Integrative genetics with high throughput, multiplex gen

点击翻译按钮获取中文摘要

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.