MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach [expression array]. Homo sapiens

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes. Overall design: In order to understand the genetic basis of NSCL/P susceptibility, we searched for genetic variants with regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle, OOM) of affected individuals. From OOM samples, which are frequently discarded during corrective surgeries that patients with orofacial clefts routinely undergo, we derived mesenchymal stem cells (OOMMSC), and correlated the individual genetic differences with gene expression. We obtained OOM samples from 43 NSCL/P-affected and four normal individuals, and established OOMMSC primary cultures according to previously published protocol. To measure transcript levels, we used Human Gene Chip 1.0 ST v1 microarrays (Affymetrix, USA), according to the manufacturer’s protocol, using 300ng of total RNA. One patient was excluded from expression analysis.

探究个体与群体遗传差异如何使个体易患疾病的有效路径，是评估遗传变异对与病理状态相关的细胞及组织类型的细胞功能（例如基因表达）的影响。非综合征性唇伴或不伴腭裂（nonsyndromic cleft lip with or without cleft palate, NSCL/P）是一种复杂且高发的先天性畸形，为解析其易感的遗传基础，我们在疾病相关组织——患者的唇肌（口轮匝肌[orbicularis oris muscle, OOM]）中搜寻具有调控功能的遗传变异。本研究使用的46份唇肌样本，通常在唇腭裂患者的常规修复手术中被废弃，我们从中分离得到间充质干细胞，并将个体遗传变异与这些培养细胞的基因表达水平进行关联分析。通过该研究策略，我们检测到了显著的顺式表达数量性状位点（cis-eQTL，即影响基因表达的DNA变异），并筛选出若干候选位点，在大型巴西队列（624名患者与668名对照）中开展关联研究。最终发现了一个全新的NSCL/P易感位点rs1063588，该位点是MRPL53基因的最优eQTL，其关联信号主要由巴西样本中的美洲原住民血统组分驱动。MRPL53（2p13.1）编码线粒体核糖体39S蛋白亚基，可与正常面部形态发生所需的转录因子MYC发生相互作用。本研究不仅凸显了混合人群采样的重要性，也证明了通过测量遗传变异对基因表达的功能效应，以解析疾病表型复杂性的研究思路的价值。实验设计概述：为解析NSCL/P易感的遗传基础，我们在疾病相关组织——患者的唇肌（口轮匝肌，OOM）中搜寻具有调控功能的遗传变异。从唇腭裂患者常规手术中废弃的唇肌样本中，我们分离得到唇肌来源间充质干细胞（OOMMSC），并将个体遗传差异与基因表达水平进行关联分析。我们共收集了43名NSCL/P患者与4名健康个体的唇肌样本，并依照已发表的实验流程建立了OOMMSC原代培养体系。为检测转录本水平，我们使用人类基因芯片1.0 ST v1微阵列（Affymetrix，美国），依照制造商的实验流程，以300ng总RNA作为起始材料。最终有1名患者被排除在表达分析之外。