Enrichment of Immune Pathways in Genes Under Geographically Restricted Adaptation in the Gullah African American Population of South Carolina Paula S. Ramos, Satria Sajuthi, Wei-Min Chen, Jasmin Divers, Jyotika K. Fernandes, Gary S. Gilkeson, Kelly J. Hunt, Diane L. Kamen, Uma Nayak, W. Timothy Garvey, Michèle M. Sale, Carl D. Langefeld

Disclosure Statement The authors have no relevant financial or commercial relationships to disclose.

Outline Disclaimer Natural selection and autoimmunity Gullah African-American (AA) population Results Discussion The reasons for the ethnic disparities in autoimmune diseases (ADs) are largely unknown. We posit that population-specific selection influencing the allele frequencies at some loci contribute to ethnic disparities. Relative to other African-Americans (AA), the Gullah population has lower European admixture and higher ancestral homogeneity from the Sierra Leone (SL) area. We sought to leverage the relative closeness between the Gullah and SL to identify autoimmune risk alleles with evidence of population differentiation that may result from geographically restricted, subtle selective pressure. We computed the fixation index (FST) between Gullah (n=277), Sierra Leonean (n=400), and Yoruba from Nigeria (n=203). We identified several regions that show evidence of selection in the Gullah, including regions known to be under selection that happened pre-admixture. We observed an enrichment of immune pathways, which suggests that autoimmune risk alleles might be present in the Gullah, as well as other AA. Given the increased prevalence of several ADs in AA, identification of regions under selection in the Gullah can further the understanding of the natural history and disease risks in AA and help explain the ethnic disparity. The goal of this research is to understand the etiology of ethnic disparities in autoimmune disease prevalence and severity.

Disclaimer I am a human geneticist.

Evolutionary and population genetics Disclaimer Classical genetics Genetic epidemiology Genomics Molecular genetics Clinical genetics Statistical genetics Bioinformatics Epigenetics Evolutionary and population genetics Cytogenetics Genetic counseling Pharmacogenetics Ethical, legal, social and policy issues Developmental genetics

There is NO biological basis for the concept of “race” Disclaimer There is NO biological basis for the concept of “race”

Measuring patterns of human genetic diversity Characterization of the ABO blood group system by the Hirszfelds in the early 1900s : Pattern of A and B blood types in World War I soldiers showed frequency gradients that correlated with the geographic origin of the soldiers. Pattern of A and B blood types in World War I soldiers showed frequency gradients that correlated with the geographic origin of the soldiers (Hirszfeld and Hirszfeld 1919). Soldiers from Western Europe (English and French) had a lower frequency of the “B” blood group, which appeared to gradually increase as one moved east toward Eastern European (Greeks, Turks, Russians) and Asian groups, suggesting that gene frequencies changed gradually across geographically defined populations.

No genetic divisions among ethnic groups. Lewontin RC No genetic divisions among ethnic groups! Lewontin RC. The apportionment of human diversity. Evolutionary biology 1972; 6:391-398. Multiple different genetic markers (blood group systems and serum protein markers). More than 100 populations sampled across seven socially constructed “racial” groups (Caucasians, Africans, East Asians, South Asians, Amerindians, Oceanians, and Australians). Vast majority of human genetic diversity (∼85%) is caused by individual differences that are shared across “all” populations and races. Despite substantial genetic variation within the human population, such variation has accumulated over time; most of this variation appeared before the expansion of Homo sapiens out of Africa and the resulting isolation of populations within continents. Genetic variation is extensive and largely shared across populations.

The considerable genetic differences we see between individuals has very little to do with so-called “racial” boundaries. Rather, it is merely the variation that was present in the original human population that seeded all the current human populations. The same genetic variants are found in most populations, although their frequencies may differ substantially. Broadly speaking, there has been too little time for the accumulation of substantive divergence in a young species such as ours. The fact that the classical model predicted extensive genetic differentiation between populations was explained by the molecular evolution pioneer Motoo Kimura, who hypothesized that most variation was selectively neutral (neither enhancing nor retarding human survival) and, therefore, largely free from the influence of Darwinian selective forces.

Lewontin’s findings… At the time of publication were controversial, but consensus gradually emerged that genetic differences among populations are modest. The observation that patterns in human genetic variation were largely gradual according to geographic boundaries and not subject to sudden population-specific changes that followed preconceived racial notions removed the biological argument for race - or it should have!

Aim Identify autoimmune risk alleles with evidence of population differentiation that may result from geographically restricted, subtle selective pressure.

Autoimmune diseases affect at least 5-8% of the population! In the US there are: 22 million people with an autoimmune disease 20 million with cancer The prevalence and severity of most autoimmune diseases (ADs) vary among ethnic groups, but the reasons for the ethnic disparities remain elusive.

Natural selection drives adaptation Skin color Malaria and sickle cell anemia Lactose intolerance Since ADs, if untreated, would affect the ability to raise offspring that successfully reproduce, alternative forces or selective pressure must exist that permit the relative high frequency of autoimmune risk alleles seen in current populations. Genetic variation Reproductive/selective advantage Increased allele frequency in population

The Sea Islands Gullah population Higher (~89%) global African ancestry than what is typically reported for AA (~80%) (Huang Y. et al, ICHG/ASHG 2011) Higher African ancestry composition from Far West Africa (Bryc K. et al, ASHG 2012) Huang et al: African populations are the most ancient and diverse on Earth, with a complex genetic substructure due to a long history of migration and admixture. African Americans (AA) are the admixed descents of African and European ancestors and are more genetically susceptible than European Americans to certain prevalent complex diseases. This renders an understanding of African ancestry critical not only in terms of evolution but also for disease based analyses. We used Multidimensional Scaling (MDS) and Principal Component Analysis (PCA) to analyze the population substructure in 384 unrelated Sierra Leone Africans and 877 unrelated Gullah AAs from Sea Island Genetic African American Family Registry (SuGAR), the Center of Biomedical Research Excellence (COBRE) for Oral Health at MUSC and the Systemic Lupus Erythematosus in Gullah Health (SLEIGH) Study. In total, 103731 LD-pruned GWAS SNPs that are also present in 130 Stanford-HGDP and 592 Hapmap III founders were included in the analysis. Four distinct linguistic and lifestyle groups were found. They include West Africans (Sierra Leone populations, Yoruba (YRI) and Mendenka), 3 distinct populations in the South and Southwest (Biaka Pygmies, Mbuti Pygmies and San), Khoisans in the East (Luhya in Webuye(LWK), Maasai in Kinyawa(MKK) and North East Bantu in Kenya) and North Africans in the Northwest (Mozabite). Substructure was also observed within Sierra Leone samples with the three largest subgroups being Mende, Temne and Creole. In addition, the Mendenka samples from Senegal were found to distinct from all other African populations in MDS analysis. Consistent with migratory and residential history, the Gullah AA samples from SuGAR, COBRE and SLEIGH exhibit similar ancestry, and all carry higher (~89%) global African ancestry than what is typically reported for AA (~80%). Compared to YRI and Sierra Leone African samples, the African ancestry of Gullah AAs is closest to Creole in Sierra Leone Africans, while genetically in-between all other Sierra Leone Africans and YRI. These findings may contribute to further understanding of the Sea Islands Gullah population’s ancestral origins. Similar finding were also obtained using model-based ancestry estimation software ADMIXTURE.  Bryc et al: African American genomes have been deeply studied with respect to the variability in their proportions of African and European ancestry, but there has been little success at using genetic data to determine the African source populations. To address these questions, we analyzed genome-wide SNP genotype data from 9,859 African Americans from six cohorts and 4,019 Africans from Sierra Leone, Gambia and Nigeria, and applied two independent methods for inferring ancestry.     First, we modeled each African American as a linear combination of European Americans and several sub-Saharan African groups, using a method that builds a linear ancestry model from population allele frequencies, and can obtain accurate estimates of ancestry proportion assuming that one has access to accurate surrogates for the true ancestral populations. However, a limitation is that it is not possible to disentangle which African populations are directly ancestral to African Americans, and which are only genetically similar to such ancestral populations.     Second we identified long segments of the genome (at least 3 centiMorgans) that are shared identical-by-descent (IBD) between African American and sub-Saharan Africans, showing that the individuals who share the segments are likely to share ancestry at that segment of the genome within the last couple of thousand years. Assuming relatively little gene flow between African tribal groups, this allows us to directly connect African American individuals to specific African tribes.     Both methods detect significant but subtle ancestry differences in sharing to Africa between African American cohorts. To our knowledge, this is the first work that has documented African ancestry differences in African Americans using genome-wide data, offering opportunities for studying history beyond what is possible with uniparentally inherited mitochondrial DNA and Y chromosome data.

Methods We had genotype data on: - 277 healthy Gullahs - 400 Sierra Leoneans (SL) - 203 Yoruba from Nigeria (YRI) HapMap3 Computed the Weir and Cockerham’s (1984) FST between Gullah and SL, and Gullah and YRI. A total of 582,100 autosomal SNPs met standard GWAS quality control. Gene regions with at least one variant within the top 0.01% of highest FST between populations were prioritized.

Selected loci with multiple SNPs within the top 0 Selected loci with multiple SNPs within the top 0.01% of highest FST between Gullah and YRI SNP Chr Position (kb) Gene region Fst rs6881896 5 55,508 ANKRD55 0.088 rs160935 55,518 0.073 rs152345 55,595 0.064 rs6457710 6 33,029 HLA 0.101 rs7744381 33,082 0.094 rs6930960 33,851 0.085 rs7767496 33,852 0.096 rs7797671 7 80,041 CD36 rs1334518 80,226 0.117 rs4728191 80,270 0.120 rs1358339 80,359 0.093 rs4507728 8 10,851 XKR6 0.083 rs4320511 10,871 0.081 rs6601550 10,888 0.104 A total of 16 gene regions in the Gullah vs. SL and 27 in the Gullah vs. YRI met the top FST criteria. The HLA and ANKRD55 are associated with multiple autoimmune diseases, XKR6 with lupus. The HLA and CD36 regions have previously been shown to be under selection that happened pre-admixture in African- Americans.

Enriched biological functions and pathways among loci with highest FST Gullah vs SL Gullah vs YRI Cellular Function and Maintenance (P=6.1E-04) Antigen presentation (P=4.93E-04) and allograft rejection signaling pathways (P=8.30E-04) Cancer functions (P=4.1E-04) CCR3 signaling in eosinophils pathway (P=4.82E-04)

Discussion These data reveal several regions consistent with evidence for selection in the Gullah. However, we cannot rule out that other population genetic factors (e.g. founder effects) contribute to these patterns in some genomic regions. The paucity of genes in the top distribution of the FST that are also associated with an autoimmune disease might be due to the lack of genetic association studies (GWAS) in African- Americans (AA). The enrichment of immune pathways suggests that autoimmune risk alleles might be present in the Gullah, as well as other AA. Given the increased prevalence of several complex traits in AA and the homogeneity of the Gullah, identification of these regions in the Gullah has the potential to elucidate complex disease risks in AA.

Goal The goal of this research is to understand the etiology of ethnic disparities in autoimmune disease prevalence and severity. Looking for AA with SLE and SSc.

Acknowledgements All the study participants. NIH grants K01 AR067280 NIH grant P60 AR062755 WFU Center for Public Health Genomics South Carolina Clinical & Translational Research (SCTR) Institute (NIH Grants UL1 RR029882 and UL1 TR000062)