目的:使用病例-亲本三重奏设计,探讨新生突变(DNM)与非综合征性唇裂伴或不伴腭裂(NSCL/P)之间的关联。
方法:对22个NSCL/P三联基因进行全外显子组测序,并使用基因组分析工具包(GATK)通过比较病例及其父母的等位基因来鉴定DNM。用SnpEff将可预测功能的信息注释到基因座上。进行了DNM的富集分析,以测试DNM的实际数量与预期数量之间的差异,并探索是否有比预期更多的DNM基因。通过文献复习,筛选出既往研究表明有确凿证据的NSCL/P相关基因。在具有蛋白质改变的DNM和NSCL/P相关基因的基因之间进行蛋白质-蛋白质相互作用分析。R包“denovolyzeR”用于富集分析(Bonferroni校正:P=0.05/n,n是整个基因组范围内的基因数量)。根据STRING数据库提供的信息,预测了具有DNM的基因和对NSCL/P危险因素有确凿证据的基因之间的蛋白质-蛋白质相互作用。
结果:共有339908个SNP合格,可用于质量控制后的后续分析。GATK鉴定的高置信度DNM数量为345。在这些DNM中,44个DNM是错义突变,一个DNM是无义突变,两个DNM是剪接位点突变,20个DNM是同义突变,其他位于内含子或基因间区域。富集分析结果表明,外显子组区域改变蛋白质的DNM数量大于预期(P<0.05),和五个基因(KRTCAP2,HMCN2,ANKRD36C,ADGRL2和DIPK2A)的DNM高于预期(P<0.05/(2×19618))。在通过文献复习选择的46个具有蛋白质改变的DNM基因和13个与NSCL/P相关的基因之间进行蛋白质-蛋白质相互作用分析。在具有DNM的基因和已知的NSCL/P相关基因之间发生了六对相互作用。测量RGPD4和SUMO1之间预测相互作用的置信水平的分数为0.868,高于其他对基因的分数。
结论:我们的研究为NSCL/P的发展提供了新的见解,并证明了携带DNM的基因的功能分析对于理解复杂疾病的遗传结构是有必要的。
OBJECTIVE: To explore the association between de novo mutations (DNM) and non-syndromic cleft lip with or without palate (NSCL/P) using
case-parent trio design.
METHODS: Whole-exome sequencing was conducted for twenty-two NSCL/P trios and Genome Analysis ToolKit (GATK) was used to identify DNM by comparing the alleles of the cases and their parents. Information of predictable functions was annotated to the locus with SnpEff. Enrichment analysis for DNM was conducted to test the difference between the actual number and the expected number of DNM, and to explore whether there were genes with more DNM than expected. NSCL/P-related genes indicated by previous studies with solid evidence were selected by literature reviewing. Protein-protein interactions analysis was conducted among the genes with protein-altering DNM and NSCL/P-related genes. R package \"denovolyzeR\" was used for the enrichment analysis (Bonferroni correction: P=0.05/n, n is the number of genes in the whole genome range). Protein-protein interactions among genes with DNM and genes with solid evidence on the risk factors of NSCL/P were predicted depending on the information provided by STRING database.
RESULTS: A total of 339 908 SNPs were qualified for the subsequent analysis after quality control. The number of high confident DNM identified by GATK was 345. Among those DNM, forty-four DNM were missense mutations, one DNM was nonsense mutation, two DNM were splicing site mutations, twenty DNM were synonymous mutations and others were located in intron or intergenic regions. The results of enrichment analysis showed that the number of protein-altering DNM on the exome regions was larger than expected (P < 0.05), and five genes (KRTCAP2, HMCN2, ANKRD36C, ADGRL2 and DIPK2A) had more DNM than expected (P < 0.05/(2×19 618)). Protein-protein interaction analysis was conducted among forty-six genes with protein-altering DNM and thirteen genes associated with NSCL/P selected by literature reviewing. Six pairs of interactions occurred between the genes with DNM and known NSCL/P-related genes. The score measuring the confidence level of the predicted interaction between RGPD4 and SUMO1 was 0.868, which was higher than the scores for other pairs of genes.
CONCLUSIONS: Our study provided novel insights into the development of NSCL/P and demonstrated that functional analyses of genes carrying DNM were warranted to understand the genetic architecture of complex diseases.