调控遗传变异对RAI1基因表达水平的影响机制及其与孤独症的关系

Retinoic acid induced-1 (RAI1) is a dose-sensitive gene located at Ch17p11.2, an unstable region that occasionally undergoes deletion or duplication during meiosis. Deletions at this locus cause Smith-Magenis syndrome (SMS) and duplications cause Potocki-Lupski syndrome (PTLS), complex developmental disorders that include autistic behaviors among their symptoms. Recent studies have identified RAI1 as the key gene in both disorders, suggesting that changes in RAI1 copy number may be a cause of autism. Because regulatory genetic variants, which influence gene expression by modulating mRNA transcription, splicing, or mRNA stability, can mimic the effects gene deletions or duplications, we hypothesize that common regulatory variants of RAI1 may contribute to sporadic autism. In the preliminary experiments, we used a novel next-generation DNA sequencing assay for allelic expression imbalance (AEI), quantitative PCR assays and mathematical modeling of population distribution to show that mRNA expression of RAI1 in human prefrontal cortex is regulated by two regulatory variants, at least one of which is located in the extended 5'-region of the gene. In this study, we propose to use electrophoretic mobility shift, chromatin immunoprecipitation and luciferase reporter assays in combination with next-generation DNA sequencing to identify the genetic variants that regulate RAI1 mRNA expression in human brain and use these variants as genetic markers in autism case-control studies to investigate possible contributions of RAI1 to autism.

视黄酸诱导基因1（RAI1）是染色体17p11.2不稳定区的剂量敏感基因，其缺失重复与多种精神疾病包括孤独症相关。该拷贝数变异对基因表达产物的剂量效应可以由调控遗传变异通过改变mRNA转录，剪切和稳定性进而改变基因表达水平来模拟，所以RAI1的调控遗传变异可能与散发孤独症不同症状程度有关。前期实验发现RAI1在人脑等位基因表达不平衡，脑表达量和基因型关联分析及群体分布数学模型提示存在两个调节RAI1表达的遗传变异。本课题拟通过凝胶迁移、染色质免疫沉淀、不同等位基因荧光素酶报告载体和第二代测序等手段，研究RAI1调控遗传变异作用机制，并应用到孤独症病例对照关联研究中，探索RAI1剂量效应与孤独症不同症状程度的关系。本研究创新性的将调控遗传变异及组合作为研究孤独症复杂症状的切入点，结合群体分布数学模型和第二代测序AEI基因型比对方法，为病例对照关联分析提供更具生物学意义和操作可行性的分子标记。

视黄酸诱导基因 1（RAI1）是染色体 17p11.2 不稳定区的一个剂量敏感基因，其缺失造成Smith-Magenis syndrome (SMS)综合症，重复造成Potocki-Lupski syndrome (PTLS) 综合症，两种罕见遗传疾病均伴有孤独症，智力迟滞等症状。本课题在中国人样本皮层两个脑区对RAI1基因的表达量进行测量，并对该区域单核苷酸多态性（SNP）进行基因基因分型和imputation，与RAI1基因表达量进行表达数量性状（expression Quantitative Trait Loci, eQTLs）关联分析，两脑区一致性的结果显示RAI1的表达受该基因5’调控区域的SNP调控。运用本实验室的△2-R2数学模型方法对RAI1基因5’调控区域的SNP进行归类分析，并用Regulomn database进行功能分析，发现SNP rs4925102和rs9907986作为常见功能性遗传变异可以调控RAI1在人脑的表达。SNP rs4925102位于视黄酸受体RARa-RXRa 结合原件RARE-DR5结合位点。视黄酸在早期胚胎和中枢神经形成中起重要作用，并且与孤独症等精神疾病有关，提示发育早期视黄酸通过结合受体RARa-RXRa RARE-DR5对RAI1的转录调控对中枢神经系统发育起重要作用。另一个数量性状位点位于与智力迟滞和孤独症有关的DEAF1 (Deformed epidermal autoregulatory factor 1)结合位点，提示DEAF1可能作为RAI1的上游调控因子，直接或间接的影响RAI1的表达水平，从而出现SMS/PTLS或智力迟滞和孤独症样症状。本课题通过凝胶迁移、染色质免疫沉淀、荧光素酶报告载体等手段，研究 RAI1 调控遗传变异作用机制，并应用到孤独症病例对照关联研究中，探索 RAI1 剂量效应与孤独症不同症状程度的关系。为病例对照关联分析提供更具生物学意义和操作可行性的分子标记。