Next-generation sequencing for D47N mutation in Cx50 analysis associated with autosomal dominant congenital cataract in a six-generation Chinese family

Background Congenital cataract is the most frequent cause of blindness during infancy or early childhood. To date, more than 40 loci associated with congenital cataract have been identified, including at least 26 genes on different chromosomes associated with inherited cataract. This present study aimed to identify the genetic mutation in a six-generation Chinese family affected with congenital cataract. Methods A detailed six-generation Chinese cataract family history and clinical data of the family members were recorded. A total of 27 family members, including 14 affected and 13 unaffected individuals were recruited. Whole exome sequencing was performed to determine the disease-causing mutation. Sanger sequencing was used to confirm the results. Results A known missense mutation, c. 139G > A (p. D47N), in Cx50 was identified. This mutation co-segregated with all affected individuals and was not observed in the unaffected family members or in 100 unrelated controls. The homology modeling showed that the structure of the mutant protein was different with that wild-type Cx50. Conclusions The missense mutation c.139G > A in GJA8 gene is associated with autosomal dominant congenital cataract in a six-generation Chinese family. The result of this present study provides further evidence that the p. D47N mutation in CX50 is a hot-spot mutation.


Background
Congenital cataract is the most frequent cause of blindness during infancy or early childhood, with an occurrence of 1-15/10,000 live births worldwide [1,2]. It explains for 10%-30% of childhood blindness [3]. Congenital cataract is characterized by the presence of an opacification of the lens at birth or during babyhood. On the basis of morphology, congenital cataract can be classified into several subtypes, including nuclear, sutural, polar, cortical cataract, etc. [4]. Congenital cataract pathogenesis involves several distinct reasons including gene defects, chromosomal abnormalities, metabolic disorders, and infections during embryogenesis. Approximately half of congenital cataracts are inherited [3]. Though autosomal recessive and X-linked inheritances have been reported, inheritance is mainly autosomal dominant [5]. Up to date, over 40 loci associated with congenital cataract have been confirmed, including no less than 26 genes on different chromosomes related to congenital cataract [6,7]. Among these mutant genes, the connexin genes and crystallin genes are the most widespread. Briefly, half of the mutations were discovered in the crystalline genes, such as alpha crystallins, beta crystallins and gamma crytallins, and approximately 25% involve mutations in membrane transport genes, such as connexin proteins (Cx43, Cx46, and Cx50) [5][6][7][8][9][10][11][12][13][14].
In current study, we utilized next-generation sequencing of whole exome to investigate genetic defects in a Chinese pedigree with congenital cataract.

Subject recruitment and DNA sampling
A six-generation Chinese cataract family was examined at the General Hospital of Daqing Oil Field, Heilongjiang province, China. Pedigree medical history was taken directly by interviewing the family members. A total of 27 family members, including 14 affected (III12, IV11, IV28, IV30, IV39, IV72,  IV73, V9, V11, V27, V28, VI3, VI9, and VI15) and 13 unaffected individuals (IV40, IV68, IV69, IV70, IV71, IV74, V10, V14, V19, V42, V57, V62, VI13) were recruited ( Fig. 1). Ethical approval for current research was obtained from the ethics committee of General Hospital of Daqing Oil Field and the study was conducted according to the Declaration of Helsinki of the World Medical Association. All members recruited in this study underwent ophthalmologic examinations, including slit lamp ophthalmoscopy, biometry, visual acuity, and fundus examination. In addition, 100 unrelated healthy subjects without cataracts were also recruited from General Hospital of Daqing Oil Field. DNA samples were extracted using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) from peripheral blood.

Short-read alignment, variant calling and annotation
Low quality reads and PCR duplicates with >5 unknown bases were eliminated [15], for insertion/deletion (indel) and single nucleotide polymorphism (SNP), respectively. Aligning between read and the National Center for Biotechnology Information human reference genome (hg 19) were performed by sequencing reads were aligned to using Burrows-Wheeler Aligner (BWA) [15] and Short Oligonucleotide Analysis Package (SOAP3) tools [16]. Indels were validated according to the alignment result with the Genome Analysis Toolkit (GATK), and SNP Fig. 1 Clinical evaluation of a Chinese pedigree with autosomal dominant congenital cataract. a Pedigree of a six-generation Chinese family with autosomal dominant congenital cataracts. The arrow indicates the proband. Squares and circles symbolize males and females, respectively. Black and white denote the status of family members affected or unaffected, respectively, by congenital cataract. b Photo was taken with a surgical microscope calling was performed with Short Oligonucleotide Analysis Package (SOAPsnp). Variants were annotated using ANNOVAR tool.

Validation of mutation by Sanger sequencing
Sanger sequencing was used to validate the variants identified by exome sequencing. Specific primers were designed by Primer Premier 3.0 software for the target region. Genomic DNA from participants and 100 normal controls was analyzed.

Bioinformatics analysis
The effects of wild-type amino acid sequences with the p. D47N mutant of Cx50 on the secondary structure were performed using Antheprot 2000 software (version 6.6.5, IBCP, Lypn, France). The solved structure of gap junction protein beta 2(Cx26) was taken as template (Protein Data Bank No.2ZW3). The model structure of homomeric wild-type and the mutant of GJA8 were modelled by Swiss-Model Server [17]. In addition, the possible functional effect of the amino acid change was predicted by PolyPhen-2 and SIFT.

Clinical evaluations
Among 171 members in this six-generation Chinese family, affected individuals account for 23.39% (Fig. 1). All affect individuals in the pedigree had bilateral cataracts. Autosomal dominant inheritance mode of the congenital cataract was ascertained by the presence of affected individuals in each generation of the family, and male-to-male transmission. The proband's son (VI 9) had been diagnosed with cataracts when he was 15 months old. Slit-lamp examination of his left eye showed perinuclear cataract.

Identification of Cx50 mutation
Whole exome sequencing was performed on genomic DNA from nine patients of congenital cataract family (III12, IV11, IV28, IV30, IV73, V9, V27, VI3, VI9 and VI15) and one unaffected individual (IV40) though nextgeneration sequencing technology. As demonstrated in Table 1, we obtained at least 64.06 million reads that mapped to targeted exome regions; more than 99.49% of the target region was covered. The mean depth of the target exome region was 180. 98×, 191 Table 2. To help identify candidate mutations, untranslated regions, variants falling within intergenic, synonymous substitutions, intronic were excluded. Then the remaining variants were filtered out in at least four public genetic variant databases, including 1000 Genomes, dbSNP, HapMap and YH. Variants with an allele frequency > 0.5% were rejected. Variants shared by 10 patients and absent from 1 unaffected individual were analyzed.
After filtering and samples comparison, one heterozygous change was confirmed in all affected individuals in congenital cataract family, G > A, at position 139 (c.139 G > A) in exon 2 of GJA8 (Cx50). This change led to the substitution of aspartic acid by asparagine at position 47 (p. D47N). This mutation was further confirmed by Sanger sequencing (Fig. 2). The D47N substitution co-segregated with all 14 affected individuals, while it was not found in the unaffected family members or in the 100 healthy controls.

Bioinformatics analysis
The potential structure and function impact of the D47N mutation was predicted to affect protein function with a score of 0.00, and could probably be damaging with a score of 1.0 by SIFT and PolyPhen-2, respectively. As shown in Fig. 3, the secondary structure of mutant Cx50 protein was different with wild type. The results stated clearly that the wild-type sheet in COOH-terminal portion is likely missing in the D47N mutant. Took the structure of Cx-26 as template, the model structure of the mutant Cx50 have distinct changes (Fig. 4). There are additional helix (red arrow) and shortened sheet (green arrow) in the D47N mutant.

Discussion
In the current study, we confirmed a missense mutation c. 139 G > A in Cx50 (GJA8) in a six-generation Chinese pedigree with congenital cataract. This mutation resulted in an asparagine substitution for aspartic at amino acid residue 47 (D47N).
Cataracts are defined as opacification of the normally transparent crystalline lens, and are the leading cause of vision loss in the world. Congenital cataract is a type of cataract that emerges at birth or during early childhood [5,18]. The abnormality of lens can interfere with normal development of eyes [5,19]. Congenital cataracts can be inherited or familial, either as an isolated lens phenotype or as part of a genetic/metabolic disorder, commonly with full penetrance and autosomal dominant transmission [19]. Genetic factors play an important role in congenital cataract [20]. Gene mutations that affecting the lens development during embryonic period are considered to be the main cause [18]. Up to now, more than 39 genes and loci have been confirmed to be involved in  Table 2 Variations identified by whole exome sequencing   Mutation type  III12  IV11  IV28  IV30  IV73  V9  V27  VI3  VI9  VI15  IV40   Indel analysis   Total  15, the formation of isolate cataract [21,22], including crystallins, such as α-, β-, γ-crystallins (e.g., CRYAA, CRYBB1, CRYBB2, CRYGD), membrane transport and channel proteins, such as α-connexins (GJA3, GJA8). Intercellular gap junction channels provide pathways for metabolic and electrical coupling between cells in different tissues, and they are permeable to ions and small solutes, such as ions (K+, Ca2+), nutrients and small metabolites [23]. Gap junction channels consist of connexin protein subunits. Connexin proteins also known as gap junction proteins have four transmembrane domins with two extracellular loops (E1 and E2) and three intracellular regions (the NH2-terminus, a cytoplasmic loop and the COOH-terminus) [24]. Three isoforms of the connexin gene family-Cx43 (GJA1), Cx46 (GJA3) and Cx50 (GJA8) are abundantly expressed in the vertebrate lens.
Cx50 is an important protein and play an important role during lens growth, maturation of lens fiber cells, and lens transparency [25]. Cx50 comprises two exons with exon-2 coding for the entire 433 amino acid residues of gap junction protein α8 (GJA8). Up to date, at least 32 mutations in Cx50 have been identified to contribute to cataract. Of the 32 coding mutations, 29 result in missense substitutions that are involved in autosomal dominant cataract, and two are frameshift mutation associated with autosomal recessive cataract [6]. The majority of missense substitution are situated in the N-terminal half of the protein, which also contains the conserved connexin domain (amino acids 3-109) [6]. Three types of mutation: D47N, D47H and D47Y indicate that the amino acid at position 47 in GJA8 is a mutational hot spot [26][27][28]. Functional findings showed that D47N mutant expressed in Xenopus oocyte pairs could not form functional gap junction channels. Moreover, co-expression of Cx50D47N with wild-type Cx50 did not inhibit the activity of wild-type Cx50 [29]. The similar behavior was also observed in the mouse Cx50D47A, a mutation underlying the cataracts in the No2 mouse [30]. D47N and D47A mutants were loss-of-function mutants. Cellular level studies showed that the mutation of Cx50 prevented its localization to the plasma membrane. And this may lead to a capacity deficiency of Connexin 50, triggering a complex sequence of events, such as disruption of transmembrane ion gradients, loss of membrane potential, decreased cell growth and subsequent decreased metabolic activity [25,31]. Cx50 is critical for balland-socket structures, actin distribution and fiber cell morphology. Cx50 gap junctional communication through ball-and-socket is important for lens development, especially during rapid, early fiber cell growth [32]. Some limitations of this study should be addressed. First, we did not collect all of pedigree samples, especially the affected individuals in the congenital cataract family. Secondly, we did not perform more experiments, such as cell function experiment of D47N mutant and animal model experiments. Both of these limit our knowledge of more information of the D47N mutant. Nonetheless, advantages in our study should also be acknowledged. Exome sequencing and next-generation sequencing provide a rational approach to screen all candidate genes for inherited cataract or other inherited disease. In addition, exome sequencing and next-generation sequencing are suitable for molecular diagnosis of hereditary diseases. Our finding supports the enormous potential of exome sequencing in molecular diagnosis of single gene disease.

Conclusions
In conclusion, the present research confirmed a recurrent mutation, c.139 G > A (p.D47N) in Cx50 in a six-generation Chinese family with autosomal dominant congenital cataract. This result provided further evidence for Cx50 in association with congenital cataract, and the amino acid at position 47 is a mutational hotspot. The function of D47N mutation needs to be further certificated in animal mode. In addition, exome sequencing and next-generation sequencing are suitable for molecular diagnosis of hereditary diseases.