify disease-causing variants in probands with CC from the southern region of Zhejiang Province, China.

This study recruited 19 families, comprising a total of 58 cases of CC, at the Eye and Vision Hospital affiliated with Wenzhou Medical University from April 2021 to April 2023. Through WES and comprehensive bioinformatics analyses, we identified variants in various genes, including GJA3, CRYGD, BFSP2, IARS2, CRYAA, CRYBA1, and ARL2. Additionally, two variants classified as “Variants of Uncertain Significance (VUS)” were identified in CRYBA4 and CRYBB3. Importantly, this study identified a novel variant in IARS2. Our findings expanded the mutation spectrum associated with congenital cataracts, facilitating early diagnosis and treatment. Targeted Whole-exome sequencing in inherited congenital cataract patients provided significant diagnostic information.

Methods

Subjects and sample collection

This study was conducted with the approval of the Ethics Committee of Wenzhou Medical University Eye Hospital (approval number 2021-239-k-209) and adhered to the guidelines of the Helsinki Declaration for researching familial relationships and DNA specimens. Each participant underwent a detailed written informed consent procedure. To ensure the accuracy and reliability of the study, all patients and their family members received comprehensive ocular examinations by professional ophthalmologists, including visual acuity, slit-lamp biomicroscopy, ophthalmoscopy, corneal evaluations, axial length measurements (IOL Master-700; Carl Zeiss Meditec AG), and fundus examination for both eyes (Table 1 and Supp.Table S1). With the cooperation of the patients and their family members, peripheral blood samples or buccal mucosal samples were collected for further research analysis. Genomic DNA was extracted using a QIAGEN Blood DNA extraction kit (QIAGEN, Germany) or the Thermo Fisher Invitrogen™ kit (MagMAX™ DNA Multi-Sample Ultra 2.0 Kit, Thermo Fisher Scientific, Norway), according to the manufacturer’s instructions.

Table 1 Clinical features of affected probands with variants identified in this study

Whole-exome sequencing and bioinformatics analysis

To conduct the analysis, DNA samples obtained from individuals affected by the condition underwent comprehensive sequencing of the entire exome (WES). This process utilized the Twist Human Core Exome Kit (Twist Bioscience, USA) and the NovaSeq 6000 platform (Illumina, San Diego, USA) for the sequencing procedure. The short-read sequence data were aligned to the hg19 human reference genome using the Burrows–Wheeler Aligner tool (BWA) [10] and variant calling with GATK [11] according to best practice guidelines. Variant annotation was performed using ANNOVAR [12], In silico prediction tools Combined Annotation-Dependent Depletion (CADD v1.7) [13], REVEL [14], SpliceAI [15], SIFT [16], MutationTaster2021 [17], PolyPhen-2 [18] and AlphaMissence [19] used to predict potential pathogenic variations; Since CCs are rare in the Chinese population, only variants with a frequency below 0.5% were chosen. The population allele frequencies of identified variants were cross-verified using data from Genome Aggregation Database (gnomAD v4.1.0) data [20], China Metabolic Analytics Project (ChinaMAP) data [21] and Westlake BioBank for Chinese (WBBC) data [22]. Then disease and phenotype databases including Online Mendelian Inheritance in Man (OMIM; http://www.omim.org), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), the Human Gene Mutation Database (HGMD; http://www.hgmd.org), and Human Phenotype Ontology (HPO; https://hpo.jax.org/app/), were used for variant interpretation. The above databases were accessed in June 2023.

Variants previously described as disease-causing in the Human Gene Mutation Database (HGMD) and literature were given the highest priority. The effects of mutations described in the HGMD were further validated by reviewing published literature reporting the variants. Only those variants with convincing evidence were selected. In addition, protein-truncation mutations, such as nonsense and frameshift (insertions or deletions), were also ranked higher in priority. Variants were analyzed for possible pathogenic significance according to the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines [23,24,25,26].

Sanger sequencing validation

Sanger sequencing validation was performed by synthesizing primers targeting the DNA fragment of interest, followed by PCR amplification. Sanger sequencing was then carried out using an ABI 3730xl sequencer (Applied Biosystems, USA). The obtained sequences were compared with the reference sequence using MutationMapper software.

Results

Clinical findings

All patients in this study had various types of congenital cataracts and no other systemic diseases. Other ophthalmic findings of the nineteen probands were listed in Table 1 and Supp.Table S1. Phenotypes of the probands with congenital cataracts were recorded (Table 1). However, two probands (Family 2: III 2, Family 9: II5) had undergone cataract surgery prior to this study, and the remaining two probands (Family 7: III 1, Family 10: II5) had not undergone surgery and did not have anterior segment photographs available. The phenotypes of these families were determined based on their medical records.

Identification of suspected causative variants

This study included a total of 58 patients from 19 pedigrees with congenital cataracts. Among these patients, likely pathogenic variants were detected in 8 pedigrees, with a positivity rate of 42.1%. Of the 8 variants in this study, the variant of IARS2 (c.1067G > T: p.G356V) was novel, with the remaining 7 variants already reported (Table 2). Additionally, two variants classified as “VUS” were identified in Family 3 and Family 10. Most families exhibited autosomal dominant inheritance, with only Family 5 being considered as having autosomal recessive inheritance. Notably, likely causative variants were identified in GJA3 in two families, and one family each for CRYGD, BFSP2, IARS2, CRYAA, CRYBA1 and ARL2 (Table 2). The specific results of the family co-segregation verification within the pedigrees in this study were detailed in Figs. 1, 2 and 3.

Table 2 Analysis of likely pathogenic variants in familial and sporadic cataracts

Fig. 1

Pedigrees and chromatograms of families 1, 2, 4, 6, 7, 8, and 9 with mutations. Squares indicate men and circles women; black and white symbols represent affected and unaffected individuals, respectively. The proband is marked with an arrow, and +/− indicates heterozygous individuals, −/− indicates individuals testing negative

Fig. 2

Pedigree and variants in IARS2. The schematic show the encoded domain structure of IARS2 (A-B). The variants found in this study are illustrated above the schematics. W1: Wild Type 1, W2: Wild Type 2, M1: Mutant Type 1, M2: Mutant Type 2; The multiple-sequence alignments from different species. (C): Arrows show conserved glycine at p.G356 and Phenylalanine at p.F859 in IARS2 protein. Abbreviation: OLIVE, Olive Baboon;

Fig. 3

Pedigrees and IGV plots of families 3 and 10 with mutations. Squares indicate men and circles women; black and white symbols represent affected and unaffected individuals, respectively. The proband is marked with an arrow, and +/− indicates heterozygous individuals

The prediction results of the variants by SIFT, Mutation Taster2021, PolyPhen-2, CADD, REVEL, SpliceAI and AlphaMissence were listed (Table 2). The pathogenic variants of family 1, 2, 4, 5, 6, 7, 8, and 9 are shown by sequencing chromatograph (Figs. 1 and 2), and the sequencing chromatograph of family 3 and 10 are depicted using Integrative Genomics Viewer (IGV) plots (Fig. 3). Given that the IARS2 (c.1067G > T: p.G356V) is novel, we performed multiple protein sequence alignments using T-Coffee (https://www.ebi.ac.uk/jdispatcher/msa/tcoffee/) [27] and Jalview (Version: 2.11.4.0) [28] to demonstrate that the variant is highly conserved across different species (Fig. 2C) [28]. To further support the pathogenicity of these variants, we constructed a comprehensive table of population mutation frequencies, including GnomAD, ChinaMAP, and WBBC (Table 2). No causative variants were identified in the remaining nine pedigrees among the cataract-related genes examined. The corresponding pedigrees of these nine families with unidentified variants are presented in Supp.Fig S1.

Five of nineteen mutations were identified in crystallin genes, while the remaining five mutations were identified in four genes. Among these, two mutations were in GJA3 (MIM: 121001), one was in cytoskeletal protein (BFSP2; MIM:107320), and the other two were located in the IARS2 (MIM: 612801) and ARL2 (MIM: 610170), respectively. In the ten mutations, six are missense mutations, one is a nonsense mutation, two are non-frameshift mutations, and one is an intronic mutation. In this study, the compound heterozygous mutation in IARS2 (c.1067G > T; p.G356V) identified in Family 5 was considered a novel variant (Table 2).

Discussion

Here, we performed targeted exome sequencing on probands from 19 families with congenital cataracts. It was discovered that 10 cases contained putative pathogenic variants in nine cataract-related genes, including missense mutations, nonsense mutations, non-frameshift deletions, and splicing mutation (Table 2). Additionally, we have elaborated on the types of cataracts and other ocular clinical manifestations involved in these families (Table 1). Therefore, by performing genetic testing on these patients from diverse families, our aim is to discover genetic variations related to the development of congenital cataracts. This will contribute to a better understanding of hereditary congenital cataracts and offer clinical guidance for better molecular diagnosis of congenital cataracts in the era of precision medicine.

Two variants were detected in the GJA3 gene, exon2: c.176 C > T in Family 1 and exon2: c.589 C > T in family 7, both of which have been previously reported [5, 29]. The variant c.176 C > T in family 1 was classified as likely pathogenic (LP) and was detected in multiple pedigrees with CCs. Li Wang et al. [30] reported that this variant (c.176 C > T) co-segregated with CCs in three affected family members. The c.589 C > T variant in Family 7 was also classified as likely pathogenic (LP). The variant (c.589 C > T) co-segregated with CCs in six affected family members [29]. The human GJA3 gene encodes a protein of 435 amino acids, located on chromosome 13q12.11 [31]. The Connexin 46 (Cx46) protein, encoded by GJA3, is primarily expressed in lens fiber cells and plays a critical role in maintaining lens transparency, particularly in mature fiber cells at the central core of the lens [32].

In this study, we have identified mutations associated with the crystallin proteins CRYGD, CRYAA, and CRYBA1 in family 2, family 6, and family 8, respectively. To date, a total of 308 disease-causing variants in crystallins have been identified, accounting for nearly 23.0% of all inherited cataract variants [2, 33]. The variant CRYGD:exon3:c.418 C > T in family 2 is classified as a pathogenic (P) variant and has been shown to co-segregate with disease in seven affected family members from two pedigrees [34, 35]. Cellular experiments have suggested that this variant causes impaires gene function [36]. The CRYGD gene that is mutated in this family encodes the crystallin protein γD, which is an important component of the lens. It possesses a specific protein structure that contributes to maintaining the normal function of the lens and plays a crucial role in lens transparency and normal visual function [37]. In family 6, the c.61 C > T:p.R21W variant was detected in the CRYAA gene of the proband’s brother and mother. This variant has been classified as P according to the American College of Medical Genetics (ACMG) guidelines. Several published studies have previously detected this variant in patients from families affected by cataracts [38,39,40,41,42], and functional experiments have indicated that this variant affects protein function [43,44,45,46]. The CRYAA gene is an important lens structural protein primarily present in lens fiber cells. It plays a role in maintaining lens transparency and stabilizing lens structure. Gene variants in CRYAA can potentially lead to endoplasmic reticulum stress (ERS)-induced unfolded protein response (UPR) during lens development, causing cellular apoptosis and subsequently leading to cataract formation [47]. The variant CRYBA1:exon4:c.272_274del in family 8 is classified as P, and has been shown to segregate with disease in 16 affected family members from three pedigrees [48, 49]. Cell transfection experiments further suggest that this variant leads to impaired gene function [49]. Variants in the CRYBA1 gene may lead to abnormalities in protein structure, preventing proper folding and assembly and thereby impacting the normal development and transparency of the lens. In addition to hereditary cataracts, CRYBA1 variants may also be associated with other ocular abnormalities such as corneal lesions and retinal diseases [50].

The BFSP2 gene encodes a protein called beaded filament structural protein 2 (BFSP2), which serves as an important structural support in the lens of the eye and other tissues and plays a vital role in maintaining the normal structure and function of the eye and nervous system [51]. The variant BFSP2: exon3: c.697_699del in family 4 was classified as LP and has been reported to segregate with disease in multiple affected family members [52, 53]. This variant is a deletion mutation that results in a change in the length of the protein without causing a change to the open reading frame [54]. The cataract phenotype resulting from the BFSP2: c.697_699del is mostly nuclear, Y-sutural, stellate, or spokelike cortical, which is consistent with the posterior polar radial cataract reported in family 4 (Fig. 4) [53]. The specific pathogenesis remains to be further fundamental research.

Fig. 4

Phenotype of nuclear cataract in families 1, 3, 4, 5, 6, and 8. Abbreviations: OD OCULUSDEXTER (Right eye), OS OCULUSSINISTER (Left eye)

In family 5, a novel compound heterozygous mutation was identified in IARS2. Specifically, the exon9: c.1067G > T was classified as VUS, and the exon21: c.2575T > C was classified as LP. This gene is a nuclear gene that encodes mitochondrial isoleucyl-tRNA synthetase [55]. The variants IARS2 (c.1067G > T: p.G356V and c.2575T > C: p.F859L) of the proband (II2) were inherited from the father and mother respectively, with both parents being heterozygous for the variants (Fig. 2A-B). Variants in the IARS2 gene typically result in an autosomal recessive inherited disease known as CAGSSS (cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal dysplasia; OMIM: 616007). Two variants were detected in IARS2, which encodes an aminoacyl-tRNA synthetase subunit, an important enzyme involved in protein synthesis. The presence of these variants may impact the normal function of the IARS2 gene, thereby adversely affecting the process of protein synthesis. Furthermore, studies have suggested that variants in the IARS2 gene are associated with various genetic disorders, including cataracts, growth hormone deficiency, sensory neuropathy, sensorineural hearing loss, and skeletal developmental abnormalities [55]. The p.G356V lies in the class Ia aminoacyl-tRNA synthetases domain, while the p.F859L localizes to the anticodon-binding domain (Fig. 2A). Multiple sequence alignments indicated that Glycine (G) at position 356 and Phenylalanine (F) at position 859 of IARS2 were highly conserved among different species (Fig. 2C). Although this variant has not been reported in previous studies, based on the location of the variant and the nature of the base change, it can be inferred that this variant might lead to structural or functional changes in the protein, thereby causing the development of diseases such as cataracts.

The variant ARL2: exon1:c.44G > T in family 9 was classified as LP. Since we were unable to collect the genetic data of the parents, co-segregation verification could not be performed for this family. Nevertheless, Xue-Bi Cai et al. [56] reported this variant in a Chinese pedigree, where it co-segregated with four affected family members. Additionally, they demonstrated that this variant impairs gene function through a transgenic mouse model. In their report, the proband and her three daughters exhibited microcornea, retinal cone dystrophy, cataracts, and posterior scleral staphyloma, which are consistent with the features of MRCS syndrome. However, in Family 9, we did not observe any of these symptoms except for cataracts. The ARL2 (ADP-ribosylation factor-like 2) gene encodes a small GTPase that plays an important role in intracellular signaling, endocytosis, and regulation of the cellular cytoskeleton (ARL2_HUMAN, P36404). The variant identified in this gene may lead to changes in protein structure or function, thereby affecting the normal signal transduction and metabolic processes within cells and potentially causing the development of cataracts.

According to the guidelines of the ACMG, the significance of the CRYBA4 variant in family 3 and the CRYBB3 variant in family 10 was classified as “Variants of Uncertain Significance”. The variant CRYBA4:exon6:c.464 C > T: p.P155L found in family 3 was a novel variant at this specific locus that is classified as of uncertain significance. This variant occurs in the beta/gamma-crystallin functional domain (PM1). This variant was not detected in ChinaMAP and WBBC. The frequencies of this variant in gnomAD were 5.5e-06 (PM2). According to various statistical methods (such as SIFT, MutationTaster, and REVEL), this variant is predicted to have deleterious effects on the gene or gene product (PP3). In family 10, the variant CRYBB3: intron1: c.-20-123G > C was a novel variant identified at this specific locus, and it is classified as of uncertain significance. The Population allele frequencies of this variant in GnomAD, ChniaMAP and WBBC were 7.2e-6, 1.5e-4 and 1.1e-4, respectively (PM2). The prediction results from SpliceAI showed that this variant may affect splicing (PP3) (Table 2). Our limitation lies in the fact that we failed to acquire the genetic collection data of the proband’s parents in family 3 for the verification of family co-segregation. In family 10, Whole Exome Sequencing (WES) indicates that the variant in CRYBB3 was detected in both the proband and the father, and both individuals are affected by congenital anterior polar cataracts (Fig. 2). However, the evidence for pathogenicity based on the current information of mutation sites in family 3 and family 10 was still insufficient, and further functional experiments are required to verify whether these mutation sites are pathogenic.

Conclusion

In conclusion, our findings revealed multiple gene variants associated with cataracts in 10 of the 19 families studied, providing clinical guidance for improved molecular diagnosis of congenital cataracts in the era of precision medicine. Additionally, large-scale exome sequencing and genetic variation analysis can enhance a deeper understanding of the pathogenesis and genetic patterns of congenital cataracts, providing a scientific foundation for the development of new therapeutic approaches and preventive measures.