Nonsynonymous variants in MYH9 and ABCA4 are the most frequent risk loci associated with nonsyndromic orofacial cleft in Taiwanese population.
Nonsynonymous variants in MYH9 and ABCA4 are the most frequent risk loci associated with nonsyndromic orofacial cleft in Taiwanese population.
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DOI：
10.1186/s12881-016-0322-2
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发表时间：
2016-08-15
影响因子：
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通讯作者：
Cheng PJ
中科院分区：
文献类型：
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作者：
Peng HH;Chang NC;Chen KT;Lu JJ;Chang PY;Chang SC;Wu-Chou YH;Chou YT;Phang W;Cheng PJ
非综合征性口面裂是一种常见的出生缺陷，病因复杂，包括多种遗传和环境危险因素。最近的全基因组分析表明，非综合征性口面裂与多达18个遗传风险位点（ABCA 4、BMP 4、CRISPLD 2、GSTT 1、FGF 8、FGFR 2、FOXE 1、IRF 6、MAFB、MSX 1、MTHFR、MYH 9、PDGFC、PVRL 1、SUMO 1、TGFA、TGFB 3和VAX 1）之间存在关联，每个位点在不同人群中具有不同的相对风险。我们评估这18个遗传风险基因座在非综合征性口面裂和正常对照中的非同义变异，以阐明台湾人群中的特定变异。我们使用下一代测序（NGS）定制面板评估了103例非综合征性口面裂和100例正常对照的这18个遗传风险位点，并基于离子激流个人基因组机器（IT-PGM）的NGS系统进行了全外显子靶向测序研究。IT-PGM数据处理，包括与人类基因组构建19参考基因组（hg 19）的比对、碱基调用、条形码化衔接子序列的修剪和不良信号读数的过滤，使用IT平台特定的管道软件Torrent Suite（版本4.2）和插件“变体调用程序”进行。进一步的高级注释是通过将导出的VCF文件从变体调用者上传到商业软件包Ion Reporter;免费的在线注释软件Vanno和Mutation Taster来促进的。良性或耐受的氨基酸变化被排除后，使用排序不耐受和多态性表型分析。使用桑格测序来验证通过NGS鉴定的显著变体。此外，使用Sequenom MassARRAY（San Diego，CA，USA）在无症状对照中确认每种变体。我们在10个研究基因（ABCA 4、MYH 9、MTHFR、CRISPLD 2、FGF 8、PVRL 1、FOXE 1、VAX 1、FGFR 2和IRF 6）中共鉴定出22种非综合征性口面裂特异性非同义变异，包括19个单核苷酸变异、2个缺失和1个重复。MYH 9和ABCA 4的非同义变异分别在6和5个个体中检测到，被确定为台湾人群中非综合征性口面裂最常见的风险位点。MYH 9和ABCA 4的非同义变异被确定为台湾人群中非综合征性口面裂最常见的风险位点。我们研究中的这些发现提供了关于不同人群中与非综合征性口面裂相关的特定变异的额外信息，并证明了我们定制的NGS面板的功能，该面板在临床上可用于同时检测与非综合征性口面裂相关的多个基因。本文的在线版本（doi：10.1186/s12881-016-0322-2）包含补充材料，可供授权用户使用。
Nonsyndromic orofacial cleft is a common birth defect with a complex etiology, including multiple genetic and environmental risk factors. Recent whole genome analyses suggested associations between nonsyndromic orofacial cleft and up to 18 genetic risk loci (ABCA4, BMP4, CRISPLD2, GSTT1, FGF8, FGFR2, FOXE1, IRF6, MAFB, MSX1, MTHFR, MYH9, PDGFC, PVRL1, SUMO1, TGFA, TGFB3, and VAX1), each of which confers a different relative risk in different populations. We evaluate the nonsynonymous variants in these 18 genetic risk loci in nonsyndromic orofacial clefts and normal controls to clarify the specific variants in Taiwanese population. We evaluated these 18 genetic risk loci in 103 cases of nonsyndromic orofacial clefts and 100 normal controls using a next-generation sequencing (NGS) customized panel and manipulated a whole-exon targeted-sequencing study based on the NGS system of an Ion Torrent Personal Genome Machine (IT-PGM). IT-PGM data processing, including alignment with the human genome build 19 reference genome (hg19), base calling, trimming of barcoded adapter sequences, and filtering of poor signal reads, was performed using the IT platform-specific pipeline software Torrent Suite, version 4.2, with the plug-in “variant caller” program. Further advanced annotation was facilitated by uploading the exported VCF file from Variant Caller to the commercial software package Ion Reporter; the free online annotation software Vanno and Mutation Taster. Benign or tolerated amino acid changes were excluded after analysis using sorting intolerant from tolerant and polymorphism phenotyping. Sanger sequencing was used to validate the significant variants identified by NGS. Furthermore, each variant was confirmed in asymptomatic controls using the Sequenom MassARRAY (San Diego, CA, USA). We identified totally 22 types of nonsynonymous variants specific in nonsyndromic orofacial clefts, including 19 single nucleotide variants, 2 deletions, and 1 duplication in 10 studied genes(ABCA4, MYH9, MTHFR, CRISPLD2, FGF8, PVRL1, FOXE1, VAX1, FGFR2, and IRF6). Nonsynonymous variants in MYH9 and ABCA4, which were detected in 6 and 5 individuals, respectively, were identified to be the most frequent risk loci in nonsyndromic orofacial clefts in the Taiwanese population. Nonsynonymous variants in MYH9 and ABCA4 were identified to be the most frequent risk loci in nonsyndromic orofacial clefts in the Taiwanese population. These findings in our study have provided additional information regarding specific variants associated with nonsyndromic orofacial clefts in different population and demonstrate the power of our customized NGS panel, which is clinically useful for the simultaneous detection of multiple genes associated with nonsyndromic orofacial clefts. The online version of this article (doi:10.1186/s12881-016-0322-2) contains supplementary material, which is available to authorized users.