Department of Child and Adolescent Psychiatry, Psychosomatics and Psychotherapy, JW Goethe University, Frankfurt am Main, Germany.
Department of Child and Adolescent Psychiatry, Saarland University, Homburg, Germany.
Mol Psychiatry. 2015 Jul;20(7):839-49. doi: 10.1038/mp.2014.103. Epub 2014 Sep 16.
Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5' promoter. Here, we directly sequenced the CNTNAP2 5' promoter region of 236 German families with one child with ASD and detected four novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG[7] with language development. The four novel and the three known minor alleles of the identified variants were predicted to alter transcription factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specific and differentiation stage-dependent effects at the level of promoter activation. The novel potential rare risk-variant M2, a G>A mutation -215 base pairs 5' of the transcriptional start site, significantly reduced promoter efficiency in HEK293T and in undifferentiated and differentiated neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus confirm the hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD.
接触蛋白相关蛋白样 2 基因 (CNTNAP2)，是神经连接素基因超家族的一员，是自闭症谱系障碍 (ASD) 中复制最好的风险基因之一。自闭症是主要由遗传决定的神经发育障碍，其特征是语言发育、社交互动和沟通受损，以及刻板行为和兴趣。尽管 CNTNAP2 的表达水平被认为会改变 ASD 的风险，但迄今为止尚无研究关注其 5'启动子。在这里，我们直接对 236 个德国家庭的 CNTNAP2 5'启动子区域进行了测序，这些家庭中有一个孩子患有 ASD，并检测到了四个新的变体。此外，我们对另外 356 个家庭中的三个最常见变体（rs150447075、rs34712024、rs71781329）进行了基因分型，并发现 rs34712024G 与 ASD 以及 rs71781329GCG[7]与语言发育有关。所鉴定的变体的四个新的和三个已知的次要等位基因被预测会改变转录因子结合位点 (TFBS)。在功能水平上，跨越这七个变体的相应序列被核因子结合。在荧光素酶启动子测定中，相应的次要等位基因在启动子激活水平上显示出线粒体特异性和分化阶段依赖性的细胞系特异性效应。新的潜在罕见风险变体 M2 是一种 -215 个碱基对位于转录起始位点 5'的 G>A 突变，在 HEK293T 以及未分化和分化的神经母细胞瘤 SH-SY5Y 细胞中显著降低了启动子效率。该变体传递给一名患有自闭症障碍的患者。相比之下，常见变体 rs34712024 的低传递、保护性的 G 等位基因增加了转录活性。这些结果得出结论，CNTNAP2 启动子变体对 ASD 风险的致病机制是通过其对 TFBS 的影响介导的，从而证实了这样一种假设，即在神经元发育过程中 CNTNAP2 水平降低会增加 ASD 的易感性。
