Genetics and Bioinformatics Core Laboratory

遗传学与生物信息学核心实验室

• 批准号: 7735226
• 负责人: Daniel Weinberger
• 金额: $ 217.31万
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 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/7735226
• 关键词: Address; Admixture; Alleles; Animal Model; Anxiety; Autopsy; Bioinformatics; Biological; Biological Assay; Biological Models; Brain; Brain Diseases; Brain region; COMT gene; Candidate Disease Gene; Cataloging; Catalogs; Catechol O-Methyltransferase; Caucasians; Caucasoid Race; Chromosomes; Clinical; Clinical Trials; Cognition; Collaborations; Complex; Computer Simulation; Computer software; DNA; DNA Sequence; Data; Data Set; Disease; Equilibrium; ErbB4 gene; Ethnic group; Exons; FGF20 gene; Family; Frequencies; Gene Combinations; Gene Frequency; Genes; Genetic; Genetic Risk; Genetic Variation; Genomics; Genotype; Gold; Haplotypes; Hereditary Disease; Human; Image; In Situ Hybridization; Individual; Introns; Laboratories; Laboratory Research; Length; Linkage Disequilibrium; Literature; Logistic Regressions; Measures; Messenger RNA; Methods; Microsatellite Repeats; Modeling; Moods; Morphologic artifacts; Mutation; National Institute of Mental Health; Neurofibromin 2; Neuropsychology; Numbers; Odds Ratio; Online Systems; Open Reading Frames; Outsourcing; PIP5K2A gene; PPP3CC gene; Parents; Phase; Phenotype; Phosphatidylinositol-4-Phosphate 5-Kinase Type II Alpha; Polymorphism Analysis; Population; Proteins; Psychotic Disorders; RNA Splicing; Race; Recombinants; Reproducibility; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Risk; Sampling; Schizophrenia; Screening procedure; Single Nucleotide Polymorphism; Site; Spottings; Stratification; Susceptibility Gene; System; Technology; Testing; Transcript; Variant; Work; base; cDNA Library; case control; clinical phenotype; data mining; follow-up; gene interaction; genetic variant; human FGF20 protein; human PPP3CC protein; novel; proband; programs; statistics; trait; transmission process

To date, we have tested numerous single nucleotide polymorphisms (SNPs) in well over 118 genes, including some of the less established but intriguing candidates such as PRODH, RGS4, CHRNA7, PIP5K2A, and PPP3CC. Among our accomplishments, we have fully sequenced the 10 exons and flanking sequences of 180 proband chromosomes for dysbindin, sequenced two exons of MRDS1, and sequenced 1.5 kb of the GAD1 upstream region. A total of 21 new SNPs were discovered in these genes, 15 of which were genotyped in the clinical samples. We have re-sequenced the exons and splice sites of GRM3 in 180 chromosomes, which led to the discovery of a few rare SNPs. We have likewise re-sequenced risk regions of KCNH2, ErbB4, PIk3d, FGF20, DAARP, and COMT and identified novel variants in these genes as well. We routinely submit our Taqman genotype assay to reproducibility checks by re-genotyping (avg. accuracy >99%) and spot accuracy checks done by double stranded sequencing (avg. >99% for most SNP assays). Genotypes are called manually within the ABI SDS software and confirmed. We perform Mendelian checks and higher order (e.g. multiple recombinants) error checking with the program MERLIN. Microsatellite genotyping has been performed in collaboration with the NIMH Mood and Anxiety Program. We measure linkage disequilibrium (LD) between markers with the D prime and r2 statistics from cases and controls in parallel using the GOLD software package. All SNPs are tested for departures from Hardy-Weinberg equilibrium. For large numbers of loci, we use SNPHAP to reconstruct haplotypes and estimate their frequencies in unrelated individuals. For family based association studies of the discrete clinical phenotype, we use the programs FBAT, TDTPHASE and TRANSMIT for unknown phase haplotype estimation. Case-control analysis of individual SNPs and SNP haplotypes is done using logistic regression in STATA and COCAPHASE. All P values are computed empirically with 10,000 permutations or bootstraps as the programs provide. Tests of association to quantitative traits such as the intermediate phenotypes are performed by the FBAT and QTDT, which allows variance-components testing of family-based samples for association and transmission disequilibrium. The orthogonal model used is robust to population stratification because, analogous to the conventional TDT, it only considers transmissions from heterozygous parents. To control for possible artifacts due to allele frequency differences across ethnic groups, analysis limited to Caucasians is performed in parallel. We have also established a panel unlinked SNPs to use as a potential genomic control panel for case control association studies, including intermediate phenotype analyses, to address potential population admixture artifacts. In our genomics project we acquire extensive genetic variation data in our susceptibility genes and complete the catalog of genetic risk genes in our datasets. As part of the GCAP program, we have greatly increased the genotyping throughput by outsourcing. We project that about every 4 months for the next 2 years we will genotype a minimum of 768 SNPs, perform follow-up work on established genes and test novel genes. In addition, we outsource the majority of re-sequencing for SNP detection to DNA sequencing companies. All exons, splice sites, and 10 kb of the upstream region will be re-sequenced in an initial pass, then some regions of some genes are sequenced further (e.g. the introns or positive haplotypes) and/or more individuals. Because most functional SNPs and mutations are not in protein coding regions, it is critical to fully characterize transcripts species in several regions of post mortem human brain. To accomplish this, we routinely execute basic mRNA transcript characterization technologies such as 5' and 3' RACE and screening of full-length transcripts, normalized cDNA libraries from multiple brain regions. This work also serves to guide quantitative RT-PCR and in situ hybridization expression studies. Another project of central importance is the statistical analyses of gene-gene interactions. It is likely that certain gene and allele combinations interact epistatically to produce risk greater than that predicted by the individual odds ratios. It is also likely that some gene combinations will increase risk even in the absence of main effects in each gene. We are using the data driven analytic approach developed at Vanderbilt called multifactor dimensionality reduction (MDR) in an attempt to detect sets of interacting alleles that predict disease status. We also engage in collaborative discussions with Salford Systems, originator of the programs CART, MARS, and TREENET, to explore and execute other data mining strategies. Our statistical geneticist uses the wealth of data to model and test complex gene-gene and gene-environmental interactions, and establish some objective criteria for integrating statistical genetic (disease and intermediate phenotype) data with convergent biological data both to gauge overall significance of given genotype/haplotype, phenotype correlations and to evaluate attributable risk.

到目前为止，我们已经在超过118个基因中测试了许多单核苷酸多态性（SNP），包括一些不太确定但有趣的候选基因，如PRODH，RGS 4，CHRNA 7，PIP 5 K2 A和PPP 3CC。在我们的成就中，我们已经完全测序了180个先证者染色体dysbindin的10个外显子和侧翼序列，测序了MRDS 1的两个外显子，并测序了1.5 kb的GAD 1上游区域。在这些基因中共发现了21个新的SNPs，其中15个在临床样本中进行了基因分型。我们对180条染色体中GRM 3的外显子和剪接位点进行了重新测序，发现了一些罕见的SNP。我们同样对KCNH 2、ErbB 4、PIK 3d、FGF 20、DAARP和COMT的风险区域进行了重新测序，并在这些基因中鉴定了新的变体。我们定期提交我们的Taqman基因型检测，通过重新基因分型（平均值）进行重现性检查。准确度>99%）和通过双链测序（avg.对于大多数SNP测定，>99%）。在ABI SDS软件中手动调用基因型并确认。我们用程序MERLIN进行孟德尔检验和高阶（例如多个重组体）错误检验。微卫星基因分型已与NIMH情绪和焦虑计划合作进行。 我们测量连锁不平衡（LD）之间的标记与D总理和r2统计的情况下，对照组平行使用GOLD软件包。测试所有SNP是否偏离Hardy-Weinberg平衡。对于大量的基因座，我们使用SNPHAP来重建单倍型，并估计它们在无关个体中的频率。对于离散临床表型的基于家族的关联研究，我们使用FBAT、TDTPHASE和TRANSMIT程序进行未知相位单倍型估计。使用STATA和COCAPHASE中的逻辑回归进行个体SNP和SNP单倍型的病例对照分析。所有P值都是根据经验计算的，程序提供了10，000个排列或自举。通过FBAT和QTDT进行与数量性状（如中间表型）的关联测试，这允许对基于家系的样本进行关联和传递不平衡的方差分量测试。所使用的正交模型是强大的人口分层，因为类似于传统的TDT，它只考虑从杂合父母的传输。为了控制由于不同种族之间等位基因频率差异而导致的可能伪影，平行进行了仅限于高加索人的分析。我们还建立了一个非连锁SNP组，用作病例对照关联研究（包括中间表型分析）的潜在基因组对照组，以解决潜在的群体混合伪影。 在我们的基因组学项目中，我们获得了易感基因中广泛的遗传变异数据，并在我们的数据集中完成了遗传风险基因的目录。作为GCAP计划的一部分，我们通过外包大大提高了基因分型的通量。我们计划在未来2年内，每4个月对至少768个SNP进行基因分型，对已建立的基因进行后续工作，并测试新基因。此外，我们将SNP检测的大部分重新测序外包给DNA测序公司。所有外显子、剪接位点和上游区域的10 kb将在初始通过中重新测序，然后进一步测序一些基因的一些区域（例如内含子或阳性单倍型）和/或更多个体。由于大多数功能性SNP和突变不在蛋白质编码区，因此充分表征死后人脑几个区域中的转录物种类至关重要。为了实现这一点，我们常规地执行基本的mRNA转录本表征技术，如5'和3' RACE和全长转录本的筛选，来自多个脑区域的标准化cDNA文库。这项工作也可用于指导定量RT-PCR和原位杂交表达研究。 另一个重要的项目是基因-基因相互作用的统计分析。很可能某些基因和等位基因组合上位性相互作用产生的风险大于个体优势比预测的风险。某些基因组合也可能会增加风险，即使每个基因中没有主效应。我们正在使用范德比尔特开发的数据驱动分析方法，称为多因素降维（MDR），试图检测预测疾病状态的相互作用等位基因集。我们还与Salford Systems（CART、MARS和TREENET计划的发起者）进行合作讨论，以探索和执行其他数据挖掘策略。我们的统计遗传学家利用丰富的数据来建模和测试复杂的基因-基因和基因-环境相互作用，并建立一些客观标准，将统计遗传（疾病和中间表型）数据与收敛生物学数据整合起来，以评估给定基因型/单倍型的总体意义、表型相关性并评估归因风险。