Faculty Bibliography

BACKGROUND:Siblings and other first-degree relatives of patients with "sporadic" (i.e., apparently nonfamilial) colorectal cancer or precursor adenomatous colon polyps have an increased risk of developing colon neoplasia. This observation suggests the presence of inherited genetic determinants for sporadic colon neoplasia. Mice homozygous for a null cyclooxygenase 2 (COX2) (also called PTGS2) allele have a dramatically reduced susceptibility to the development of intestinal adenomas. In humans, use of pharmacologic inhibitors of COX2 enzyme activity are associated with reduced risk of colon neoplasia. This study examined whether the human COX2 locus may be linked to colon neoplasia in humans. METHODS:We used the affected sibling-pair method to test for linkage of the human COX2 locus to colon neoplasia. RESULTS:We examined 74 concordantly affected sibling pairs from 46 sibships with colon neoplasia. One hundred five siblings from these sibships were diagnosed with either colorectal cancer or colon adenomatous polyps before age 65 years. No linkage between COX2 and colon neoplasia was found by use of a multipoint model-free linkage analysis (estimate of allele sharing was 0.44; standard error = +/-0.04; 95% confidence interval = 0.36 to 0.52). Moreover, even allowing for heterogeneity, the potential that a COX2 colon neoplasia susceptibility variant was present within a substantial subset of these sibships was strongly excluded under either a recessive or a dominant inheritance model (95% confidence to exclude a model in which 2.7% or more of the sibling pairs harbor a dominant susceptibility allele). CONCLUSIONS:This study of concordantly affected sibling pairs thus demonstrates that variations in the COX2 gene are unlikely to be a source of individual susceptibility to colon neoplasia in humans.

The human genome provides a reference sequence, which is a template for resequencing studies that aim to discover and interpret the record of common ancestry that exists in extant genomes. To understand the nature and pattern of variation and linkage disequilibrium comprising this history, we present a study of approximately 31 kb spanning an approximately 70 kb region of FMR1, sequenced in a sample of 20 humans (worldwide sample) and four great apes (chimp, bonobo, and gorilla). Twenty-five polymorphic sites and two insertion/deletions, distributed in 11 unique haplotypes, were identified among humans. Africans are the only geographic group that do not share any haplotypes with other groups. Parsimony analysis reveals two main clades and suggests that the four major human geographic groups are distributed throughout the phylogenetic tree and within each major clade. An African sample appears to be most closely related to the common ancestor shared with the three other geographic groups. Nucleotide diversity, pi, for this sample is 2.63 +/- 6.28 x 10(-4). The mutation rate, mu is 6.48 x 10(-10) per base pair per year, giving an ancestral population size of approximately 6200 and a time to the most recent common ancestor of approximately 320,000 +/- 72,000 per base pair per year. Linkage disequilibrium (LD) at the FMR1 locus, evaluated by conventional LD analysis and by the length of segment shared between any two chromosomes, is extensive across the region.

The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of approximately 50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read approximately 80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically.

OBJECTIVE:To evaluate the association between the angiotensinogen-6 polymorphism (AGT-6) and blood pressure levels. DESIGN/METHODS:Data were analysed from the first 4,322 subjects of the NHLBI Family Blood Pressure Program (FBPP), consisting of four networks (GenNet, GENOA, HyperGEN and SAPPHIRe), each conducting a multicentre observational family study to identify and characterize the genetic determinants of hypertension and blood pressure. The four studies use different designs (concordant sibpairs, discordant pairs, sibships, extended pedigrees), target different ethnic groups (Caucasian, African-American, Japanese, Chinese), and have different inclusion/exclusion criteria. However, the protocols and definitions were standardized across networks before data collection to allow maximum poolability. METHODS:Each network/racial group was analysed separately, using generalized linear models that accounted for the non-independence of family members and/or the confounding of anti-hypertensive medications as needed. The results were also pooled using a pre-planned meta-analysis technique. RESULTS:AGT-6 was not significantly associated with blood pressure in any network/racial group. In the meta-analysis, the pooled effect of AGT-6 was small [hazard ratio = 1.10, 95% confidence interval (CI) = 0.99-1.22, P= 0.0647 for systolic; hazard ratio = 1.04, 95% CI = 0.89-1.21, P= 0.6383 for diastolic]. A post-hoc analysis restricting to subjects meeting JNC VI criteria for Stage I hypertension (blood pressure > 140/90 mmHg or medicated) showed a stronger statistically significant relationship for systolic blood pressure (hazard ratio = 1.44, 95% CI = 1.04-2.00, P= 0.0283). CONCLUSIONS:AGT-6 has minimal to no effect on the inter-individual variation of blood pressure levels, and is at best a 'minor gene' for blood pressure in the population as a whole.

Population-based candidate gene association analyses are becoming increasingly popular as a result of a greater number of genes and gene polymorphisms having been identified for which some functional information is available. Because many biochemical and physiologic systems impact blood pressure regulation and hypertension susceptibility, many of these identified genes and polymorphisms are candidates for population-level association studies involving blood pressure levels or hypertension status. Recent studies have suggested that the alpha-adducin gene may harbor polymorphisms that influence blood pressure level. Therefore, we embarked on a study to test one such polymorphism in two large US samples: one from an urban African American population (Maywood, IL) and another from a rural white population (Tecumseh, MI). We used both family-based association tests and tests that consider the impact of additional measured factors beyond adducin gene variation on blood pressure levels. We found no evidence for a significant effect of the chosen adducin polymorphism on blood pressure variation in either sample. We also found no association between Adducin genotypes and antihypertensive use. These facts, together with similar findings in companion studies, suggest that the alpha-adducin gene polymorphism does not have a pronounced effect on blood pressure variation in the populations studied. This does not suggest, however, that the alpha-adducin gene does not have a role in blood pressure regulation and hypertension susceptibility.

The origin of human triploidy is controversial. Early cytogenetic studies found the majority of cases to be paternal in origin; however, recent molecular analyses have challenged these findings, suggesting that digynic triploidy is the most common source of triploidy. To resolve this dispute, we examined 91 cases of human triploid spontaneous abortions to (1) determine the mechanism of origin of the additional haploid set, and (2) assess the effect of origin on the phenotype of the conceptus. Our results indicate that the majority of cases were diandric in origin because of dispermy, whereas the maternally-derived cases mainly originated through errors in meiosis II. Furthermore, our results indicate a complex relationship between phenotype and parental origin: paternally-derived cases predominate among "typical" spontaneous abortions, whereas maternally-derived cases are associated with either early embryonic demise or with relatively late demise involving a well-formed fetus. As the cytogenetic studies relied on analyses of the former type of material and the molecular studies on the latter sources, the discrepancies between the data sets are explained by differences in ascertainment. In studies correlating the origin of the extra haploid set with histological phenotype, we observed an association between paternal-but not maternal-triploidy and the development of partial hydatidiform moles. However, only a proportion of paternally derived cases developed a partial molar phenotype, indicating that the mere presence of two paternal genomes is not sufficient for molar development.

Linkage disequilibrium (LD) mapping has been applied to many simple, monogenic, overtly Mendelian human traits, with great success. However, extensions and applications of LD mapping approaches to more complex human quantitative traits have not been straightforward. In this article, we consider the analysis of biallelic DNA marker loci and human quantitative trait loci in settings that involve sampling individuals from opposite ends of the trait distribution. The purpose of this sampling strategy is to enrich samples for individuals likely to possess (and not possess) trait-influencing alleles. Simple statistical models for detecting LD between a trait-influencing allele and neighboring marker alleles are derived that make use of this sampling scheme. The power of the proposed method is investigated analytically for some hypothetical gene-effect scenarios. Our studies indicate that LD mapping of loci influencing human quantitative trait variation should be possible in certain settings. Finally, we consider possible extensions of the proposed methods, as well as areas for further consideration and improvement.

X inactivation is a chromosome-specific form of genetic regulation in which thousands of genes on one homologue become silenced early in female embryogenesis. Although many aspects of X inactivation are now understood, the spread of the X inactivation signal along the entire length of the chromosome remains enigmatic. Extending the Gartler-Riggs model [Gartler, S. M. & Riggs, A. D. (1983) Annu. Rev. Genet. 17, 155-190], Lyon recently proposed [Lyon, M. F. (1998) Cytogenet. Cell Genet. 80, 133-137] that a nonrandom organization of long interspersed element (LINE) repetitive sequences on the X chromosome might be responsible for its facultative heterochromatization. In this paper, we present data indicating that the LINE-1 (L1) composition of the human X chromosome is fundamentally distinct from that of human autosomes. The X chromosome is enriched 2-fold for L1 repetitive elements, with the greatest enrichment observed for a restricted subset of LINE-1 elements that were active <100 million years ago. Regional analysis of the X chromosome reveals that the most significant clustering of these elements is in Xq13-Xq21 (the center of X inactivation). Genomic segments harboring genes that escape inactivation are significantly reduced in L1 content compared with X chromosome segments containing genes subject to X inactivation, providing further support for the association between X inactivation and L1 content. These nonrandom properties of L1 distribution on the X chromosome provide strong evidence that L1 elements may serve as DNA signals to propagate X inactivation along the chromosome.

Reduced penetrance in genetic disorders may be either dependent or independent of the genetic background of gene carriers. Hirschsprung disease (HSCR) demonstrates a complex pattern of inheritance with approximately 50% of familial cases being heterozygous for mutations in the receptor tyrosine kinase RET. Even when identified, the penetrance of RET mutations is only 50-70%, gender-dependent, and varies with the extent of aganglionosis. We searched for additional susceptibility genes which, in conjunction with RET, lead to phenotypic expression by studying 12 multiplex HSCR families. Haplotype analysis and extensive mutation screening demonstrated three types of families: six families harboring severe RET mutations (group I); and the six remaining families, five of which are RET-linked families with no sequence alterations and one RET-unlinked family (group II). Although the presence of RET mutations in group I families is sufficient to explain HSCR inheritance, a genome scan reveals a new susceptibility locus on 9q31 exclusively in group II families. As such, the gene at 9q31 is a modifier of HSCR penetrance. These observations imply that identification of new susceptibility factors in a complex disease may depend on classification of families by mutational type at known susceptibility genes.