George Argyropoulos

Skin pigmentation, biogeographical ancestry and admixture mapping

Ancestry informative markers (AIMs) are genetic loci showing alleles with large frequency differences between populations. AIMs can be used to estimate biogeographical ancestry at the level of the population, subgroup (e.g. cases and controls) and individual. Ancestry estimates at both the subgroup and individual level can be directly instructive regarding the genetics of the phenotypes that differ qualitatively or in frequency between populations. These estimates can provide a compelling foundation for the use of admixture mapping (AM) methods to identify the genes underlying these traits. We present details of a panel of 34 AIMs and demonstrate how such studies can proceed, by using skin pigmentation as a model phenotype. We have genotyped these markers in two population samples with primarily African ancestry, viz. African Americans from Washington D.C. and an African Caribbean sample from Britain, and in a sample of European Americans from Pennsylvania. In the two African population samples, we observed significant correlations between estimates of individual ancestry and skin pigmentation as measured by reflectometry (R2=0.21, P<0.0001 for the African-American sample and R2=0.16, P<0.0001 for the British African-Caribbean sample). These correlations confirm the validity of the ancestry estimates and also indicate the high level of population structure related to admixture, a level that characterizes these populations and that is detectable by using other tests to identify genetic structure. We have also applied two methods of admixture mapping to test for the effects of three candidate genes (TYR, OCA2, MC1R) on pigmentation. We show that TYR and OCA2 have measurable effects on skin pigmentation differences between the west African and west European parental populations. This work indicates that it is possible to estimate the individual ancestry of a person based on DNA analysis with a reasonable number of well-defined genetic markers. The implications and applications of ancestry estimates in biomedical research are discussed.

Ancestral proportions and admixture dynamics in geographically defined African Americans living in South Carolina

We analyzed admixture in samples of six different African-American populations from South Carolina: Gullah-speaking Sea Islanders in coastal South Carolina, residents of four counties in the Low Country (Berkeley, Charleston, Colleton, and Dorchester), and persons living in the city of Columbia, located in central South Carolina. We used a battery of highly informative autosomal, mtDNA, and Y-chromosome markers. Two of the autosomal markers (FY and AT3) are linked and lie 22 cM apart on chromosome 1. The results of this study indicate, in accordance with previous historical, cultural, and anthropological evidence, a very low level of European admixture in the Gullah Sea Islanders (m = 3.5 +/- 0.9%). The proportion of European admixture is higher in the Low Country (m ranging between 9. 9 +/- 1.8% and 14.0 +/- 1.9%), and is highest in Columbia (m = 17.7 +/- 3.1%). A sex-biased European gene flow and a small Native American contribution to the African-American gene pool are also evident in these data. We studied the pattern of pairwise allelic associations between the FY locus and the nine other autosomal markers in our samples. In the combined sample from the Low Country (N = 548), a high level of linkage disequilibrium was observed between the linked markers, FY and AT3. Additionally, significant associations were also detected between FY and 4 of the 8 unlinked markers, suggesting the existence of significant genetic structure in this population. A continuous gene flow model of admixture could explain the observed pattern of genetic structure. A test conditioning on the overall admixture of each individual showed association of ancestry between the two linked markers (FY and AT3), but not between any of the unlinked markers, as theory predicts. Thus, even in the presence of genetic structure due to continuous gene flow or some other factor, it is possible to differentiate associations due to linkage from spurious associations due to genetic structure.

Evidence for balancing selection from nucleotide sequence analyses of human G6PD.

Glucose-6-phosphate dehydrogenase (G6PD) mutations that result in reduced enzyme activity have been implicated in malarial resistance and constitute one of the best examples of selection in the human genome. In the present study, we characterize the nucleotide diversity across a 5.2-kb region of G6PD in a sample of 160 Africans and 56 non-Africans, to determine how selection has shaped patterns of DNA variation at this gene. Our global sample of enzymatically normal B alleles and A, A-, and Med alleles with reduced enzyme activities reveals many previously uncharacterized silent-site polymorphisms. In comparison with the absence of amino acid divergence between human and chimpanzee G6PD sequences, we find that the number of G6PD amino acid polymorphisms in human populations is significantly high. Unlike many other G6PD-activity alleles with reduced activity, we find that the age of the A variant, which is common in Africa, may not be consistent with the recent emergence of severe malaria and therefore may have originally had a historically different adaptive function. Overall, our observations strongly support previous genotype-phenotype association studies that proposed that balancing selection maintains G6PD deficiencies within human populations. The present study demonstrates that nucleotide sequence analyses can reveal signatures of both historical and recent selection in the genome and may elucidate the impact that infectious disease has had during human evolution.

Large-scale SNP analysis reveals clustered and continuous patterns of human genetic variation

Understanding the distribution of human genetic variation is an important foundation for research into the genetics of common diseases. Some of the alleles that modify common disease risk are themselves likely to be common and, thus, amenable to identification using gene-association methods. A problem with this approach is that the large sample sizes required for sufficient statistical power to detect alleles with moderate effect make gene-association studies susceptible to false-positive findings as the result of population stratification [1, 2]. Such type I errors can be eliminated by using either family-based association tests or methods that sufficiently adjust for population stratification [3–5]. These methods require the availability of genetic markers that can detect and, thus, control for sources of genetic stratification among populations. In an effort to investigate population stratification and identify appropriate marker panels, we have analysed 11,555 single nucleotide polymorphisms in 203 individuals from 12 diverse human populations. Individuals in each population cluster to the exclusion of individuals from other populations using two clustering methods. Higher-order branching and clustering of the populations are consistent with the geographic origins of populations and with previously published genetic analyses. These data provide a valuable resource for the definition of marker panels to detect and control for population stratification in population-based gene identification studies. Using three US resident populations (European-American, African-American and Puerto Rican), we demonstrate how such studies can proceed, quantifying proportional ancestry levels and detecting significant admixture structure in each of these populations.

Extent of linkage disequilibrium between the androgen receptor gene CAG and GGC repeats in human populations: implications for prostate cancer risk.

While studies have implicated alleles at the CAG and GGC trinucleotide repeats of the androgen receptor gene with high-grade, aggressive prostate cancer disease, little is known about the normal range of variation for these two loci, which are separated by about 1.1xa0kb. More importantly, few data exist on the extent of linkage disequilibrium (LD) between the two loci in different human populations. Here we present data on CAG and GGC allelic variation and LD in six diverse populations. Alleles at the CAG and GGC repeat loci of the androgen receptor were typed in over 1000 chromosomes from Africa, Asia, and North America. Levels of linkage disequilibrium between the two loci were compared between populations. Haplotype variation and diversity were estimated for each population. Our results reveal that populations of African descent possess significantly shorter alleles for the two loci than non-African populations (P<0.0001). Allelic diversity for both markers was higher among African Americans than any other population, including indigenous Africans from Sierra Leone and Nigeria. Analysis of molecular variance revealed that approx. 20% of CAG and GGC repeat variance could be attributed to differences between the populations. All non-African populations possessed the same common haplotype while the three populations of African descent possessed three divergent common haplotypes. Significant LD was observed in our sample of healthy African Americans. The LD observed in the African American population may be due to several reasons; recent migration of African Americans from diverse rural communities following urbanization, recurrent gene flow from diverse West African populations, and admixture with European Americans. This study represents the largest genotyping effort to be performed on the two androgen receptor trinucleotide repeat loci in diverse human populations.

A functional SNP in the promoter of the SERPINH1 gene increases risk of preterm premature rupture of membranes in African Americans

Prematurity is more prevalent in African Americans than in European Americans. We investigated the contribution of a functional SNP in the promoter of the SERPINH1 gene, enriched among those of African ancestry, to preterm premature rupture of membranes (PPROM), the leading identifiable cause of preterm birth. SERPINH1 encodes heat-shock protein 47, a chaperone essential for collagen synthesis. The SERPINH1 −656 minor T allele had a greater frequency in African populations and African Americans than in European Americans (12.4% vs. 4.1%). The −656 T allele displayed significantly reduced promoter activity compared to the major −656 C allele in amnion fibroblasts, which lay down the fibrillar collagen that gives tensile strength to the amnion. An initial case-control study demonstrated that the −656 T allele is significantly more frequent in African-American neonates (P < 0.0009) born from pregnancies complicated by PPROM compared with controls (odds ratio of 3.22, 95% confidence interval 1.50, 7.22). There was no significant difference in ancestry among cases and controls using a dihybrid model based on 29 ancestry-informative markers. Adjusting the results of the case-control study for admixture still yielded a statistically significant association between the −656 T allele and PPROM (P < 0.002). A follow-up case-control study gave similar results. The combined case-control findings showed a highly significant (P < 0.0000045) association between the −656 T allele and PPROM. The SERPINH1 −656 T allele is the first example of an ancestry-informative marker associated with preterm birth in African Americans.

Three Novel mtDNA Restriction Site Polymorphisms Allow Exploration of Population Affinities of African Americans

To develop informative tools for the study of population affinities in African Americans, we sequenced the hypervariable segments I and II (HVS I and HVS II) of mitochondrial DNA (mtDNA) from 96 Sierra Leoneans; European Americans; rural, Gullah-speaking African Americans; urban African Americans living in Charleston, South Carolina; and Jamaicans. We identified single nucleotide polymorphisms (SNPs) exhibiting ethnic affinities, and developed restriction endonuclease tools to screen these SNPs. Here we show that three HVS restriction site polymorphisms (RSPs), EcoRV, FokI, and MfeI, exhibit appreciable differences in frequency (average d = 0.4165) between putative African American parental populations (i.e., extant Africans living in Sierra Leone and European Americans). Estimates of European American mtDNA admixture, calculated from haplotypes composed of these three novel RSPs, show a cline of increasing admixture from Gullah-speaking African American (m = 0.0300) to urban Charleston African American (m = 0.0689) to West Coast African American (m = 0.1769) populations. This haplotype admixture in the Gullahs is the lowest recorded to date among African Americans, consistent with previous studies using autosomal markers. These RSPs may become valuable new tools in the study of ancestral affinities and admixture dynamics of African Americans.

Gestational diabetes mellitus and gene mutations which affect insulin secretion.

We investigated whether genetic mutations known to impair insulin secretion and glucose tolerance are operative in a group of American women with gestational diabetes mellitus. Study groups were comprised of elderly non-diabetic controls (n = 55) with normal glucose tolerance and patients with gestational diabetes (n = 50), together with one family with maturity-onset diabetes of the young (three controls and three affected). No mutations were detected in any exon of the human glucokinase gene or the mitochondrial tRNA[Leu](UUR) gene by single strand conformational analysis and direct exon sequencing. Also, chi2 analysis showed no significant association with gestational diabetes for a polymorphism at position -30 (G --> A) of the beta-cell-specific glucokinase gene promoter. We have determined that glucokinase and mitochondrial tRNA[Leu](UUR) gene mutations, which are known to impair insulin secretion are relatively uncommon and do not constitute a large component of genetic risk for gestational diabetes in the study population.

Endogenous mutations in human uncoupling protein 3 alter its functional properties

Human uncoupling protein 3 (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity. Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G>A) on the uncoupling activity of UCP3. It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (ΔΨ), possibly conferring susceptibility to develop metabolic diseases.

Characterization of a human import component of the mitochondrial outer membrane, TOMM70A.

Functional mitochondria require up to 1000 proteins to function properly, with 99% synthesized as precursors in the cytoplasm and transported into the mitochondria with the aid of cytosolic chaperones and mitochondrial translocators (import components). Proteins to be imported are chaperoned to the mitochondria by the cytosolic heat shock protein (cHSP70) and are immediately pursued by Translocators of the Outer Membrane (TOMs), followed by transient interactions of the unfolded proteins with Translocators of the Inner Membrane (TIMs). In the present study, we describe a human gene, TOMM70A, orthologous to the yeast Tom70 import component. TOMM70A is ubiquitously expressed in human tissues, maps on chromosome 3q13.1-q13.2 and consists of 12 coding exons spanning over 37 kb. TOMM70A localizes in the mitochondria of COS-7 cells, and in organello import assays confirmed its presence in the Outer Mitochondrial membrane (OM) of rat liver mitochondria. TOMM70A could play a significant role in the import of nuclear-encoded mitochondrial proteins with internal targeting sites such as ADP/ATP carriers and the uncoupling proteins.