1 Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, China.
2 Shanghai Stomatological Hospital, Shanghai, China.
J Dent Res. 2018 Apr;97(4):388-394. doi: 10.1177/0022034517743930. Epub 2017 Dec 5.
Nonsyndromic orofacial clefts (NSOCs) are congenital newborn malformations. Myosin heavy chain 9 ( MYH9) is a candidate gene of NSOCs. To investigate the associations between single-nucleotide polymorphisms (SNPs) of MYH9 and NSOC susceptibility, a 2-stage case-control study was designed and 4 potentially functional SNPs of MYH9 (rs12107, rs2269529, rs9619601, rs5756130) were selected and genotyped by iPLEX Sequenom MassARRAY and TaqMan assay in the first stage (599 NSOC cases and 590 controls). The significant SNPs in the first stage were replicated in the second stage (676 NSOC cases and 705 controls) by TaqMan assay. Reverse transcription polymerase chain reaction, cell transfection, and luciferase assay were performed accordingly to explore their functionality. In stage I, rs12107 was nominally associated with NSOCs, whereas rs2269529 showed a significant association (rs12107: P = 0.028; rs2269529: P = 0.001). In stage II, rs12107 was nominally associated with NSOCs, and rs2269529 showed a significant association (rs12107: P = 0.014; rs2269529: P = 0.006). In combined stages, these 2 SNPs gained significant associations (rs12107: P = 0.004; rs2269529: P = 4.4 × 10). In subphenotype analysis, these 2 SNPs were associated with cleft lip only (CLO) and cleft lip with palate (CLP), and rs2269529 was also associated with cleft palate only (CPO). Haplotype analysis revealed associations between rs12107-G/rs2269529-T and NSOC susceptibility ( P = 0.011). Combined analysis of rs12107 and rs2269529 indicated the risk of NSOCs increased with the number of risk alleles (rs12107-G and rs2269529-T, P for trend = 0.008). MYH9 SNP rs12107 AG + GG and rs2269529 CT + TT were associated with higher MYH9 expression in lip tissues compared with their corresponding wild-type homozygote. For rs12107, higher luciferase activities of G allele than A allele were observed in the luciferase assay. MYH9 was downregulated when transfecting its putative binding target miR-196b-3p into human embryo plate mesenchyme (HEPM) and C2C12 cell lines. For rs2269529, C > T contributed to increased MYH9 messenger RNA. In conclusion, rs12107 and rs2269529 were associated with the expression of MYH9 and contributed to the susceptibility of NSOCs.
非综合征性口面裂（NSOC）是一种先天性新生儿畸形。肌球蛋白重链 9（MYH9）是 NSOC 的候选基因。为了研究 MYH9 单核苷酸多态性（SNP）与 NSOC 易感性之间的关系，进行了一项两阶段病例对照研究，并选择了 MYH9 的 4 个潜在功能 SNP（rs12107、rs2269529、rs9619601、rs5756130），并在第一阶段（599 例 NSOC 病例和 590 例对照）通过 iPLEX Sequenom MassARRAY 和 TaqMan 检测进行基因分型。在第二阶段（676 例 NSOC 病例和 705 例对照）通过 TaqMan 检测对第一阶段的显著 SNP 进行了复制。相应地进行了逆转录聚合酶链反应、细胞转染和荧光素酶检测，以探讨其功能。在第一阶段，rs12107 与 NSOC 呈名义关联，而 rs2269529 则表现出显著关联（rs12107：P=0.028；rs2269529：P=0.001）。在第二阶段，rs12107 与 NSOC 呈名义关联，rs2269529 也表现出显著关联（rs12107：P=0.014；rs2269529：P=0.006）。在联合阶段，这两个 SNP 获得了显著的关联（rs12107：P=0.004；rs2269529：P=4.4×10）。在亚表型分析中，这两个 SNP 与唇裂（CLO）和唇裂伴腭裂（CLP）有关，rs2269529 也与单纯腭裂（CPO）有关。单体型分析显示 rs12107-G/rs2269529-T 与 NSOC 易感性之间存在关联（P=0.011）。rs12107 和 rs2269529 的联合分析表明，随着风险等位基因数量的增加（rs12107-G 和 rs2269529-T，P 趋势=0.008），NSOC 的风险增加。MYH9 SNP rs12107 AG+GG 和 rs2269529 CT+TT 与唇组织中 MYH9 表达升高有关，与相应的野生型纯合子相比。对于 rs12107，荧光素酶检测显示 G 等位基因的荧光素酶活性高于 A 等位基因。将其假定的结合靶标 miR-196b-3p 转染到人胚胎盘间充质（HEPM）和 C2C12 细胞系中时，下调了 MYH9。对于 rs2269529，C>T 导致 MYH9 信使 RNA 增加。总之，rs12107 和 rs2269529 与 MYH9 的表达有关，并导致 NSOC 的易感性。
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