Objectives: To study the association of ventricular septal defect (VSD) with rare variations in the promoter region of HAND2 gene, as well as related molecular mechanisms.
Methods: Blood samples were collected from 349 children with VSD and 345 healthy controls. The target fragments were amplified by polymerase chain reaction and sequenced to identify the rare variation sites in the promoter region of the HAND2 gene. Dual-luciferase reporter assay was used to perform a functional analysis of the variation sites. Electrophoretic mobility shift assay (EMSA) was used to investigate related molecular mechanisms. TRANSFAC and JASPAR databases were used to predict transcription factors.
Results: Sequencing revealed that three variation sites (g.173530852A>G, g.173531173A>G, and g.173531213C>G) were only observed in the promoter region of the HAND2 gene in 10 children with VSD, among whom 4 children had only one variation site. The dual-luciferase reporter assay revealed that g.173531213C>G reduced the transcriptional activity of the HAND2 gene promoter. EMSA and transcription factor prediction revealed that g.173531213C>G created a binding site for transcription factor.
Conclusions: The rare variation, g.173531213C>G, in the promoter region of the HAND2 gene participates in the development and progression of VSD possibly by affecting the binding of transcription factors.
目的: 对室间隔缺损（ventricular septal defect，VSD）与HAND2基因启动子区罕见变异的关联性进行分析，并对相关分子机制进行初步探索。方法: 收集349例VSD组患儿和345例健康对照组儿童血液样本，通过聚合酶链反应扩增目的片段并进行测序，确定HAND2基因启动子区的罕见变异位点。使用双荧光素酶检测进行变异位点功能分析，并通过电泳迁移率变动分析进行分子机制研究，同时使用TRANSFAC和JASPAR数据库预测转录因子。结果: 通过测序发现3个变异位点（g.173530852A>G、g.173531173A>G和g.173531213C>G）只出现在10例VSD患儿的HAND2基因启动子上，其中4例患儿仅有1个变异位点。双荧光素酶检测显示g.173531213C>G降低了HAND2基因启动子转录活性。电泳迁移率变动分析和转录因子预测提示g.173531213C>G产生了1个转录因子结合位点。结论: HAND2基因启动子区的罕见变异g.173531213C>G可能通过影响转录因子的结合，进而参与VSD的发生发展。.
Keywords: Child; HAND2 gene; Rare variation; Ventricular septal defect.