Integrative genetics with high throughput, multiplex gen

具有高通量、多重基因的整合遗传学

• 批准号: 7317402
• 负责人: David Goldman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7317402
• 关键词: Integrative; genetics; throughput; multiplex; gen

HIGH THROUGHPUT SNP IDENTIFICATION. LNG has discovered over 100 single nucleotide polymorphisms (SNPs) in more than 50 neuro-genetic candidate genes for addictive behaviors, including alcoholism. Approximately 20% of these SNPs result in non-synonymous amino acid changes that may alter function of the encoded protein or alterations in expression of the gene. The implications of detected sequence variation have been profound: for example a rare serotonin transporter variant we found was shown to be functional and discovered to lead to severe pathology: Asperger's syndrome, treatment resistant OCD, and anorexia nervosa, in two families in which it is segregating, and a third common allele of the serotonin transporter promoter polymorphism (HTTLPR) which leads to altered function and enabled detection of linkage of the gain-of-function LA allele to OCD in two populations. For example, the common HTR2C Ser23Cys and HTR2A Asn452His alleles have been shown to be functional and linked to the clozapine responsiveness of schizophrenics. Genes for SNP screening were selected on the basis of postulated roles in alcohol, treatment response, identification in whole genome or candidate linkage or association study, the availability of genomic sequence data and expression studies. Because a goal of the project is to determine the role of SNPs in complex genetic disorders, we focused our screening in efforts on protein coding portions of the candidate gene and possible regulatory regions within and flanking the protein coding regions. We used a DNA panel composed of 477 genomic DNAs enriched for clinical and ethnic diversity. HIGH THROUGHPUT GENOTYPING. Central issues in high throughput genotyping procedures are accuracy, flexibility, and cost. Because of its assay design flexibility, low error rate, and potential for performing 96 to 384 assays simultaneously, we have used some 1,000 individual 5' exonuclease assays. We also use SNPlex, a DNA ligation method, in which pools of 48 SNPs are simultaneously genotyped, with detection on an ABI capillary sequencer and DNA preparation using robotics. Regarding array-based genotyping, we have created a 1536 SNP ?Addictions Array.? This array enables haplotype-based and candidate locus coverage of some 130 genes, including genes important in the domains of alcohol metabolism, stress/anxiety, monoamine function, and signaling. The array also includes 186 genomic control loci for detecting, and correcting, ethnic stratification in case control studies. Use of the ?Addictions Array? by Extramural investigators has been facilitated such that the array is being genotyped on some 20,000 individuals including sample collections from Yale, Emory University, the Rockefeller University, Columbia University, the University of Colorado, Medical College of Virginia, Washington University, and NIDCR. CLINICAL GENOTYPING certified to CLIA (Clinical Laboratory Improvement Act) standard is being performed for certain studies.

高吞吐量SNP识别。液化天然气在50多个神经遗传候选基因中发现了100多种单核苷酸多态性（SNP），包括酗酒，包括酗酒。这些SNP中约有20％会导致非同义氨基酸变化，这些变化可能会改变编码蛋白的功能或基因表达的改变。检测到的序列变化的含义是深刻的：例如，我们发现我们发现的一种罕见的5-羟色胺转运蛋白变异蛋白变异被证明是功能性的，并发现导致严重的病理学：阿斯伯格综合征，抗治疗性强迫症和神经性厌食性神经性神经性，在两个家族中，它是隔离的两个家族，是链球菌的第三个常见的促进剂促进剂（促进剂）的等位基因（促进剂）的促进剂（促进剂）（促进剂）（促进剂）（在两个人群中，启用了功能障碍LA等位基因与OCD的联系。例如，已证明常见的HTR2C SER23CYS和HTR2A ASN452HIS等位基因具有功能性，并与精神分裂症患者的氯氮平反应性联系在一起。根据假定在酒精，治疗反应，整个基因组或候选联系或关联研究中的鉴定，基因组序列数据和表达研究的可用性，选择用于SNP筛查的基因。因为该项目的目标是确定SNP在复杂的遗传疾病中的作用，所以我们将筛查重点放在候选基因的蛋白质编码部分以及蛋白质编码区域内和侧翼的蛋白质编码部分上。我们使用了由477个基因组DNA组成的DNA面板，该基因组DNA富含临床和种族多样性。高通量基因分型。高通量基因分型程序中的中心问题是准确性，灵活性和成本。由于其测定的设计灵活性，低错误率和同时执行96至384分测定的潜力，因此我们使用了约1,000个单独的5'外核酸酶测定法。我们还使用SNPLEX（一种DNA连接方法），其中同时对48个SNP的池进行了基因分型，并在ABI毛细管序列仪上检测到使用机器人技术进行DNA制备。关于基于数组的基因分型，我们创建了一个1536 SNP？成瘾阵列。该阵列使基于单倍型的基因座覆盖了一些130个基因，其中包括在酒精代谢，压力/焦虑，单胺功能和信号传导领域重要的基因。该阵列还包括186个基因组控制基因座，用于检测和纠正案例控制研究中的种族分层。使用“成瘾阵列”？由校外调查人员进行了促进，以至于将大约20,000名个人进行基因分型，包括耶鲁大学，埃默里大学，洛克菲勒大学，哥伦比亚大学，科罗拉多大学，科罗拉多大学，弗吉尼亚大学，华盛顿大学和NIDCR。在某些研究中，正在对CLIA认证的临床基因分型（临床实验室改进法）进行标准。