Identification of a novel GJA3 mutation in a large Chinese family with congenital cataract using targeted exome sequencing

RESEARCH ARTICLE Identification of a novel GJA3 mutation in a large Chinese family with congenital cataract using targeted exome sequencing Yihua Yao1, Xuedong Zheng1, Xianglian Ge2, Yanghui Xiu1, Liu Zhang1, Weifang Fang1, Junzhao Zhao3, Feng Gu2*, Yihua Zhu1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, China, 2 School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, State Key Laboratory Cultivation Base and Key Laboratory of Vision Science, Ministry of Health and Zhejiang Provincial Key Laboratory of Ophthalmology and Optometry, Wenzhou, Zhejiang, China, 3 The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China * (YHZ); (FG) Abstract OPEN ACCESS Citation: Yao Y, Zheng X, Ge X, Xiu Y, Zhang L, Fang W, et al. (2017) Identification of a novel GJA3 mutation in a large Chinese family with congenital cataract using targeted exome sequencing. PLoS ONE 12(9): e0184440. https://doi.org/10.1371/ journal.pone.0184440 Editor: Yong-Bin Yan, Tsinghua University School of Life Sciences, CHINA Received: June 23, 2017 Accepted: August 23, 2017 Published: September 6, 2017 Copyright: © 2017 Yao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Autosomal dominant congenital cataract (ADCC) is a clinically and genetically heterogeneous ocular disease in children that results in serious visual impairments or even blindness. Targeted exome sequencing (TES) is an efficient method used for genetic diagnoses of inherited diseases. In the present study, we used a custom-made TES panel to identify the genetic defect of a four-generation Chinese family with bilateral pulverulent nuclear cataracts. A novel heterozygous missense mutation c.443C>T (p. T148I) in GJA3 was identified. The results of the bioinformatic analysis showed that the mutation was deleterious to the structure and hemichannel function of Cx46 encoded by GJA3. Plasmids expressing wildtype and mutant human Cx46 were constructed and ectopically expressed in human lens epithelial cells (HLECs) or human embryonic kidney (HEK-293) cells. Fluorescent images indicated aggregated signals of mutant protein in the cytoplasm, and a higher protein level was also detected in T148I stable cell lines. In summary, we identified a novel mutation in GJA3 for ADCC, which provided molecular insights into the pathogenic mechanism of ADCC. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction Funding: This work was supported by grants from Natural Science Foundation of China (81270999/ H1204, YHZ, http://www.nsfc.gov.cn/), Professor Academic Development Fund of Fujian Medical University (JS14019, YHZ, http://www.nsfc.gov.cn/ ), the Chinese National Program on Key Basic Research Project (973 Program, 2013CB967502, FG, http://program.most.gov.cn/), Natural Science Congenital cataract (CC) is a common and severe hereditary ocular disease in children, leading to serious visual impairments or even blindness[1, 2]. The main characteristic of CC is lens opacity and abnormal ocular development from birth or during infancy, childhood or adolescence. Approximately 1 to 6 cases per 10,000 live births develop non-syndromic CC in industrialized countries, whereas these proportions are estimated to be 5 to 15 per 10,000 live births in developing countries[3–5]. Approximately 8% to 25% of isolated CC are considered to be hereditary diseases, accounting for nearly 70% of congenital cataracts[6]. Autosomal dominant PLOS ONE | https://doi.org/10.1371/journal.pone.0184440 September 6, 2017 1 / 15 A novel GJA3 mutation causing congenital cataract Foundation of China (81201181/H1818, FG, http:// www.nsfc.gov.cn/), Zhejiang Provincial & Ministry of Health research fund for medical sciences (201339279, FG,http://www.zjkjt.gov.cn/), Wenzhou Medical University PI Start-Up Grant (QTJ12011, FG, http://www.wmu.edu.cn/). Competing interests: The authors have declared that no competing interests exist. congenital cataract (ADCC) is the most common trait of CC patients, and the disease can also be inherited through autosomal recessive or X-linked models[7]. To date, more than 60 genetic loci for inherited CCs have been mapped. Among these, more than 20 causative genes for these loci have been associated with ADCC, and the number of the identified genes is increasing[7, 8]. Half of the identified mutations in the ADCC family are crystallin genes, including CRYAA, CRYAB, CRYBA1/A3, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD and CRYGS, another one-quarter are gap junction (GJ) genes, including GJA3 and GJA8[9]. The remaining genes are MIP, LIM2, MP19, BFSP1/2, HSF4, MAF, PITX3 and EPHA2[7, 9–11]. GJs are transport channels that function by forming extensive intercellular communication channels in the avascular lens; each channel is created by two hemichannels, also known as connexons, between adjacent cells[12]. GJs provide an intracellular and extracellular transmembrane network for the transportation of small molecules, such as second messengers, nutrients and ions. The functions of GJs are not only crucial to the maintenance of lens homeostasis and transparency but also important for lens development and differentiation [13]. Cx43, Cx46 and Cx50, encoded by GJA1, GJA3 and GJA8, respectively, are mainly located in lens fiber cells as well as partly in HLECs[14, 15]. Mutations of GJA3 and GJA8 are linked to ADCC through different mechanisms, including the formation of inefficient GJ channels [16–18], abnormal expression in the cytoplasm or nucleus[19, 20], changes in channel or hemichannel functions[21], alterations in electrophysiological characteristics[22], and dominant negative mutants on wild-type (WT) GJs[23, 24]. Therefore, mutated GJ genes (including GJA3 identified in this study) are the main disease-causing genes in CC patients. In this study, the genetic defect of a four-generation Chinese family with pulverulent nuclear ADCC were identified via TES and Sanger sequencing. Using this approach, a novel heterozygous mutation in GJA3 was revealed in this family. Silico prediction and experimental studies were performed to dissect the pathogenic mechanism of this mutation associated with ADCC. Materials and methods Ethics statement This study was approved by the ethics committee of the First Affiliated Hospital of Fujian Medical University, Fuzhou, China, on March 9, 2016. Participants were recruited from April to June 2016 and provided written informed consent consistent with the tenets of the Declaration of Helsinki. Patients and preparation of genomic DNA A four-generation Chinese family with ADCC was recruited for clinic (...truncated)