中国人群中非综合征耳聋相关MARVELD2 (DFNB49)基因新单核苷酸多态性位点分析

New SNP variants of MARVELD2 ( DFNB49 ) associated with non-syndromic hearing loss in Chinese population # # 中国人群中非综合征耳聋相关 MARVELD2 ( DFNB49 )基因新单核苷酸多态性位点分析 1 2 first-author 1 2 2 2 3 3 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:yechency@zju.edu.cn †E-mail:yechency@zju.edu.cn yechency@zju.edu.cn 1 2 Division of Medical Genetics and Genomics, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Division of Medical Genetics and Genomics, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Institute of Genetics, Zhejiang University, Hangzhou 310058, China Institute of Genetics, Zhejiang University, Hangzhou 310058, China Attardi Institute of Mitochondrial Biomedicine, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China Attardi Institute of Mitochondrial Biomedicine, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China Non-Syndromic Hearing Loss (NSHL) is a common defect in humans. Variants of MARVELD2 at the DFNB49 locus have been shown to cause bilateral, moderate to profound NSHL. However, the role of MARVELD2 in NSHL susceptibility in the chinese population has not been studied. Here we conducted a case-control study in an eastern chinese population to profile the spectrum and frequency of MARVELD2 variants, as well as the association of MARVELD2 gene variants with NSHL. Our results showed that variants identified in the chinese population are significantly different from those reported in Slovak, Hungarian, and Czech Roma, as well as Pakistani families. We identified 11 variants in a cohort of 283 NSHL cases. Through Sanger sequencing and bioinformatics analysis, we found that c.730G>A variant has detrimental effects in the eastern chinese population, and may have relatively high correlation with NSHL pathogenicity. 目的 2 探究 MARVELD2 在中国非综合征耳聋(NSHL)人群中的突变频谱和突变频率. 创新点 2 发现 MARVELD2 突变频谱具有明显种族特异性.中国NSHL人群中的突变位点及频率不同于已报道的其他人群,并首次筛选到新致聋候选突变 MARVELD2 c.730G > A.本研究有助于进一步阐释 MARVELD2 在NSHL中的作用. 方法 2 收集283例NSHL患者外周血,提取基因组DNA,涉及9对引物覆盖 MARVELD2 基因编码区,经聚合酶链反应(PCR)扩增后Sanger测序.测序结果与参考序列比对,获得的 MARVELD2 变异位点通过正常人群频率比较、氨基酸保守性分析、氨基酸性质分析、SIFT和PolyPhen有害性预测及蛋白结构功能预测分析等进一步筛选得到耳聋候选突变位点. 结论 2 中国NSHL人群的 MARVELD2 突变位点与巴基斯坦人群,以及斯洛伐克、匈牙利和捷克罗马人群不同,具有明显的种族特异性.本研究在283个NSHL病例中共鉴定了11个变异位点.其中,c.730G > A突变可能影响MARVELD2蛋白的正常功能,与NSHL致病有较高的相关性,是一个候选致聋突变. Chinese population MARVELD2 Non-Syndromic Hearing Loss SNP variants MARVELD2 非综合征耳聋(NSHL) 单核苷酸多态性位点 Non-syndromic hearing loss (NSHL) is a common defect in humans. Variants of MARVELD2 at the DFNB49 locus have been shown to cause bilateral, moderate to profound NSHL. However, the role of MARVELD2 in NSHL susceptibility in the Chinese population has not been studied. Here we conducted a case-control study in an eastern Chinese population to profile the spectrum and frequency of MARVELD2 variants, as well as the association of MARVELD2 gene variants with NSHL. Our results showed that variants identified in the Chinese population are significantly different from those reported in Slovak, Hungarian, and Czech Roma, as well as Pakistani families. We identified 11 variants in a cohort of 283 NSHL cases. Through Sanger sequencing and bioinformatics analysis, we found that c.730G > A variant has detrimental effects in the eastern Chinese population, and may have relatively high correlation with NSHL pathogenicity. NSHL is a common human sensory defect (Smith et al., 2005 2005 ; Morton and Nance, 2006 2006 ). Up to now, more than 50 genes with autosomal recessive inheritance and 30 genes with autosomal dominant inheritance have been reported to be associated with NSHL (Dror and Avraham, 2009 2009 , 2010 2010 ; Schraders et al., 2012 2012 ). MARVELD2 , which encodes tricellulin, is located on chromosome 5q13.2 and linked to the DFNB49 locus (Ramzan et al., 2005 2005 ; Riazuddin et al., 2006 2006 ; Mašindová et al., 2015 2015 ). In the human inner ear, there are many fluid-filled compartments of different ionic compositions. The strict compartmentalization plays a significant role in hearing. Tricellulin, together with other tight junction proteins, functions as a seal control lateral ion diffusion to ensure normal hearing (Sterkers et al., 1988 1988 ; Kitajiri et al., 2004 2004 ). Thus, it is easy to understand that variants in MARVELD2 may cause hearing loss to varying degrees (Chishti et al., 2008 2008 ). Up to now, seven single nucleotide polymorphism (SNP) variants of MARVELD2 have been reported to cause human NSHL, including c.1183-1G > A, c.1331+1G > A, c.1331+2T > C, c.1331+2deITGAG, c.1498C > T, c.1543delA, and p.C395-Q501del (Riazuddin et al., 2006 2006 ; Chishti et al., 2008 2008 ; Babanejad et al., 2012 2012 ; Šafka Brožková et al., 2012 2012 ; Mašindová et al., 2015 2015 ; Nayak et al., 2015 2015 ). However, the mutational spectrum and frequency of MARVELD2 in the Chinese NSHL population are still poorly understood. In the present study, a systematic mutational screening of MARVELD2 was performed in a Chinese NSHL cohort. A total of 283 genetically unrelated NSHL Chinese subjects were recruited. Informed consent, blood samples, and clinical evaluations were obtained from all participants under protocols approved by the Ethics Committee of both Zhejiang University and Wenzhou Medical University, China. Genomic DNA was isolated from peripheral blood as detailed previously (Zheng et al., 2015 2015 ). Nine pairs of primers were used to amplify the coding sequence of MARVELD2 (Table S1 S1 ). Polymerase chain reaction (PCR) products were identified by agarose gel electrophoresis, and the purified fragments were then analyzed by direct sequencing. Subsequently, bidirectional sequencing results were compared with a reference sequence from GenBank (NM_001244734). A filter was created to analyze candidate variants, which consists of five rounds of screening, including allele frequency, amino acid conservation, amino acid substitution, detrimental prediction, and protein domain and structure analysis. The protein three dimensional (3D) structure and function were predicted by iterative threading assembly refinement (I-TASSER) (Yang et al., 2015 2015 ). All the statistical analyses were carried out by SPSS software (Version 21.0, SPSS Inc., Chicago, IL, USA). The chi-square test or Fisher’s exact test was performed to evaluate the detrimental effect of all variants detected in the affected subjects, and P -value of < 0.05 was regarded as statistically significant. In order to study the mutation spectrum of MARVELD2 in the Chinese population, we recruited 283 NSHL subjects from Zhejiang Province in China. This population consisted of 163 males and 120 females, including 191 profound, 47 severe, 37 moderate, and 8 mild hearing loss. Their ages of onset ranged from congenital to 60 years, with an average of 6.8 years. DNA fragments spanning the entire coding region of MARVELD2 were amplified by PCR. As shown in Table 1 1 , 11 variants localized in exons were identified and all are heterozygous in affected subjects. Except for c.1499G > A, all variants are in exon 2 (Fig. 1a 1a ) which is the largest exon in tricellulin and includes the MARVEL domain. Variants that passed the filter perfectly were denoted by asterisks. Statistical analysis of the variants detected in both controls and affected subjects was carried out by chi-square test; P -value of < 0.05 was considered statistically significant. SIFT: scale-invariant feature transform; ExAC: the Exome Aggregation Consortium 2 Variant 2 Amino acid substitution 2 Amino acid property change 2 Exon 2 Detrimental effect (SIFT) 2 Detrimental effect(PolyPhen) 2 Conservation (%) 2 Allele frequency in 283 affected subjects (%) 2 Allele frequency in 252 controls (%) 2 P -value b b (Fisher’s exact test) 2 Allele frequency in ExAC (%) East Asian Total *c.730G > A a a p.G244R Non-polar aliphatic to alkaline 2/7 Damaging Probably damaging 100.00 1.06 0 0.032 *c.772G>A p.V258M Non-polar aliphatic to polar neutral 2/7 Damaging Probably damaging 100.00 0.18 0 1.000 0 0 *c.1006C>T p.R336W Alkaline to aromatic 2/7 Damaging Probably damaging 100.00 0.18 0 1.000 0 0 c.98C>T p.T33I Polar to non-polar 2/7 Tolerated Benign 81.16 34.81 50.42 0 48.62 49.97 c.127G>A p.A43T Non-polar aliphatic to polar neutral 2/7 Damaging Benign 85.19 0.18 0 1.000 0.01 0 c.130G>A p.D44N Acidic to polar neutral 2/7 Damaging Probably damaging 88.89 0.18 0.42 0.604 0.12 0.01 c.230C>T p.P77L Non-polar aliphatic to non-polar aliphatic 2/7 Damaging Probably damaging 100.00 0.18 0 1.000 c.592G>A p.V198M Non-polar aliphatic to polar neutral 2/7 Tolerated Probably damaging 85.88 0.53 0.85 0.713 0.24 0.02 c.949C>G p.R317G Alkaline to non-polar aliphatic 2/7 Damaging Probably damaging 100.00 0.35 0.42 1.000 0.05 0 c.1109C>T p.A370V Non-polar aliphatic to non-polar aliphatic 2/7 Tolerated Benign 85.19 0.18 0 1.000 0.07 0 c.1499G>A p.R500Q Alkaline to polar neutral 5/7 Tolerated Benign 62.96 0.35 0 0.501 0.13 0.01 (a) The proximate locations of 11 selected sense variants are shown; almost all these variants are in exon 2, only c.1499G > A in exon 5. Representative sequencing chromatograms of three variants which passed the filter are listed. (b) Protein structure of human tricellulin. MARVEL domains are shown in purple, and the occludin- ELL domain is in yellow. Red residues indicate the positions of mutations which pass the filter. (c) 3D structure of human tricellulin was predicted using the I-TASSER software. Protein domains are shown in grey, and ligands in green. Red spheres indicate the positions of mutations which pass the filter (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) In order to comprehensively analyze these variants, a sophisticated filter was created. The cut-down thresholds of frequency are less than 0.05 in both population groups (1000 Genomes, as well as the In-House Database provided by BGI China that is composed of 252 unaffected Chinese subjects). Next, we filtered out neutral or quiet mutations. The changes of the amino acid sequence of the expressed protein may not necessarily affect protein structure or function. For example, the replaced amino acid may have very similar chemical properties to the amino acid of the original allele, in which case, the protein may still function normally. In this study, the c.230C > T and c.1109C > T variants caused amino acid change from Pro to Leu at position 77 and from Ala to Val at position 370, respectively. All these amino acids have the non-polar aliphatic side chain. Thus these variants were filtered out (Chasman and Adams, 2001 2001 ; Teng et al., 2008 2008 ). In addition, c.127G > A, c.1499G > A, c.1109C > T, c.98C > T, and c.592G > A did not pass the screen of bioinformatic tools (scale-invariant feature transform (SIFT) and PolyPhen-2) (Ng and Henikoff, 2003 2003 ; Adzhubei et al., 2013 2013 ). Finally, three variants c.772G > A, c.730G > A, and c.1006C > T passed the filter (Fig. 1b 1b ), and could be potential pathogenic variants responsible for NSHL. In addition, only c.730G > A had the P -value < 0.05 among the three putative pathogenic variants filtered by our criteria when calculated by Fisher’s exact test. For further confirmation, we used I-TASSER to predict the structure and function of MARVELD2 (Yang and Zhang, 2015 2015 ; Wang et al., 2017 2017 ). As shown in Fig. 1c 1c , c.730G > A causes a substitution of 244th glycine to arginine, which is predicted as a ligand binding site. This may lead to an abnormal interactive function of MARVELD2 and eventually cause NSHL. MARVELD2 encodes tricellulin, which is an important component of tricellular tight junctions and is involved in the formation of tricellular contacts (Ramzan et al., 2005 2005 ; Riazuddin et al., 2006 2006 ). In tight junctions, tricellulin minimizes permeability to macromolecules but not to ions, and this minimization is a significant factor in normal hearing (Krug et al., 2009 2009 ). Raleigh et al. ( 2010 2010 ) showed that MARVEL, occludin, and tricellulin have distinct but overlapping functions at the tight junction. Oda et al. ( 2014 2014 ) reported that tricellulin also regulates F-actin organization through Cdc42 during cell–cell junction formation. It had been reported that variants in MARVELD2 cause bilateral, moderate to profound NSHL (Riazuddin et al., 2006 2006 ; Chishti et al., 2008 2008 ). A gene-targeted knock-in (TricR497X/R497X) mouse was generated by Nayak et al. ( 2015 2015 ), which aimed to mimic the pathology of a human MARVELD2 variant. The results showed that deafness appears to be caused either by an increase in K + ion concentration around the basolateral surfaces or by an increase in small molecules such as adenosine triphosphate (ATP) around the hair bundle, leading to cellular dysfunction and degeneration (Higashi et al., 2013 2013 ). Recently, Nayak et al. ( 2015 2015 ) reported that MARVELD2 variants are responsible for about 1.5% of NSHL in Pakistani families. In this study, we analyzed MARVELD2 variants in the Chinese population and identified an SNP variation (c.730G > A) which may have a correlation with NSHL pathogenicity in that population. The c.730G > A variant showed a significantly higher frequency in the patient population (1.06%) than in controls (0.00%). In addition, c.730G > A caused a substitution for glycine to arginine, which may result in severe change of structure and function in the ligand binding domain and eventually lead to NSHL. Thus, our results indicated c.730G > A as a pathogenic variant of NSHL pathogenicity. Up to now, seven SNP variants of MARVELD2 causing NSHL have been reported in families of Pakistan, Slovak, Hungarian, and Czech Roma origins (Riazuddin et al., 2006 2006 ; Chishti et al., 2008 2008 ; Babanejad et al., 2012 2012 ; Šafka Brožková et al., 2012 2012 ; Mašindová et al., 2015 2015 ; Nayak et al., 2015 2015 ), as shown in Table 2 2 . However, the MARVELD2 variants identified in this study were not found in any of the previously reported studies. Our work profiles the spectrum and frequency of MARVELD2 variants in the Chinese population, and this expands the MARVELD2 variants pool. In addition, the study increases knowledge on MARVELD2 variants causing NSHL, and this could be important for clinical diagnosis. Mutation Amino acids substitution Origin of family Reference c.1183-1G>A Pakistani Riazuddin et al., 2006 2006 c.1331+1G>A Pakistani Chishti et al., 2008 2008 c.1331+2T>C Pakistani, Czech Roma, Slovak Roma, Hungarian Roma Riazuddin et al., 2006 2006 ; Šafka Brožková et al., 2012 2012 ; Mašindová et al., 2015 2015 c.1331+2deITGAG Pakistani Riazuddin et al., 2006 2006 c.1498C>T p.R500X Pakistani Riazuddin et al., 2006 2006 c.1543delA p.K517RfsX16 Iranian Babanejad et al., 2012 2012 Exons 4–5 deletion p.C395-Q501del Pakistani Nayak et al., 2015 2015 c.1006C>T p.R336W Chinese This report c.730G>A p.G244R Chinese This report c.772G>A p.V258M Chinese This report c.949C>G p.R317G Chinese This report References I Adzhubei , DM Jordan , SR Sunyaev . Predicting functional effect of human missense mutations using polyphen-2 . Curr Protoc Hum Genet , 2013 . 76 ( 1 ): 7.20.1 - 72041 . DOI: 10.1002/0471142905.hg0720s76 http://doi.org/10.1002/0471142905.hg0720s76 . Predicting functional effect of human missense mutations using polyphen-2 M Babanejad , Z Fattahi , N Bazazzadegan , 等 . A comprehensive study to determine heterogeneity of autosomal recessive nonsyndromic hearing loss in Iran . Am J Med Genet A , 2012 . 158A ( 10 ): 2485 - 2492 . 22903915 DOI: 10.1002/ajmg.a.35572 http://doi.org/10.1002/ajmg.a.35572 . A comprehensive study to determine heterogeneity of autosomal recessive nonsyndromic hearing loss in Iran D Chasman , RM Adams . Predicting the functional consequences of non-synonymous single nucleotide polymorphisms: structure-based assessment of amino acid variation . J Mol Biol , 2001 . 307 ( 2 ): 683 - 706 . 11254390 DOI: 10.1006/jmbi.2001.4510 http://doi.org/10.1006/jmbi.2001.4510 . Predicting the functional consequences of non-synonymous single nucleotide polymorphisms: structure-based assessment of amino acid variation MS Chishti , A Bhatti , S Tamim , 等 . Splice-site mutations in the TRIC gene underlie autosomal recessive nonsyndromic hearing impairment in Pakistani families . J Hum Genet , 2008 . 53 ( 2 ): 101 - 105 . 18084694 DOI: 10.1007/s10038-007-0209-3 http://doi.org/10.1007/s10038-007-0209-3 . Splice-site mutations in the TRIC gene underlie autosomal recessive nonsyndromic hearing impairment in Pakistani families AA Dror , KB Avraham . Hearing loss: mechanisms revealed by genetics and cell biology . Annu Rev Genet , 2009 . 43 411 - 437 . 19694516 DOI: 10.1146/annurev-genet-102108-134135 http://doi.org/10.1146/annurev-genet-102108-134135 . Hearing loss: mechanisms revealed by genetics and cell biology AA Dror , KB Avraham . Hearing impairment: a panoply of genes and functions . Neuron , 2010 . 68 ( 2 ): 293 - 308 . 20955936 DOI: 10.1016/j.neuron.2010.10.011 http://doi.org/10.1016/j.neuron.2010.10.011 . Hearing impairment: a panoply of genes and functions T Higashi , DR Lenz , M Furuse , 等 . A “Tric” to tighten cell–cell junctions in the cochlea for hearing . J Clin Invest , 2013 . 123 ( 9 ): 3712 - 3715 . 23979155 DOI: 10.1172/JCI69651 http://doi.org/10.1172/JCI69651 . A “Tric” to tighten cell–cell junctions in the cochlea for hearing SI Kitajiri , M Furuse , K Morita , 等 . Expression patterns of claudins, tight junction adhesion molecules, in the inner ear . Hear Res , 2004 . 187 ( 1-2 ): 25 - 34 . 14698084 DOI: 10.1016/s0378-5955(03)00338-1 http://doi.org/10.1016/s0378-5955(03)00338-1 . Expression patterns of claudins, tight junction adhesion molecules, in the inner ear SM Krug , S Amasheh , JF Richter , 等 . Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability . Mol Biol Cell , 2009 . 20 ( 16 ): 3713 - 3724 . 19535456 DOI: 10.1091/mbc.E09-01-0080 http://doi.org/10.1091/mbc.E09-01-0080 . Tricellulin forms a barrier to macromolecules in tricellular tight junctions without affecting ion permeability I Mašindová , A Šoltýsová , L Varga , 等 . MARVELD2 ( DFNB49 ) mutations in the hearing impaired central European Roma population–prevalence, clinical impact and the common origin . PLoS ONE , 2015 . 10 ( 4 ): e0124232 25885414 DOI: 10.1371/journal.pone.0124232 http://doi.org/10.1371/journal.pone.0124232 . MARVELD2 (DFNB49) mutations in the hearing impaired central European Roma population–prevalence, clinical impact and the common origin CC Morton , WE Nance . Newborn hearing screening–a silent revolution . New Engl J Med , 2006 . 354 ( 20 ): 2151 - 2164 . 16707752 DOI: 10.1056/NEJMra050700 http://doi.org/10.1056/NEJMra050700 . Newborn hearing screening–a silent revolution G Nayak , L Varga , C Trincot , 等 . Molecular genetics of MARVELD2 and clinical phenotype in Pakistani and Slovak families segregating DFNB49 hearing loss . Hum Genet , 2015 . 134 ( 4 ): 423 - 437 . 25666562 DOI: 10.1007/s00439-015-1532-y http://doi.org/10.1007/s00439-015-1532-y . Molecular genetics of MARVELD2 and clinical phenotype in Pakistani and Slovak families segregating DFNB49 hearing loss PC Ng , S Henikoff . SIFT: predicting amino acid changes that affect protein function . Nucleic Acids Res , 2003 . 31 ( 13 ): 3812 - 3814 . 12824425 DOI: 10.1093/nar/gkg509 http://doi.org/10.1093/nar/gkg509 . SIFT: predicting amino acid changes that affect protein function Y Oda , T Otani , J Ikenouchi , 等 . Tricellulin regulates junctional tension of epithelial cells at tricellular contacts through Cdc42 . J Cell Sci , 2014 . 127 ( Pt 19 ): 4201 - 4212 . 25097232 DOI: 10.1242/jcs.150607 http://doi.org/10.1242/jcs.150607 . Tricellulin regulates junctional tension of epithelial cells at tricellular contacts through Cdc42 DR Raleigh , AM Marchiando , Y Zhang , 等 . Tight junction-associated marvel proteins MarvelD3, tricellulin, and occludin have distinct but overlapping functions . Mol Biol Cell , 2010 . 21 ( 7 ): 1200 - 1213 . 20164257 DOI: 10.1091/mbc.E09-08-0734 http://doi.org/10.1091/mbc.E09-08-0734 . Tight junction-associated marvel proteins MarvelD3, tricellulin, and occludin have distinct but overlapping functions K Ramzan , RS Shaikh , J Ahmad , 等 . A new locus for nonsyndromic deafness DFNB49 maps to chromosome 5q12.3-q14.1 . Hum Genet , 2005 . 116 ( 1-2 ): 17 - 22 . 15538632 DOI: 10.1007/s00439-004-1205-8 http://doi.org/10.1007/s00439-004-1205-8 . A new locus for nonsyndromic deafness DFNB49 maps to chromosome 5q12.3-q14.1 S Riazuddin , ZM Ahmed , AS Fanning , 等 . Tricellulin is a tight-junction protein necessary for hearing . Am J Hum Genet , 2006 . 79 ( 6 ): 1040 - 1051 . 17186462 DOI: 10.1086/510022 http://doi.org/10.1086/510022 . Tricellulin is a tight-junction protein necessary for hearing D Šafka Brožková , J Laštůvková , H Štěpánková , 等 . DFNB49 is an important cause of non-syndromic deafness in Czech Roma patients but not in the general Czech population . Clin Genet , 2012 . 82 ( 6 ): 579 - 582 . 22097895 DOI: 10.1111/j.1399-0004.2011.01817.x http://doi.org/10.1111/j.1399-0004.2011.01817.x . DFNB49 is an important cause of non-syndromic deafness in Czech Roma patients but not in the general Czech population M Schraders , L Ruiz-Palmero , E Kalay , 等 . Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment . Am J Hum Genet , 2012 . 91 ( 5 ): 883 - 889 . 23122587 DOI: 10.1016/j.ajhg.2012.09.012 http://doi.org/10.1016/j.ajhg.2012.09.012 . Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment RJH Smith , JF Jr Bale , KR White . Sensorineural hearing loss in children . Lancet , 2005 . 365 ( 9462 ): 879 - 890 . 15752533 DOI: 10.1016/S0140-6736(05)71047-3 http://doi.org/10.1016/S0140-6736(05)71047-3 . Sensorineural hearing loss in children O Sterkers , E Ferrary , C Amiel . Production of inner ear fluids . Physiol Rev , 1988 . 68 ( 4 ): 1083 - 1128 . 3054946 DOI: 10.1152/physrev.1988.68.4.1083 http://doi.org/10.1152/physrev.1988.68.4.1083 . Production of inner ear fluids S Teng , E Michonova-Alexova , E Alexov . Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions . Curr Pharm Biotechnol , 2008 . 9 ( 2 ): 123 - 133 . 18393868 DOI: 10.2174/138920108783955164 http://doi.org/10.2174/138920108783955164 . Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions Y Wang , J Virtanen , ZD Xue , 等 . I-TASSER-MR: automated molecular replacement for distant-homology proteins using iterative fragment assembly and progressive sequence truncation . Nucleic Acids Res , 2017 . 45 ( W1 ): W429 - W434 . 28472524 DOI: 10.1093/nar/gkx349 http://doi.org/10.1093/nar/gkx349 . I-TASSER-MR: automated molecular replacement for distant-homology proteins using iterative fragment assembly and progressive sequence truncation JY Yang , Y Zhang . I-TASSER server: new development for protein structure and function predictions . Nucleic Acids Res , 2015 . 43 ( W1 ): W174 - W181 . 25883148 DOI: 10.1093/nar/gkv342 http://doi.org/10.1093/nar/gkv342 . I-TASSER server: new development for protein structure and function predictions JY Yang , RX Yan , A Roy , 等 . The I-TASSER suite: protein structure and function prediction . Nat Methods , 2015 . 12 ( 1 ): 7 - 8 . 25549265 DOI: 10.1038/nmeth.3213 http://doi.org/10.1038/nmeth.3213 . The I-TASSER suite: protein structure and function prediction J Zheng , ZB Ying , ZY Cai , 等 . GJB2 mutation spectrum and genotype-phenotype correlation in 1067 Han Chinese subjects with non-syndromic hearing loss . PLoS ONE , 2015 . 10 ( 6 ): e0128691 26043044 DOI: 10.1371/journal.pone.0128691 http://doi.org/10.1371/journal.pone.0128691 . GJB2 mutation spectrum and genotype-phenotype correlation in 1067 Han Chinese subjects with non-syndromic hearing loss We thank the patients and their families for the participation in this study. Compliance with ethics guidelines Jing ZHENG, Wen-fang MENG, Chao-fan ZHANG, Han-qing LIU, Juan YAO, Hui WANG, Ye CHEN, and Min-xin GUAN declared that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. 10.1631/jzus.B1700185 Project supported by the National Basic Research Priorities Program of China (Nos. 2014CB541702 and 2014CB541704), the National Natural Science Foundation of China (Nos. 81470685 and 81600817), and the Zhejiang Provincial Public Welfare Technology Applied Research Project (No. 2016C33148), China 2017-12-20 2018-03-07 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2019 2019 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 20