W. Scott Watkins

Human Population Genetic Structure and Inference of Group Membership

A major goal of biomedical research is to develop the capability to provide highly personalized health care. To do so, it is necessary to understand the distribution of interindividual genetic variation at loci underlying physical characteristics, disease susceptibility, and response to treatment. Variation at these loci commonly exhibits geographic structuring and may contribute to phenotypic differences between groups. Thus, in some situations, it may be important to consider these groups separately. Membership in these groups is commonly inferred by use of a proxy such as place-of-origin or ethnic affiliation. These inferences are frequently weakened, however, by use of surrogates, such as skin color, for these proxies, the distribution of which bears little resemblance to the distribution of neutral genetic variation. Consequently, it has become increasingly controversial whether proxies are sufficient and accurate representations of groups inferred from neutral genetic variation. This raises three questions: how many data are required to identify population structure at a meaningful level of resolution, to what level can population structure be resolved, and do some proxies represent population structure accurately? We assayed 100 Alu insertion polymorphisms in a heterogeneous collection of approximately 565 individuals, approximately 200 of whom were also typed for 60 microsatellites. Stripped of identifying information, correct assignment to the continent of origin (Africa, Asia, or Europe) with a mean accuracy of at least 90% required a minimum of 60 Alu markers or microsatellites and reached 99%-100% when >/=100 loci were used. Less accurate assignment (87%) to the appropriate genetic cluster was possible for a historically admixed sample from southern India. These results set a minimum for the number of markers that must be tested to make strong inferences about detecting population structure among Old World populations under ideal experimental conditions. We note that, whereas some proxies correspond crudely, if at all, to population structure, the heuristic value of others is much higher. This suggests that a more flexible framework is needed for making inferences about population structure and the utility of proxies.

The Simons Genome Diversity Project: 300 genomes from 142 diverse populations

Here we report the Simons Genome Diversity Project data set: high quality genomes from 300 individuals from 142 diverse populations. These genomes include at least 5.8 million base pairs that are not present in the human reference genome. Our analysis reveals key features of the landscape of human genome variation, including that the rate of accumulation of mutations has accelerated by about 5% in non-Africans compared to Africans since divergence. We show that the ancestors of some pairs of present-day human populations were substantially separated by 100,000 years ago, well before the archaeologically attested onset of behavioural modernity. We also demonstrate that indigenous Australians, New Guineans and Andamanese do not derive substantial ancestry from an early dispersal of modern humans; instead, their modern human ancestry is consistent with coming from the same source as that of other non-Africans.

A strong signature of balancing selection in the 5' cis-regulatory region of CCR5.

CCR5 encodes a cell surface chemokine receptor molecule that serves as the principal coreceptor, with CD4, for HIV-type 1 (HIV-1). Varied HIV-1 susceptibility and time to progression to AIDS have been associated with polymorphisms in CCR5. Many of these polymorphisms are located in the 5′ cis-regulatory region of CCR5, suggesting that it may have been a target of natural selection. We characterized CCR5 sequence variation in this region in 400 chromosomes from worldwide populations and compared it to a genome-wide analysis of 100 Alu polymorphisms typed in the same populations. Variation was substantially higher than expected and characterized by an excess of intermediate-frequency alleles. A genealogy of CCR5 haplotypes had deep branch lengths despite markedly little differentiation among populations. This finding suggested a deviation from neutrality not accounted for by population structure, which was confirmed by tests for natural selection. These results are strong evidence that balancing selection has shaped the pattern of variation in CCR5 and suggest that HIV-1 resistance afforded by CCR5 5′ cis-regulatory region haplotypes may be the consequence of adaptive changes to older pathogens.

Microsatellites as EWS/FLI response elements in Ewing's sarcoma

The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma. However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewings sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewings sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChIP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NR0B1, a gene required for Ewings sarcoma oncogenesis. The microsatellite in the NR0B1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis.

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.

Global diversity, population stratification, and selection of human copy-number variation.

Duplications and deletions in the human genome Duplications and deletions can lead to variation in copy number for genes and genomic loci among humans. Such variants can reveal evolutionary patterns and have implications for human health. Sudmant et al. examined copy-number variation across 236 individual genomes from 125 human populations. Deletions were under more selection, whereas duplications showed more population-specific structure. Interestingly, Oceanic populations retain large duplications postulated to have originated in an ancient Denisovan lineage. Science, this issue 10.1126/science.aab3761 Copy-number variation reveals how selection affects the human genome across the globe. INTRODUCTION Most studies of human genetic variation have focused on single-nucleotide variants (SNVs). However, copy-number variants (CNVs) affect more base pairs of DNA among humans, and yet our understanding of CNV diversity among human populations is limited. RATIONALE We aimed to understand the pattern, selection, and diversity of copy-number variation by analyzing deeply sequenced genomes representing the diversity of all humans. We compared the selective constraints of deletions versus duplications to understand population stratification in the context of the ancestral human genome and to assess differences in CNV load between African and non-African populations. RESULTS We sequenced 236 individual genomes from 125 distinct human populations and identified 14,467 autosomal CNVs and 545 X-linked CNVs with a sequence read-depth approach. Deletions exhibit stronger selective pressure and are better phylogenetic markers of population relationships than duplication polymorphisms. We identified 1036 population-stratified copy-number–variable regions, 295 of which intersect coding regions and 199 of which exhibit extreme signatures of differentiation. Duplicated loci were 1.8-fold more likely to be stratified than deletions but were poorly correlated with flanking genetic diversity. Among these, we highlight a duplication polymorphism restricted to modern Oceanic populations yet also present in the genome of the archaic Denisova hominin. This 225–kilo–base pair (kbp) duplication includes two microRNA genes and is almost fixed among human Papuan-Bougainville genomes. The data allowed us to reconstruct the ancestral human genome and create a more accurate evolutionary framework for the gain and loss of sequences during human evolution. We identified 571 loci that segregate in the human population and another 2026 loci of fixed-copy 2 in all human genomes but absent from the reference genome. The total deletion and duplication load between African and non-African population groups showed no difference after we account for ancestral sequences missing from the human reference. However, we did observe that the relative number of base pairs affected by CNVs compared to single-nucleotide polymorphisms is higher among non-Africans than Africans. CONCLUSION Deletions, duplications, and CNVs have shaped, to different extents, the genetic diversity of human populations by the combined forces of mutation, selection, and demography. Figure Global human CNV diversity and archaic introgression of a chromosome 16 duplication. (Left) The geographic coordinates of populations sampled are indicated on a world map (colored dots). The pie charts show the continental population allele frequency of a single ~225-kbp duplication polymorphism found exclusively among Oceanic populations and an archaic Denisova. (Right) The ancestral structure of this duplication locus (1) and the Denisova duplication structure (2) are shown in relation to their position on chromosome 16. We estimate that the duplication emerged ~440 thousand years ago (ka) in the Denisova and then introgressed into ancestral Papuan populations ~40 ka. In order to explore the diversity and selective signatures of duplication and deletion human copy-number variants (CNVs), we sequenced 236 individuals from 125 distinct human populations. We observed that duplications exhibit fundamentally different population genetic and selective signatures than deletions and are more likely to be stratified between human populations. Through reconstruction of the ancestral human genome, we identify megabases of DNA lost in different human lineages and pinpoint large duplications that introgressed from the extinct Denisova lineage now found at high frequency exclusively in Oceanic populations. We find that the proportion of CNV base pairs to single-nucleotide–variant base pairs is greater among non-Africans than it is among African populations, but we conclude that this difference is likely due to unique aspects of non-African population history as opposed to differences in CNV load.

Genotype – phenotype analysis of angiotensinogen polymorphisms and essential hypertension: the importance of haplotypes

Objectives To better understand the relationship between angiotensinogen (AGT) genetic variation and essential hypertension, AGT genotypes and haplotypes were tested for association with hypertensive endophenotypes and essential hypertension. Methods Two hundred and fifty-six Hypertensive Pathotype (HyperPATH)/Specialized Center of Research (SCOR) cases and 126 controls were genotyped for 24 single-nucleotide polymorphisms (SNPs) in the AGT gene. SNPs and AGT haplotypes were tested for association with plasma AGT, renal plasma flow (RPF), and essential hypertension. Results New associations between essential hypertension, plasma AGT, and RPF are reported for alleles −1178G, 6066A, 6152A, 6233C, and 12822C. The maximum odds ratio for association of hypertension and AGT genetic variation was 2.3 [95% confidence interval (CI) 1.5–3.8; P < 0.0003] for allele 6233C. Previous associations for −1074T, −532T, −217A, −6A, and 4072C are confirmed (P < 0.05). Sodium depletion enhances associations between AGT SNPs and plasma AGT. Most individually associated SNPs, including −6A and 4072C, are found on a common complete AGT haplotype, H4 (frequency = 0.09). Individuals with haplotype H4 have significantly higher plasma AGT and reduced RPF (P < 0.003 and P < 0.0002, respectively). Other common haplotypes are not associated with increased plasma AGT levels in this data set despite the presence of the −6A and 4072C alleles, suggesting that AGT haplotype H4 is more predictive of elevated plasma AGT than is −6A or 4072C. Conclusion This study demonstrates the importance of analyzing haplotypes in addition to single genotypes in association studies. By demonstrating the dependence of AGT associations on sodium depletion status, it helps to explain previous conflicting association results.

Fine-scaled human genetic structure revealed by SNP microarrays

We report an analysis of more than 240,000 loci genotyped using the Affymetrix SNP microarray in 554 individuals from 27 worldwide populations in Africa, Asia, and Europe. To provide a more extensive and complete sampling of human genetic variation, we have included caste and tribal samples from two states in South India, Daghestanis from eastern Europe, and the Iban from Malaysia. Consistent with observations made by Charles Darwin, our results highlight shared variation among human populations and demonstrate that much genetic variation is geographically continuous. At the same time, principal components analyses reveal discernible genetic differentiation among almost all identified populations in our sample, and in most cases, individuals can be clearly assigned to defined populations on the basis of SNP genotypes. All individuals are accurately classified into continental groups using a model-based clustering algorithm, but between closely related populations, genetic and self-classifications conflict for some individuals. The 250K data permitted high-level resolution of genetic variation among Indian caste and tribal populations and between highland and lowland Daghestani populations. In particular, upper-caste individuals from Tamil Nadu and Andhra Pradesh form one defined group, lower-caste individuals from these two states form another, and the tribal Irula samples form a third. Our results emphasize the correlation of genetic and geographic distances and highlight other elements, including social factors that have contributed to population structure.

Maximum-likelihood estimation of recent shared ancestry (ERSA)

Accurate estimation of recent shared ancestry is important for genetics, evolution, medicine, conservation biology, and forensics. Established methods estimate kinship accurately for first-degree through third-degree relatives. We demonstrate that chromosomal segments shared by two individuals due to identity by descent (IBD) provide much additional information about shared ancestry. We developed a maximum-likelihood method for the estimation of recent shared ancestry (ERSA) from the number and lengths of IBD segments derived from high-density SNP or whole-genome sequence data. We used ERSA to estimate relationships from SNP genotypes in 169 individuals from three large, well-defined human pedigrees. ERSA is accurate to within one degree of relationship for 97% of first-degree through fifth-degree relatives and 80% of sixth-degree and seventh-degree relatives. We demonstrate that ERSAs statistical power approaches the maximum theoretical limit imposed by the fact that distant relatives frequently share no DNA through a common ancestor. ERSA greatly expands the range of relationships that can be estimated from genetic data and is implemented in a freely available software package.

Toward a more uniform sampling of human genetic diversity: A survey of worldwide populations by high-density genotyping

High-throughput genotyping data are useful for making inferences about human evolutionary history. However, the populations sampled to date are unevenly distributed, and some areas (e.g., South and Central Asia) have rarely been sampled in large-scale studies. To assess human genetic variation more evenly, we sampled 296 individuals from 13 worldwide populations that are not covered by previous studies. By combining these samples with a data set from our laboratory and the HapMap II samples, we assembled a final dataset of ~250,000 SNPs in 850 individuals from 40 populations. With more uniform sampling, the estimate of global genetic differentiation (F(ST)) substantially decreases from ~16% with the HapMap II samples to ~11%. A panel of copy number variations typed in the same populations shows patterns of diversity similar to the SNP data, with highest diversity in African populations. This unique sample collection also permits new inferences about human evolutionary history. The comparison of haplotype variation among populations supports a single out-of-Africa migration event and suggests that the founding population of Eurasia may have been relatively large but isolated from Africans for a period of time. We also found a substantial affinity between populations from central Asia (Kyrgyzstani and Mongolian Buryat) and America, suggesting a central Asian contribution to New World founder populations.