Identification of Functional Variants for Cleft Lip with or without Cleft Palate in or near PAX7, FGFR2, and NOG by Targeted Sequencing of GWAS Loci
Identification of Functional Variants for Cleft Lip with or without Cleft Palate in or near PAX7, FGFR2, and NOG by Targeted Sequencing of GWAS Loci
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DOI：
10.1016/j.ajhg.2015.01.004
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发表时间：
2015-03-05
影响因子：
9.8
通讯作者：
Murray, Jeffrey C.
中科院分区：
文献类型：
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作者：
Leslie, Elizabeth J.;Taub, Margaret A.;Murray, Jeffrey C.
尽管非综合征性口面部裂的全基因组关联研究（GWASs）已经确定了多个强烈相关的区域，但因果变异尚不清楚。为了解决这个问题，我们从GWASs和其他研究中选择了13个区域，对1409个亚洲和欧洲的三人组进行了靶向测序，并进行了一系列统计和功能分析。在NOG附近的一组强烈相关的常见变异中，我们发现rs227727破坏了增强子活性。我们进一步在NTN1和NOG附近发现了显著的非编码罕见变异簇，并发现了几个可能影响蛋白质功能的罕见编码变异，包括ARHGAP29中的四个无义变异。我们确认了48个新生突变，并根据现有的最佳生物学证据，选择了其中两个进行功能分析。在体外实验中，PAX7的一个突变破坏了编码转录因子的DNA结合。第二种是一种非编码突变，在体外和体内都破坏了FGFR2下游的神经嵴增强子的活性。这项靶向测序研究提供了强有力的功能证据，表明几种特定的变异是人类非综合征性唇裂的主要风险等位基因。
Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.