Integrative genetics with high throughput, multiplex gen

高通量SNP鉴定。LNG已经在包括酗酒在内的成瘾行为的50多个神经遗传候选基因中发现了100多个单核苷酸多态性（SNPs）。大约20%的snp导致非同义氨基酸的改变，这可能改变编码蛋白的功能或基因表达的改变。检测到的序列变异的影响是深远的：例如，我们发现的一种罕见的血清素转运体变异被证明是功能性的，并被发现导致严重的病理。阿斯伯格综合症，治疗抵抗性强迫症和神经性厌食症，在两个分离的家族中，以及血清素转运子启动子多态性（HTTLPR）的第三个常见等位基因，它导致功能改变，并使检测功能获得性LA等位基因与强迫症的联系成为可能。例如，常见的HTR2C Ser23Cys和HTR2A Asn452His等位基因已被证明具有功能性，并与精神分裂症患者的氯氮平反应性有关。用于SNP筛选的基因是根据酒精中的假定作用、治疗反应、全基因组或候选连锁或关联研究中的鉴定、基因组序列数据的可用性和表达研究来选择的。由于该项目的目标是确定snp在复杂遗传疾病中的作用，因此我们将筛选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两侧可能的调控区域上。我们使用了一个由477个基因组DNA组成的DNA面板，这些基因组DNA丰富了临床和种族多样性。高通量基因分型。高通量基因分型程序的中心问题是准确性、灵活性和成本。由于其分析设计的灵活性，低错误率，以及同时进行96至384次分析的潜力，我们已经使用了大约1000个单独的5'外切酶测定。我们还使用了SNPlex，一种DNA连接方法，其中48个snp池同时进行基因分型，在ABI毛细管测序仪上进行检测，并使用机器人技术进行DNA制备。关于基于阵列的基因分型，我们已经创建了1536 SNP ？上瘾数组。该阵列可实现约130个基因的单倍型和候选基因座覆盖，包括在酒精代谢、应激/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个基因组控制位点，用于在病例对照研究中检测和纠正种族分层。？上瘾数组?在校外调查人员的协助下，正在对包括耶鲁大学、埃默里大学、洛克菲勒大学、哥伦比亚大学、科罗拉多大学、弗吉尼亚医学院、华盛顿大学和NIDCR收集的样本在内的约20 000个人进行基因分型。通过CLIA（临床实验室改进法案）标准认证的临床基因分型正在进行某些研究。

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.