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European Americans Nov 13, 2023 22:00:38 GMT

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Post by Admin on Nov 13, 2023 22:00:38 GMT

Unappreciated subcontinental admixture in Europeans and European Americans and implications for genetic epidemiology studies

Abstract
European-ancestry populations are recognized as stratified but not as admixed, implying that residual confounding by locus-specific ancestry can affect studies of association, polygenic adaptation, and polygenic risk scores. We integrate individual-level genome-wide data from ~19,000 European-ancestry individuals across 79 European populations and five European American cohorts. We generate a new reference panel that captures ancestral diversity missed by both the 1000 Genomes and Human Genome Diversity Projects. Both Europeans and European Americans are admixed at the subcontinental level, with admixture dates differing among subgroups of European Americans. After adjustment for both genome-wide and locus-specific ancestry, associations between a highly differentiated variant in LCT (rs4988235) and height or LDL-cholesterol were confirmed to be false positives whereas the association between LCT and body mass index was genuine. We provide formal evidence of subcontinental admixture in individuals with European ancestry, which, if not properly accounted for, can produce spurious results in genetic epidemiology studies.

Introduction
Human genetic studies have primarily considered admixed populations to have resulted from interbreeding between two or more continentally separated populations1,2,3. However, continental ancestry is not necessarily a single homogenous component of genetic diversity, but rather can be a composite of diverse subcontinental ancestries4,5. In some instances, differentiation between intra-continental populations is on par with or higher than differentiation between inter-continental populations1,6. Also, there are examples from pharmacogenetics of variants that are differentiated at the intra-continental level, such as in the case of abacavir hypersensitivity syndrome, for which the causal allele (HLA-B*5701) has a prevalence of 13.6% among Maasai in Kenya but a prevalence of ~0% among Yoruba in Nigeria7.

Despite genetic studies highlighting a clear pattern of North-to-South genetic variation in Europe8,9,10 and strong evidence of admixture within Europe by ancient DNA analysis11,12, European-ancestry populations are generally treated in association models as stratified but not as admixed at the subcontinental level. As a result, genetic epidemiology studies of Europeans or European Americans usually control for potential confounding effects of population stratification using genome-wide ancestry estimated by principal components analysis13, but do not control for locus-specific ancestry, which is inherent to admixed populations14. Potential consequences are that detection of causal genetic variation is hampered and estimation of effect sizes can be biased, leading to further negative consequences such as misestimation of polygenic adaptation15 and poor predictive performance of polygenic risk scores16.

Recently developed approaches have enabled the use of genome-wide data (either array-based genotype or whole genome sequence data) to assess admixture at two levels: genome-wide ancestry (also known as global ancestry)13,17,18, which is the individual’s ancestry averaged across the entire genome, and locus-specific ancestry (also known as local ancestry)19,20,21, which allows for inference of an individual’s ancestry at each locus. The power, resolution, and specificity of disease or trait mapping studies can be improved by leveraging both genome-wide and locus-specific ancestries3,22,23. To assess both genome-wide and locus-specific ancestries in admixed individuals, present-day populations are used as proxies for ancestral populations that serve as references for ancestry estimation. Considering that ~96% of participants in genome-wide association studies (GWAS) have European ancestry24, a comprehensive analysis is needed to evaluate the adequacy of European reference panels for ancestry analysis using European-ancestry individuals.

The prevalence of lactase persistence varies widely across Europe and the most strongly associated variant rs4988235 in the lactase gene (LCT) has been reported to be under positive selection and associated with height, body mass index (BMI), and low-density lipoprotein (LDL)25,26,27,28. The SNP rs4988235 is one of the most highly differentiated variants in Europe29, with derived allele (A) frequencies ranging from 93.1% in Swedes to 2.9% in Sardinians30. Importantly, rs4988235 and height are well known to covary following a north-to-south axis31, and the association between rs4988235 and height has been suggested to be spurious based on attenuation following adjustment for genome-wide ancestry27. Nonetheless, there are no association studies in European-ancestry populations that control for confounding at both the genome-wide and locus-specific ancestry levels to test the validity of the association between rs4988235 and reported associated traits.

To test for the existence of subcontinental ancestries within Europe, we integrated genome-wide data from 1,216 individuals across 79 European populations. Then, to examine population structure and admixture, we integrated genome-wide data from 17,669 European Americans from five genetic epidemiology cohorts in the US. Finally, to illustrate the potential implications of confounding by subcontinental ancestry and admixture, we interrogated the association between rs4988235 and height, LDL-cholesterol, and BMI.

We found that the 1000 Genomes and Human Genome Diversity Projects provided incomplete coverage of European ancestries, so we generated a new reference panel to capture additional European ancestral diversity. Our admixture analyses yielded formal evidence that European-ancestry individuals are admixed at the subcontinental level, with admixture dates differing among European American subgroups. After adjustment for both genome-wide and locus-specific ancestry, previously reported associations between rs4988235 and height or LDL were no longer statistically significant, strongly supporting that they are false positives due to uncorrected stratification. We observed that better fits can be obtained when models were adjusted for principal components (PCs) derived from projection of European Americans onto our new reference panel, rather than for PCs derived from study-specific unsupervised analysis. Altogether, this study indicates that full adjustment for subcontinental European admixture (at both genome-wide and locus-specific levels) should become best practice in genetic association studies using European-ancestry individuals, including the UK Biobank32 in Europe and the All of Us Research Program33 and the VA Million Veteran Program34 in the United States.

www.nature.com/articles/s41467-023-42491-0

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European Americans Nov 14, 2023 21:14:03 GMT

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Post by Admin on Nov 14, 2023 21:14:03 GMT

Results
Reference panels of European diversity
We generated a new reference panel capturing genetic diversity from 79 European populations from five population genetics studies: the 1000 Genomes Project35, the Human Genome Diversity Project (HGDP)36, the Human Origins dataset37, a study of the Caucasus Mountains38, and a study of the Jewish Diaspora39 (Fig. 1A and Supplementary Data 1). After quality control to reduce batch effects, our European panel included 1,216 unrelated individuals and 104,414 genotyped SNPs. Principal component analysis (PCA)13 showed that North Europeans (e.g., Finnish, Lithuanian, and Estonian) vs Southeast Europeans (e.g., Armenian, Georgian Jew, and Georgian Megrel) represented the extremes along the first principal component (Fig. 1B). Along the second principal component, Southwest Europeans (e.g., Sardinian, Basque, and Spanish) vs Southeast Europeans (e.g., South Caucasus) represented the extremes. Subsequent principal components separated population-specific genetic variability (Fig. S1). To compare our panel with commonly used European reference panels from the Human Genome Diversity Project (HGDP)36 and the 1000 Genomes Project35,36, we calculated convex hull areas40 defined by the first two principal components (Fig. 1B, C), which captured 1.06% of the genetic variance. Compared to our panel, the 1000 Genomes Project and the HGDP covered 26.8% and 61.3% of European diversity, respectively, while the combination of the 1000 Genomes Project and the HGDP covered 77.3% (Fig. 1C). These results indicate that the 1000 Genomes Project and the HGDP, separately and combined, provide incomplete coverage of European genetic diversity.

Fig. 1: European reference panels and coverage of European genetic diversity.

A) Map of Europe showing the geographic location of samples from 79 European populations. The map was drawn using the R package “maps” version 3.4.1. B) The first two principal components (PC1 and PC2) of genetic diversity and the percent variance explained. C) Coverage of genetic diversity over the first two principal components (convex hull area). 1000G = 1000 Genomes Project, HGDP Human Genome Diversity Project.

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European Americans Jan 6, 2024 18:38:44 GMT

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Post by Admin on Jan 6, 2024 18:38:44 GMT

Subcontinental stratification in individuals with European ancestry
To expand and refine our understanding of subcontinental stratification and admixture in European-ancestry populations, we integrated genome-wide genotype data from approximately 19,000 European-ancestry individuals (Fig. 2). These data included our European panel (1216 unrelated individuals) and 17,669 European Americans from five genetic epidemiology cohorts in the US: Atherosclerosis Risk in Communities (ARIC, n = 9633), Coronary Artery Risk Development in Young Adults (CARDIA, n = 1675), Framingham Heart Study (FHS, n = 2451), Genetic Epidemiology Network of Arteriopathy (GENOA, n = 1384), and Multi-Ethnic Study of Atherosclerosis (MESA, n = 2526). To assess continental-level structure, we evaluated our European-ancestry dataset with a worldwide reference panel (Fig. S2). Most Europeans formed a discrete cluster along the first two principal components, as previously observed35,36. Similarly, by projecting European Americans (Supplementary Data 2) onto the worldwide reference panel, we observed that >99% of European Americans clustered with European reference individuals, with few individuals distributed along the first principal component (European-African gradient) or the second principal component (European-Asian gradient). These results suggest that the Europeans included in our panel represent a cluster in relation to worldwide genetic diversity and that European Americans in genetic epidemiology cohorts in the US have small to negligible population stratification at the inter-continental scale.

Fig. 2: Projection analysis of European Americans onto our European reference panel.

We plotted the convex hull area for all cohorts combined and for each European American cohort. The full legend as well as the geographic location of samples from 79 European populations can be found in Fig. 1. Convex hull area = Coverage of genetic diversity over the first two principal components. European Americans cohorts in the US: Atherosclerosis Risk in Communities (ARIC), Coronary Artery Risk Development in Young Adults (CARDIA), Framingham Heart Study (FHS), Genetic Epidemiology Network of Arteriopathy (GENOA), and Multi-Ethnic Study of Atherosclerosis (MESA).

Next, to evaluate European subcontinental stratification in European American cohorts, we projected individuals from each European American cohort onto our European reference panel, represented by the first two principal components. We calculated that European American cohorts collectively covered 68.2% of European diversity in our panel (Fig. 2), with differential coverage by cohort: 55.7% in MESA, 51.2% in ARIC, 44.1% in CARDIA, 28.4% in FHS, and 9.7% in GENOA. The ARIC, CARDIA, FHS, and MESA individuals formed at least three clusters: one with North Europeans (e.g., British and Scandinavian), one with Southeast Europeans (e.g., Ashkenazi Jew and Romanian Jew), and one overlapping Finnish individuals. GENOA individuals mostly overlapped British or Scandinavian reference individuals, with few individuals overlapping South Europeans. Most FHS samples overlapped with or were between North and South Europeans, with a large number of individuals clustering with Italian reference individuals. Most European Americans clustering with Finnish reference samples were from the ARIC cohort.

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European Americans Jan 8, 2024 21:17:01 GMT

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Post by Admin on Jan 8, 2024 21:17:01 GMT

Subcontinental admixture in individuals with European ancestry
Unsupervised analysis with ADMIXTURE17 using our European reference panel identified the most likely number of ancestry clusters as three (Fig. 3A), suggesting that Europeans have three-way admixture among North, Southwest, and Southeast Europeans. The stacked bar plot of mixture proportions showed that the North European-associated ancestry cluster decreased along the north-to-south geographic direction (Fig. 3A). Formal correlation tests between population ancestry means and geographic coordinates revealed that latitude was significantly correlated (p < 2.85 × 10−8) with the North European-associated ancestry cluster (Spearman’s rho = 0.814), and longitude was correlated with Southwest- (Spearman’s rho = −0.859) and Southeast-associated (Spearman’s rho = 0.579) European ancestry clusters (Fig. 3B). We observed similar levels of genetic differentiation (FST) between the inferred European ancestry clusters: FST = 0.033 between North and Southwest, FST = 0.032 between North and Southeast, and FST = 0.028 between Southwest and Southeast. For these comparisons, European-associated ancestry clusters are genetically homogeneous populations identified by ADMIXTURE, not real-world populations. To put these amounts of genetic differentiation into perspective, FST estimates between European ancestry clusters are comparable to FST between British (GBR) and either Mexican (MXL, which have ~50% Native American ancestry, FST = 0.031) or Punjabi in Pakistan (PJL, who have >70% South Asia Ancestry, FST = 0.027) samples (Supplementary Data 3). Additionally, FST estimates between European ancestry clusters are at least three-fold higher than FST between Mandenka from Gambia in West Africa and Luhya from Kenya from East Africa (FST = 0.011, Supplementary Data 3). Even when comparing real-world European populations, FST estimates between Finnish in North Europe and Armenians or Georgians in South Europe are approximately twofold higher (FST ~ 0.02) than FST between Mandenka and Luhya (FST = 0.011), i.e., between West and East Africans, and not as high as FST between inferred European ancestry clusters. The FST estimates in our analysis were in agreement with FST values reported by the 1000 Genomes Project35.

Fig. 3: Subcontinental ancestries in Europe and correlation of ancestry with geography.

A) Bar plot showing ancestry proportions in the European populations and a cross-validation plot supporting K = 3 as the most likely number of ancestry clusters. Purple, orange, and cyan colors represent ancestry clusters associated with North, Southwest, and Southeast European populations, respectively. Individual Bar plots were sorted in descending order of the amount of North European ancestry (Purple), and populations are sorted in descending order of the average of North European ancestry. B) Correlation plots depicting Spearman’s rho between ancestry proportions and geographic coordinates. Colored lines represent fitted linear regressions. p value was derived from a two-sided test.

Supervised ADMIXTURE17 analysis of European Americans (using individuals with ≥90% of one of three geography-associated European ancestry clusters as parentals) showed patterns of European ancestry clusters that differed by cohort (Fig. 4 and Supplementary Data 4). GENOA had the highest mean proportion of the North European ancestry cluster (44%, SE = 3.9%) and the lowest proportion of the Southeast European ancestry cluster (7%, SE = 3%), while FHS had the lowest mean proportion of the North European ancestry cluster (29.9%, SE = 3.7%). MESA had the highest proportion of the Southeast European ancestry cluster (25.4%, SE = 3.1%), followed by FHS (19.7% SE = 3%). The admixture patterns in the European American cohorts were consistent with the projection analysis (Fig. 2), e.g., the GENOA individuals clustered tightly with British and Scandinavian individuals on the first principal component. By testing genetic admixture using f3 statistics41, using Europeans as admixture sources and European Americans as admixture targets, we obtained formal evidence for admixture in the history of European Americans (Supplementary Data 5A–E). Also, we observed positive correlation between FST (a measurement of North-South European differentiation) and FIT (a measurement of inbreeding) at SNPs throughout the genome in European American cohorts, consistent with subcontinental ancestry-related assortative mating in European Americans (Supplementary Data 6). Our results confirm the presence of subcontinental population structure in both Europeans and European Americans, that this structure reflects mixed ancestry in the vast majority of individuals, and that mixed ancestry reflects admixture rather than discrete subpopulations in Europe.

Last Edit: Jan 8, 2024 21:17:30 GMT by Admin

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European Americans Jan 11, 2024 19:27:23 GMT

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Fig. 4: Ancestry proportions in European Americans.

Bar plot representation of individual ancestry proportions inferred from supervised analysis. Purple, orange, and cyan colors represent ancestry clusters associated with North, Southwest, and Southeast European populations, respectively. Individual Bar plots were sorted in descending order of the amount of North ancestry cluster (Purple). European Americans cohorts in the US: Atherosclerosis Risk in Communities (ARIC), Coronary Artery Risk Development in Young Adults (CARDIA), Framingham Heart Study (FHS), Genetic Epidemiology Network of Arteriopathy (GENOA), and Multi-Ethnic Study of Atherosclerosis (MESA).

Admixture dating in European Americans
To date admixture in European Americans, we first applied a clustering approach42 to the first two principal components and inferred that European Americans likely cluster within three subgroups of individuals (Fig. 5A and Fig. S3). Projection analysis of European Americans onto our European reference panel revealed that European Americans were widely distributed across a north-south axis, with centroids of inferred subgroups related to North (Subgroup N), Southwest (Subgroup SW), and Southeast (Subgroup SE) Europeans (Fig. 5B). The highest proportion of ancestry in Subgroup N individuals was North European ancestry (54.5%). Similarly, the highest proportions of ancestry in Subgroup SW and Subgroup SE individuals were Southwest European ancestry (53.7%) and Southeast European ancestry (71.2%), respectively. Next, we used MALDER43 to infer admixture times for individuals within each of the three subgroups of European Americans. We observed significant admixture dating for all three subgroups, with subgroup SE yielding an admixture date ~10 generations more recent (42.00 generations, SE = 6.82) than admixture dates for subgroup SW (54.28 generations, SE = 10.43) and subgroup N (50.89 generations, SE = 14.26). As a confirmatory analysis, we used LaNeta44 for admixture dating in European Americans, and we observed similar admixture times as inferred by MALDER (Supplementary Data 7).

Fig. 5: Substructure and admixture dating in European Americans.

A) The number of clusters (k) was estimated using gap statistics (the error bars represent 95% confidence intervals), based on the first two principal components (PCs) derived from the B) projection analysis of European Americans (15,917 unrelated individuals). We estimated that European Americans are distributed across three subgroups representing North (N), Southwest (SW), and Southeast (SE) Europeans. C) Bar plot representing ancestry profiles within each estimated cluster of European Americans. The error bars represent standard deviation. D) Admixture dating across clusters of European Americans. Point estimates and standard errors (represented by the error bars) of statistically significant admixture dates are shown on the horizontal axis.