Faculty Bibliography

Large scale human genetic studies require technologies for generating millions of genotypes with relative ease but also at a reasonable cost and with high accuracy. We describe a highly parallel method for genotyping single nucleotide polymorphisms (SNPs), using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligonucleotide tags. First, marker-specific primers are used in PCR amplifications of genomic regions containing SNPs. Second, the amplification products are used as templates in single base extension (SBE) reactions using chimeric primers with 3' complementarity to the specific SNP loci and 5' complementarity to specific probes, or tags, synthesized on the array. The SBE primers, terminating one base before the polymorphic site, are extended in the presence of labeled dideoxy NTPs, using a different label for each of the two SNP alleles, and hybridized to the tag array. Third, genotypes are deduced from the fluorescence intensity ratio of the two colors. This approach takes advantage of multiplexed sample preparation, hybridization, and analysis at each stage. We illustrate and test this method by genotyping 44 individuals for 142 human SNPs identified previously in 62 candidate hypertension genes. Because the hybridization results are quantitative, this method can also be used for allele-frequency estimation in pooled DNA samples.

In this study we quantify the features of meiotic recombination on the long arm of human chromosome 21. We constructed a 67. 3-centimorgan (cM) high-resolution, comprehensive, and accurate genetic linkage map of chromosome 21q using 187 highly polymorphic markers covering almost the entire long arm; 46 loci, consisting of mutually recombining marker sets, were ordered with greater than 1000:1 odds and with average interlocus distance of 1.46 cM. These markers were used to accurately identify all exchanges in 186 female and 160 male meioses and to show (1) significant excess of recombination in female versus male meioses, (2) an overall decline in female:male recombination between the centromere and the telomere, (3) greater positive chiasma interference in male than in female meioses, and (4) lack of correlation between exchange frequency and parental age. By comparing the genetic map with the 21q sequence map, we show a general trend of increasing male, but near-constant female, recombination versus physical distance across 21q, explaining the gender-specific recombination effect. The recombination rate varies considerably between genders across 21q but is the greatest (eightfold) in the pericentromeric region, with a rate of approximately 250 kb/cM in females and approximately 2125 kb/cM in males. We used information on the locations of all exchanges to construct an empirical map function that confirms the statistical findings of positive interference. These analyses reveal that occurrence of recombination on 21q is not only gender-specific but also region-specific and that recombination suppression at the centromere is not universal. We also find evidence that male exchange location is highly correlated with gene density.

This review discusses the prospects for understanding the genetic basis of complex traits in humans. We take the view that work done on Drosophila melanogaster can serve as a model for understanding complex traits in humans, and the literature on this model system, as well as on humans, is reviewed. The prospects for success in understanding the genetic basis of complex traits depend, in part, on the nature of the forces acting on genetic variation. We suggest that different experimental approaches should be undertaken for traits caused by common genetic variants versus those arising from rare genetic variants.

There is considerable controversy regarding a putative association between schizophrenia and a biallelic BalI polymorphism in the first exon of the dopamine D3 receptor gene (DRD3), although meta-analyses of published data suggest an association. If such an association exists, it may be detectable at markers physically close to DRD3. Accordingly, we conducted a case-control association study using D3S1310, a short tandem repeat polymorphism located approximately 700 kb telomeric to DRD3 on chromosome 3q13.3. The subjects were Swedish patients with schizophrenia (DSM III-R criteria, n = 110) and screened adult controls (n = 83). A trend for a negative association with the 141 bp allele was detected (chi2 = 7.6, d.f. = 1, P = 0.006; odds ratio 0.46, 95% confidence intervals 0.26, 0.81). However, following corrections for multiple comparisons using subgroups (n = 15) the difference was not significant. Also, due to the risk for population stratification in case-control association studies the results must be treated as tentative. If replicated the results may lend further support for the proposition of an association between schizophrenia and DRD3 or a gene in close proximity to DRD3 on chromosome 3q.

The complete human genome nucleotide sequence and technologies for assessing sequence variation on a genome-scale will prompt comprehensive studies of comparative genomic diversity in human populations across the globe. These studies, besides rejuvenating population genetics and our interest in how genetic variation is created and maintained, will provide the intellectual basis for understanding the genetic basis for complex diseases and traits.

As the human genome is sequenced and annotated, an important step in future genetic studies of complex traits and diseases will be the identification of relevant candidate genes. To enable such compilations, it would be useful to collate all necessary and available genetic information for each candidate gene. To this end, we have created a web tool (http://genome.cwru.edu/gist/gist.html+ ++) to allow the rapid cataloging of currently available genetic data. This tool, called GIST (or "Gene Information Search Tool"), allows an investigator to search the major genomic databases containing gene and marker information from a single query point. To prove the utility of GIST, a catalog of 150 hypertension candidate genes was created. This resource collates all available nucleotide and amino acid sequence data, expression data, chromosomal map location, and genetic marker interval for each gene, collected from on-line databases. These data can be used to guide genetic studies of hypertension.