机构：[1]Peking Union Med Coll, Grad Sch, Beijing 100021, Peoples R China;[2]Lanzhou Univ, Sch Life Sci, Lanzhou 730000, Peoples R China;[3]Huazhong Univ Sci & Technol, Key Lab Mol Biophys, Coll Life Sci & Technol, Wuhan 430074, Peoples R China;[4]Huazhong Univ Sci & Technol, Ctr Human Genome Res, Wuhan 430074, Peoples R China;[5]Capital Med Univ, Beijing Childrens Hosp, Beijing Pediat Inst, Publ Cent Lab, Beijing 100045, Peoples R China;科研平台儿科研究所首都医科大学附属北京儿童医院[6]Qingdao Univ, Qingdao Med Coll, Affiliated Hosp, Qingdao, Peoples R China;[7]Natl Res Inst Family Planning, Beijing 100081, Peoples R China;[8]WHO, Collaborating Ctr Res Human Reprod, Beijing, Peoples R China;[9]Peoples Hosp Gansu Prov, Lanzhou 730000, Gansu, Peoples R China
Congenital heart defect Connexon43 Mutation Congenital heart disease (CHD) affects 1–2% of all children and is the leading cause of death in infants under 1 year of age [1] . The majority of congenital heart malformations do not segregate in Mendelian ratios, although they show familial aggregation, which suggests that genetic factors play a role in their development. Almost 30% of major cardiac malformations are associated with additional developmental abnormalities and result from a recognized chromosomal anomaly or occur as part of a syndrome [2] . Connexin43 ( Cx43 ) is one of the most prevalent connexin proteins. Connexin43 has previously been shown to be vital for normal murine heart development, as demonstrated by GJA1 knockout mice, which die at birth as the result of a severe heart malformation [3] . To form a functioning gap junction channel, six connexin subunits form a hemichannel, or connexon, in the plasma membrane of a cell. Two connexons from adjacent cells dock to form an entire channel, which allows for the passage of molecules up to 1.2 kDa between the cytoplasm of the two cells [4] . Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development [5] . Therefore, the goal of this study was to investigate whether the GJA1 gene, which encodes Cx43 , is involved with development and function of the heart. Blood samples were taken from a total of 418 patients with congenital heart defects and 250 controls who were collected from three centers (Lanzhou University, Huazhong University of Science and Technology, and Beijing Children's Hospital). All patients signed the informed consent voluntarily. Clinical assessment of the patients was performed, which included anthropometric measurement, physical examination for dysmorphism and malformation, and radiological evaluation. The patients also underwent a chest X-ray examination, electrocardiogram, and ultrasonic echocardiogram. Genomic DNA from 418 CHD patients and 250 healthy controls was extracted from peripheral blood leukocytes using a standard method. The coding regions and their flanking introns of Cx43 were then amplified by polymerase chain reaction (PCR) in all patients and control individuals using the following primers: 5′GAAATACGTGAAACCGTTGG3′ and 5′CCTGGTGCACTTTCTACAGC3′. The product was tested and directly sequenced on an automated sequencer ABI 3730XL (Applied Biosystems, Foster City, CA, USA) to perform mutation analysis. The CLC DNA workbench 5 system was used to analyze the conversion of the Cx43 protein sequence. Mutation analysis of the entire coding region of ). Nucleotide variation was very prominent in CHD patients but the same changes were not detected in 250 normal controls. Cx43 revealed heterozygous missense mutations in all individuals diagnosed with CHD who were analyzed in the current study by sequencing ( Fig. 1 The mutations were distributed throughout the ; ). We also found two novel silent mutations, c.330G>A (E110E) and c.456G>A (R239R), as well as two previously reported polymorphisms (rs2227888 and rs57946868). Cx43 protein. Mutation 1 (c. 458G>A, resulting in R153Q) was detected in the third transmembrane domain, and mutation 2 (c. 781G>T, resulting in G261W) and mutation 3 (c.968 C>T, resulting in A323V) were detected in the second cytoplasmic region of Cx43 ( Fig. 2 Table 1 We found three novel missense variants (c.458G>A, c.781G>T, and c.968 C>T) in 418 patients with CHD. We also showed that the c.458G>A heterozygous variant lies in the third transmembrane domain, and we speculate that there is a relationship to the combination between Cx43 gene and the epicytes. It is also noteworthy that the two variants c.781G>T and c.968C>T are located in the second cytoplasmic region, which is the Cx43 C-terminal. The Cx43 protein is considered to play a crucial role in development and function because it allows direct cell-to-cell exchange of molecules, which mediate multiple signaling events. Gap junctions, clusters of cell-to-cell channels composed of connexins, mediate the orderly spread of electrical excitation throughout the heart. Experimental evidence from transgenic murine models [6–9] combined with findings from the diseased human heart [10–13] indicate that altered ventricular gap junction organization and connexin expression are key contributors to rhythm disturbances and contractile dysfunction. The ). In contrast, the cytoplasmic domains are unique to each connexin; the cytoplasmic loop and the carboxy terminus vary extensively in length and amino acid composition Cx43 C-terminal sequence contains a proline-rich region corresponding to the consensus of a protein–protein interaction PY-motif (xPPxY), and an overlapping putative tyrosine-based sorting signal (Yxxf; f=hydrophobic), known to play a role in the intracellular trafficking of many membrane proteins [14] . Therefore, we believe that the variants in our study are more likely to lead to alterations of the mature protein. Most importantly, the three non-synonymous variations are altitudinally conservative in many species ( Homo sapiens , Rattus norvegicus , Mus musculus , Gallus gallus , Equus caballus ) ( Fig. 3 [15] . The missense mutations may change certain structures of Cx43 and influence the interaction with other connexons to form a normal membrane channel. In our study, most patients suffer from ventricular septal defect (VSD) ( ). The incidence rate of VSD in newborn infants varies from 2% to 5% Table 2 [16,17] . Ewart [18] and Sullivan [19] prove that abnormal expression of Cx43 can cause abnormalities of outflow tract of right ventricle. Two patients with mutations suffer from VSD in the research. In conclusion, the present mutational analysis of Cx43 gene in 418 Chinese CHD patients revealed three novel non-synonymous variants close to hydrophobic C-terminus. To our knowledge, the quantity of sample in our study is the biggest for the first time. At the same time, the alteration in Cx43 may affect the protein structure to influence the channel gating. Evidence from the previous and present studies has made us firmly believe that Cx43 gene plays an important role in human cardiac development and the pathogenesis of CHD. This work was supported by the National Basic Research Program of China (2007CB511905), the National Infrastructure Program of Chinese Genetic Resources (2006DKA21300), and the Program for Youth Innovation Talents of Gansu Province, from government of Gansu Province. We would like to thank the CHD patients for their involvement in this study. We also would like to thank the clinicians who provided the patients for this study. At the same time, the authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [20] .
第一作者机构：[1]Peking Union Med Coll, Grad Sch, Beijing 100021, Peoples R China;[7]Natl Res Inst Family Planning, Beijing 100081, Peoples R China;
通讯作者：
通讯机构：[1]Peking Union Med Coll, Grad Sch, Beijing 100021, Peoples R China;[7]Natl Res Inst Family Planning, Beijing 100081, Peoples R China;[8]WHO, Collaborating Ctr Res Human Reprod, Beijing, Peoples R China;
推荐引用方式(GB/T 7714)：
Wang Binbin,Wen Qiaolian,Xie XiaoDong,et al.Mutation analysis of Connexon43 gene in Chinese patients with congenital heart defects[J].INTERNATIONAL JOURNAL OF CARDIOLOGY.2010,145(3):487-489.doi:10.1016/j.ijcard.2009.06.026.
APA：
Wang, Binbin,Wen, Qiaolian,Xie, XiaoDong,Liu, Shiguo,Liu, Mugen...&Ma, Xu.(2010).Mutation analysis of Connexon43 gene in Chinese patients with congenital heart defects.INTERNATIONAL JOURNAL OF CARDIOLOGY,145,(3)
MLA：
Wang, Binbin,et al."Mutation analysis of Connexon43 gene in Chinese patients with congenital heart defects".INTERNATIONAL JOURNAL OF CARDIOLOGY 145..3(2010):487-489
