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

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) [5‐14].

In current study, we utilized next-generation sequencing of whole exome to investigate genetic defects in a Chinese pedigree with congenital cataract.

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 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 [26‐28]. 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 ball-and-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.

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 hot-spot. 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.