Genomic Profiling and Functional Mutation Analysis in Autism Spectrum Disorders

描述（由申请人提供）：本提案是对“精神障碍基因组分析”申请请求的回应，并提出了接触蛋白相关蛋白2 （CNTNAP2）以及突触前细胞基质蛋白Piccolo （PCLO）的密集基因组分析和功能分析，以阐明它们在自闭症谱系障碍（ASD）中的作用。此外，该应用程序还建议对Contactin和Contactin相关途径中的基因以及编码已知与PCLO相互作用的蛋白质的基因进行深度测序。我们建议利用耶鲁大学State、De Camilli和Giraldez实验室之间正在进行的合作，允许对已确定的罕见序列突变进行快速功能分析（体外和体内），这将用于澄清基因组发现并确认罕见突变与疾病风险的相关性。支持这一应用的组织原则如下：1)罕见的遗传变异可能在ASD的病因学中发挥重要作用；2)在大量患者和对照中检测罕见（MAF<5%）和非常罕见（MAF<1%）变异的方法现在在经济和技术上都是可行的；3)罕见和非常罕见的变异，包括在高度保守的编码区域的变异，在受影响和未受影响个体的基因组中广泛存在；4)罕见和非常罕见的中间效应等位基因可能需要关联而不是连锁策略来评估疾病风险；5)在精神障碍基因组图谱成功的这个阶段，最紧迫的问题是将疾病相关突变与低频中性突变区分开来，并解决在罕见和非常罕见等位基因的情况下评估疾病关联的方法学挑战。该应用程序建议解决这些问题如下：1)使用下一代技术在大量，非常充分地表征和仔细匹配种族的病例对照样本中识别罕见和非常罕见的突变；2)在分类诊断的基础上，采用定量的ASD表型分型，以增强罕见变异的病例对照相关性；3)利用完善的关联方法，包括基因组控制、非常罕见突变的突变负担分析、多重比较和复制样本的校正，以减少1型错误；4)使用体外和体内方法评估在病例和对照组中发现的罕见突变的功能后果，特别是在CNTNAP2和PCLO基因中；重要的是，5)为了验证这样一个假设，即功能分析将CNTNAP2的罕见功能变异与中性变异分层，将澄清关联分析的结果，正如在其他罕见变异研究中成功应用的那样(1)。

DESCRIPTION (provided by applicant): This proposal is in response to a request for applications to address "Genomic Profiling of Mental Disorders," and proposes intensive genomic profiling and functional analysis of Contactin Associated Protein 2 (CNTNAP2) as well as the presynaptic cytomatrix protein Piccolo (PCLO), in an effort to clarify their roles in Autism Spectrum Disorders (ASD). In addition, the application proposes to deeply sequence genes in the Contactin and Contactin Associated pathways, as well as genes coding for proteins known to interact with PCLO. We propose to leverage ongoing collaborations among the State, De Camilli and Giraldez labs at Yale to allow for rapid functional assays (both in vitro and in vivo) of identified rare sequence mutations, which will be used to clarify genomic findings and to confirm the relevance of rare mutations for disease risk. The organizing principles supporting this application are as follows: 1) Rare genetic variation is likely to play a significant role in the etiology of ASD; 2) Methods of detecting rare (MAF<5%) and very rare (MAF<1%) variation in large numbers of patients and controls are now economically and technically feasible; 3) Rare and very rare variations, including those in highly conserved coding regions, are widespread throughout the genomes of both affected and unaffected individuals; 4) Rare and very rare alleles of intermediate effect will likely require association as opposed to linkage strategies to evaluate disease risk; and 5) That among the most pressing issues at this stage in the success of genomic profiling of mental disorders are differentiating disease-related mutations from low frequency neutral mutations and addressing the methodolgical challenges of assessing disease association in the case of rare and very rare alleles. This application proposes to address these issues as follows: 1) Deep-resequencing using next generation technologies to identify rare and very rare mutations in a large, extremely well-characterized and carefully ethnically matched case-control sample; 2) To use quantitative ASD phenotyping in addition to categorical diagnoses in an effort to enhance the power of case-control association of rare variants; 3) To leverage well established association methodologies, including genomic control, mutation burden analyses for very rare mutations, correction for multiple comparisons and replication samples, to decrease type 1 error; 4) To use both in vitro and in vivo methods to evaluate the functional consequences of rare mutations identified in cases and controls, specifically in the genes CNTNAP2 and PCLO; and importantly, 5) To test the hypothesis that functional assays stratifying rare functional from neutral variation in CNTNAP2 will clarify the results of association analyses, as has been successfully employed in other rare variant studies (1). PUBLIC HEALTH RELEVANCE: This study seeks to identify the role of specific genes in Autism Spectrum Disorders. The research plan utilizes high throughput sequencing technologies and combines these with molecular in vitro and in vivo biological studies in an effort to clarify the precise contribution of two brain expressed molecules Contactin Associated Protein 2 and Picollo as well as related genes to Autism and related conditions.