The population genetics characteristics of a 90 locus panel of microhaplotypes.

Single-nucleotide polymorphisms (SNPs) and small genomic regions with multiple SNPs (microhaplotypes, MHs) are rapidly emerging as novel forensic investigative tools to assist in individual identification, kinship analyses, ancestry inference, and deconvolution of DNA mixtures. Here, we analyzed information for 90 microhaplotype loci in 4009 individuals from 79 world populations in 6 major biogeographic regions. The study included multiplex microhaplotype sequencing (mMHseq) data analyzed for 524 individuals from 16 populations and genotype data for 3485 individuals from 63 populations curated from public repositories. Analyses of the 79 populations revealed excellent characteristics for this 90-plex MH panel for various forensic applications achieving an overall average effective number of allele values (Ae) of 4.55 (range 1.04-19.27) for individualization and mixture deconvolution. Population-specific random match probabilities ranged from a low of 10-115 to a maximum of 10-66. Mean informativeness (In) for ancestry inference was 0.355 (range 0.117-0.883). 65 novel SNPs were detected in 39 of the MHs using mMHseq. Of the 3018 different microhaplotype alleles identified, 1337 occurred at frequencies > 5% in at least one of the populations studied. The 90-plex MH panel enables effective differentiation of population groupings for major biogeographic regions as well as delineation of distinct subgroupings within regions. Open-source, web-based software is available to support validation of this technology for forensic case work analysis and to tailor MH analysis for specific geographical regions.

Selecting microhaplotypes optimized for different purposes.

Massively parallel sequencing is transforming forensic work by allowing various useful forensic markers, such as STRPs and SNPs, to be multiplexed providing information on ancestry, individual and familial identification, phenotypes for eye/hair/skin pigmentation, and the deconvolution of mixtures. Microhaplotypes also become feasible with massively parallel sequencing, these are DNA segments (smaller than 300 nucleotides) that are selected to contain multiple SNPs unambiguously defining three or more haplotype alleles occurring at common frequencies. The physical extent of a microhaplotype can thus be covered by a single sequence read making these loci phase-known codominant genetic systems. Such microhaplotypes supply significantly more information than a single SNP can. Our efforts to develop useful sets of microhaplotypes have already identified 182 such loci that we have studied on a large number of human populations from around the world. We present various analyses on 83 populations in our ongoing study for a subset of the best microhaplotypes currently available illustrating their characteristics and potential utility for ancestry, identification, and mixture deconvolution.

Ancestry inference of 96 population samples using microhaplotypes.

Microhaplotypes have become a new type of forensic marker with a great ability to identify and deconvolute mixtures because massively parallel sequencing (MPS) allows the alleles (haplotypes) of the multi-SNP loci to be determined directly for an individual. As originally defined, a microhaplotype locus is a short segment of DNA with two or more SNPs defining three or more haplotypes. The length is short enough, less than about 300 bp, that the read length of current MPS technology can produce a phase-known sequence of each chromosome of an individual. As part of the discovery phase of our studies, data on 130 microhaplotype loci with estimates of haplotype frequency data on 83 populations have been published. To provide a better picture of global allele frequency variation, we have now tested 13 more populations for 65 of the microhaplotype loci from among those with higher levels of inter-population gene frequency variation, including 8 loci not previously published. These loci provide clear distinctions among 6 biogeographic regions and provide some information distinguishing up to 10 clusters of populations.

Genetic variation in Tunisia in the context of human diversity worldwide.

OBJECTIVES: North Africa has a complex demographic history of migrations from within Africa, Europe, and the Middle East. However, population genetic studies, especially for autosomal genetic markers, are few relative to other world regions. We examined autosomal markers for eight Tunisian and Libyan populations in order to place them in a global context. MATERIALS AND METHODS: Data were collected by TaqMan on 399 autosomal single nucleotide polymorphisms on 331 individuals from Tunisia and Libya. These data were combined with data on the same SNPs previously typed on 2585 individuals from 57 populations from around the world. Where meaningful, close by SNPs were combined into multiallelic haplotypes. Data were evaluated by clustering, principal components, and population tree analyses. For a subset of 102 SNPs, data from the literature on seven additional North African populations were included in analyses. RESULTS: Average heterozygosity of the North African populations is high relative to our global samples, consistent with a complex demographic history. The Tunisian and Libyan samples form a discrete cluster in the global and regional views and can be separated from sub-Sahara, Middle East, and Europe. Within Tunisia the Nebeur and Smar are outlier groups. Across North Africa, pervasive East-West geographical patterns were not found. DISCUSSION: Known historical migrations and invasions did not displace or homogenize the genetic variation in the region but rather enriched it. Even a small region like Tunisia contains considerable genetic diversity. Future studies across North Africa have the potential to increase our understanding of the historical demographic factors influencing the region. Am J Phys Anthropol 161:62-71, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.

The distribution and most recent common ancestor of the 17q21 inversion in humans.

The polymorphic inversion on 17q21, sometimes called the microtubular associated protein tau (MAPT) inversion, is an approximately 900 kb inversion found primarily in Europeans and Southwest Asians. We have identified 21 SNPs that act as markers of the inverted, i.e., H2, haplotype. The inversion is found at the highest frequencies in Southwest Asia and Southern Europe (frequencies of approximately 30%); elsewhere in Europe, frequencies vary from < 5%, in Finns, to 28%, in Orcadians. The H2 inversion haplotype also occurs at low frequencies in Africa, Central Asia, East Asia, and the Americas, though the East Asian and Amerindian alleles may be due to recent gene flow from Europe. Molecular evolution analyses indicate that the H2 haplotype originally arose in Africa or Southwest Asia. Though the H2 inversion has many fixed differences across the approximately 900 kb, short tandem repeat polymorphism data indicate a very recent date for the most recent common ancestor, with dates ranging from 13,600 to 108,400 years, depending on assumptions and estimation methods. This estimate range is much more recent than the 3 million year age estimated by Stefansson et al. in 2005.

Exploring genomic structure differences and similarities between the Greek and European HapMap populations: implications for association studies.

Studies of the genomic structure of the Greek population and Southeastern Europe are limited, despite the central position of the area as a gateway for human migrations into Europe. HapMap has provided a unique tool for the analysis of human genetic variation. Europe is represented by the CEU (Northwestern Europe) and the TSI populations (Tuscan Italians from Southern Europe), which serve as reference for the design of genetic association studies. Furthermore, genetic association findings are often transferred to unstudied populations. Although initial studies support the fact that the CEU can, in general, be used as reference for the selection of tagging SNPs in European populations, this has not been extensively studied across Europe. We set out to explore the genomic structure of the Greek population (56 individuals) and compare it to the HapMap TSI and CEU populations. We studied 1112 SNPs (27 regions, 13 chromosomes). Although the HapMap European populations are, in general, a good reference for the Greek population, regions of population differentiation do exist and results should not be light-heartedly generalized. We conclude that, perhaps due to the individual evolutionary history of each genomic region, geographic proximity is not always a perfect guide for selecting a reference population for an unstudied population.

A global view of the OCA2-HERC2 region and pigmentation.

Mutations in the gene OCA2 are responsible for oculocutaneous albinism type 2, but polymorphisms in and around OCA2 have also been associated with normal pigment variation. In Europeans, three haplotypes in the region have been shown to be associated with eye pigmentation and a missense SNP (rs1800407) has been associated with green/hazel eyes (Branicki et al. in Ann Hum Genet 73:160-170, 2009). In addition, a missense mutation (rs1800414) is a candidate for light skin pigmentation in East Asia (Yuasa et al. in Biochem Genet 45:535-542, 2007; Anno et al. in Int J Biol Sci 4, 2008). We have genotyped 3,432 individuals from 72 populations for 21 SNPs in the OCA2-HERC2 region including those previously associated with eye or skin pigmentation. We report that the blue-eye associated alleles at all three haplotypes were found at high frequencies in Europe; however, one is restricted to Europe and surrounding regions, while the other two are found at moderate to high frequencies throughout the world. We also observed that the derived allele of rs1800414 is essentially limited to East Asia where it is found at high frequencies. Long-range haplotype tests provide evidence of selection for the blue-eye allele at the three haplotyped systems but not for the green/hazel eye SNP allele. We also saw evidence of selection at the derived allele of rs1800414 in East Asia. Our data suggest that the haplotype restricted to Europe is the strongest marker for blue eyes globally and add further inferential evidence that the derived allele of rs1800414 is an East Asian skin pigmentation allele.

North Asian population relationships in a global context.

Population genetic studies of North Asian ethnic groups have focused on genetic variation of sex chromosomes and mitochondria. Studies of the extensive variation available from autosomal variation have appeared infrequently. We focus on relationships among population samples using new North Asia microhaplotype data. We combined genotypes from our laboratory on 58 microhaplotypes, distributed across 18 autosomes, on 3945 individuals from 75 populations with corresponding data extracted for 26 populations from the Thousand Genomes consortium and for 22 populations from the GenomeAsia 100 K project. A total of 7107 individuals in 122 total populations are analyzed using STRUCTURE, Principal Component Analysis, and phylogenetic tree analyses. North Asia populations sampled in Mongolia include: Buryats, Mongolians, Altai Kazakhs, and Tsaatans. Available Siberians include samples of Yakut, Khanty, and Komi Zyriane. Analyses of all 122 populations confirm many known relationships and show that most populations from North Asia form a cluster distinct from all other groups. Refinement of analyses on smaller subsets of populations reinforces the distinctiveness of North Asia and shows that the North Asia cluster identifies a region that is ancestral to Native Americans.

Genetic relationships of Southwest Asian and Mediterranean populations.

The Southwest Asian, circum-Mediterranean, and Southern European populations (collectively, SWAMSE) together with Northern European populations form one of five "continental" groups of global populations in many analyses of population relationships. This region is of great anthropologic and forensic interest but relationships of large numbers of populations within the region have not been able to be cleanly resolved with autosomal genetic markers. To examine the genetic boundaries to the SWAMSE region and whether internal structure can be detected we have assembled data for a total of 151 separate autosomal genetic markers on populations in this region and other parts of the world for a global set of 95 populations. The markers include 83 ancestry informative SNPs as singletons and 68 microhaplotype loci defined by 204 SNPs. The 151 loci are ancestry informative on a global scale, identifying at least five biogeographic clusters. One of those clusters is a clear grouping of 37 populations containing the SWAMSE plus northern European populations to the exclusion of populations in South Central Asia and populations from farther East. A refined analysis of the 37 populations shows the northern European populations clustering separately from the SWAMSE populations. Within Southwest Asia the Samaritans and Shabaks are distinct outliers. The Yemenite Jews, Saudi, Kuwaiti, Palestinian Arabs, and Southern Tunisians cluster together loosely while the remaining populations from Northern Iraq, Mediterranean Europe, the Caucasus region, and Iran cluster in a more complex graded fashion. The majority of the SWAMSE populations from the mainland of Southwest Asia form a cluster with little internal structure reflecting a very complex history of endogamy and migrations. The set of 151 DNA polymorphisms not only distinguishes major geographical regions globally but can distinguish ancestry to a small degree within geographical regions such as SWAMSE. We discuss forensic characteristics of the polymorphisms and also identify those that rank highest by Rosenberg's In measure for the SWAMSE region populations and for the global set of populations analyzed. DATA AVAILABILITY: Genotypes on all 151 markers for all 3790 individuals typed in the Kidd Lab on the 72 Kidd lab populations have been deposited in the Zenodo archive and can be freely accessed at https://doi.org/10.5281/zenodo.4658892. Some of the data has been made public previously as supplemental files appended to publications. Data for the additional individuals included in the analyses was taken from already public datasets as indicated in the text.

Population-specific variation in haplotype composition and heterozygosity at the POLB locus.

DNA polymerase beta plays a central role in base excision repair (BER), which removes large numbers of endogenous DNA lesions from each cell on a daily basis. Little is currently known about germline polymorphisms within the POLB locus, making it difficult to study the association of variants at this locus with human diseases such as cancer. Yet, approximately thirty percent of human tumor types show variants of DNA polymerase beta. We have assessed the global frequency distributions of coding and common non-coding SNPs in and flanking the POLB gene for a total of 14 sites typed in approximately 2400 individuals from anthropologically defined human populations worldwide. We have found a marked difference between haplotype frequencies in African populations and in non-African populations.

SNPs for a universal individual identification panel.

An efficient method to uniquely identify every individual would have value in quality control and sample tracking of large collections of cell lines or DNA as is now often the case with whole genome association studies. Such a method would also be useful in forensics. SNPs represent the best markers for such purposes. We have developed a globally applicable resource of 92 SNPs for individual identification (IISNPs) with extremely low probabilities of any two unrelated individuals from anywhere in the world having identical genotypes. The SNPs were identified by screening over 500 likely/candidate SNPs on samples of 44 populations representing the major regions of the world. All 92 IISNPs have an average heterozygosity [0.4 and the F(st) values are all\0.06 on our 44 populations making these a universally applicable panel irrespective of ethnicity or ancestry. No significant linkage disequilibrium (LD) occurs for all unique pairings of 86 of the 92 IISNPs (median LD = 0.011) in all of the 44 populations. The remaining 6 IISNPs show strong LD in most of the 44 populations for a small subset (7) of the unique pairings in which they occur due to close linkage. 45 of the 86 SNPs are spread across the 22 human autosomes and show very loose or no genetic linkage with each other. These 45 IISNPs constitute an excellent panel for individual identification including paternity testing with associated probabilities of individual genotypes less than 10(-15), smaller than achieved with the current panels of forensic markers. This panel also improves on an interim panel of 40 IISNPs previously identified using 40 population samples. The unlinked status of the subset of 45 SNPs we have identified also makes them useful for situations involving close biological relationships. Comparisons with random sets of SNPs illustrate the greater discriminating power, efficiency, and more universal applicability of this IISNP panel to populations around the world. The full set of 86 IISNPs that do not show LD can be used to provide even smaller genotype match probabilities in the range of 10(-31)-10(-35) based on the 44 population samples studied.

Validation of novel forensic DNA markers using multiplex microhaplotype sequencing.

Microhaplotypes (MH) are comprised of multiple single nucleotide polymorphisms (SNPs) that are located within 300 bases of genomic sequence. Improved tools are needed to facilitate broader application of microhaplotypes in a diverse range of populations and forensic settings. We designed an assay for multiplex sequencing of 90 microhaplotypes (mMHseq) that include 46 MH loci with high Effective Number of Alleles (Ae) from previous studies [1], and 44 high Ae MH loci containing between four to fourteen SNPs that were identified from the 1000 Genomes (1KG) Project. The unique design of mMHseq integrates a novel method for multiplex amplification from small DNA amounts, and multiplex sequencing of 48 samples in a single MiSeq run to detect all relevant MH variation. Assay performance was evaluated in a cohort of 156 individuals from seven different world populations from Africa, Asia, and Europe. Three of those populations from East Africa (Chagga, Sandawe, and Zaramo) and one from Eastern Europe (Adygei) had sufficient individuals sequenced by the assay to be included in statistical analyses with the 26 1KG populations. For those 30 populations the mean global average Ae was 5.08 (range: 2.7-11.54) and mean informativeness for biogeographic variation (In) was 0.30 (range: 0.08-0.70). Eighty-five novel SNPs were detected in 58 of the 90 microhaplotypes. Open-source, web-based software was developed to visualize haplotype phase data for each microhaplotype and individual. Our approach for multiplex microhaplotype sequencing can be customized and expanded as novel loci are being discovered.

The distinctive geographic patterns of common pigmentation variants at the OCA2 gene.

Oculocutaneous Albinism type 2 (OCA2) is a gene of great interest because of genetic variation affecting normal pigmentation variation in humans. The diverse geographic patterns for variant frequencies at OCA2 have been evident but have not been systematically investigated, especially outside of Europe. Here we examine population genetic variation in and near the OCA2 gene from a worldwide perspective. The very different patterns of genetic variation found across world regions suggest strong selection effects may have been at work over time. For example, analyses involving the variants that affect pigmentation of the iris argue that the derived allele of the rs1800407 single nucleotide polymorphism, which produces a hypomorphic protein, may have contributed to the previously demonstrated positive selection in Europe for the enhancer variant responsible for light eye color. More study is needed on the relationships of the genetic variation at OCA2 to variation in pigmentation in areas beyond Europe.

Global variation in CYP2C8-CYP2C9 functional haplotypes.

We have studied the global frequency distributions of 10 single nucleotide polymorphisms (SNPs) across 132 kb of CYP2C8 and CYP2C9 in approximately 2500 individuals representing 45 populations. Five of the SNPs were in noncoding sequences; the other five involved the more common missense variants (four in CYP2C8, one in CYP2C9) that change amino acids in the gene products. One haplotype containing two CYP2C8 coding variants and one CYP2C9 coding variant reaches an average frequency of 10% in Europe; a set of haplotypes with a different CYP2C8 coding variant reaches 17% in Africa. In both cases these haplotypes are found in other regions of the world at <1%. This considerable geographic variation in haplotype frequencies impacts the interpretation of CYP2C8/CYP2C9 association studies, and has pharmacogenomic implications for drug interactions.

Population relationships based on 170 ancestry SNPs from the combined Kidd and Seldin panels.

The benefits of ancestry informative SNP (AISNP) panels can best accrue and be properly evaluated only as sufficient reference population data become readily accessible. Ideally the set of reference populations should approximate the genetic diversity of human populations worldwide. The Kidd and Seldin AISNP sets are two panels that have separately accumulated thus far the largest and most diverse collections of data on human reference populations from the major continental regions. A recent tally in the ALFRED allele frequency database finds 164 reference populations available for all the 55 Kidd AISNPs and 132 reference populations for all the 128 Seldin AISNPs. Although much more of the genetic diversity in human populations around the world still needs to be documented, 81 populations have genotype data available for all 170 AISNPs in the union of the Kidd and Seldin panels. In this report we examine admixture and principal component analyses on these 81 worldwide populations and some regional subsets of these reference populations to determine how well the combined panel illuminates population relationships. Analyses of this dataset that focused on Native American populations revealed very strong cluster patterns associated with many of the individual populations studied.

Haplotype evolution of SLITRK1, a candidate gene for Gilles de la Tourette syndrome.

Gilles de la Tourette syndrome (GTS) is a complex disorder with a clear genetic component but no clearly identified genes with variation of etiologic relevance. Various candidate regions and genes show some evidence of affecting risk, though clearly not all patients/families can be explained by any one of them. Resequencing one candidate gene, SLITRK1, has identified four new variants. Including them, we have typed over 2,300 normal individuals from 44 populations for 11 SNPs spanning the gene. The unusual global pattern seen is that one non-ancestral haplotype is the single most common haplotype worldwide. Other haplotypes appear to result from accumulation of mutations with no evidence of historical recombination. Although there is no evidence of selection, the haplotype frequency variation seen around the world will need to be considered in any future association studies of this locus with GTS or any other neuropsychiatric disorder.

Recent Selection on a Class I ADH Locus Distinguishes Southwest Asian Populations Including Ashkenazi Jews.

The derived human alcohol dehydrogenase (ADH)1B*48His allele of the ADH1B Arg48His polymorphism (rs1229984) has been identified as one component of an East Asian specific core haplotype that underwent recent positive selection. Our study has been extended to Southwest Asia and additional markers in East Asia. Fst values (Sewall Wright's fixation index) and long-range haplotype analyses identify a strong signature of selection not only in East Asian but also in Southwest Asian populations. However, except for the ADH2B*48His allele, different core haplotypes occur in Southwest Asia compared to East Asia and the extended haplotypes also differ. Thus, the ADH1B*48His allele, as part of a core haplotype of 10 kb, has undergone recent rapid increases in frequency independently in the two regions after divergence of the respective populations. Emergence of agriculture may be the common factor underlying the evident selection.

Improving ancestry distinctions among Southwest Asian populations.

The Kidd Lab panel of 55 AISNPs can provide up to 10 statistically relevant biogeographic groupings of a global set of populations. A second-tier panel would be useful for increasing the accuracy for further differentiation of populations within a specific global grouping. Because recent advances in massively parallel sequencing (MPS) methods allow the genotyping of many more SNPs, we are now identifying additional SNPs to provide refined discrimination among regional subsets of populations; Southwest Asia and the nearby Mediterranean region (SWA) is our current target for such a "second tier" panel. We selected the potentially best SNPs from various sources: our own laboratory database (>4600 SNPs), AISNP panels (Kidd 55 and Seldin 128 SNP panels), and published papers reporting European and SW Asian populations. Rosenberg's Informativeness, Fst, and allele frequency heatmap matrices are used to determine the best SNPs for the region. A total of 2568 individuals, from 39 different populations ranging from North-East Africa through the SW Asia and Europe to the Ural Mountains, were included in the refinement processes and analyses. Heatmap, PCA, Structure (K = 4), and ancestry inference for selected individuals with an in-lab version of FROG-kb analyses indicate that these 86 AISNPs provide the basis for building an improved, optimized panel of AISNPs that collectively provide additional information on differences among populations in that part of the world. Testing this panel with additional populations from the area and with new SNPs and/or microhaplotypes is expected to improve the panel.

Significant variation in haplotype block structure but conservation in tagSNP patterns among global populations.

The initial belief that haplotype block boundaries and haplotypes were largely shared across populations was a foundation for constructing a haplotype map of the human genome using common SNP markers. The HapMap data document the generality of a block-like pattern of linkage disequilibrium (LD) with regions of low and high haplotype diversity but differences among the populations. Studies of many additional populations demonstrate that LD patterns can be highly variable among populations both across and within geographic regions. Because of this variation, emphasis has shifted to the generalizability of tagSNPs, those SNPs that capture the bulk of variation in a region. We have examined the LD and tagSNP patterns based upon over 2000 individual samples in 38 populations and 134 SNPs in 10 genetically independent loci for a total of 517 kb with an average density of 1 SNP/5 kb. Four different 'block' definitions and the pairwise LD tagSNP selection algorithm have been applied. Our results not only confirm large variation in block partition among populations from different regions (agreeing with previous studies including the HapMap) but also show that significant variation can occur among populations within geographic regions. None of the block-defining algorithms produces a consistent pattern within or across all geographic groups. In contrast, tagSNP transferability is much greater than the similarity of LD patterns and, although not perfect, some generalizations of transferability are possible. The analyses show an asymmetric pattern of tagSNP transferability coinciding with the subsetting of variation attributed to the spread of modern humans around the world.

Conservative evolution in duplicated genes of the primate Class I ADH cluster.

Humans have seven alcohol dehydrogenase genes (ADH) falling into five classes. Three out of the seven genes (ADH1A, ADH1B and ADH1C) belonging to Class I are expressed primarily in liver and code the main enzymes catalyzing ethanol oxidization. The three genes are tandemly arrayed within the ADH cluster on chromosome 4 and have very high nucleotide similarity to each other (exons: >90%; introns: >70%), suggesting the genes have been generated by duplication event(s). One explanation for maintaining similarity of such clustered genes is homogenization via gene conversion(s). Alternatively, recency of the duplications or some other functional constraints might explain the high similarities among the genes. To test for gene conversion, we sequenced introns 2, 3, and 8 of all three Class I genes (total>15.0 kb) for five non-human primates--four great apes and one Old World Monkey (OWM)--and compared them with those of humans. The phylogenetic analysis shows each intron sequence clusters strongly within each gene, giving no evidence for gene conversion(s). Several lines of evidence indicate that the first split was between ADH1C and the gene that gave rise to ADH1A and ADH1B. We also analyzed cDNA sequences of the three genes that have been previously reported in mouse and Catarrhines (OWMs, chimpanzee, and humans) and found that the synonymous and non-synonymous substitution (dN/dS) ratios in all pairs are less than 1 representing purifying selection. This suggests that purifying selection is more important than gene conversion(s) in maintaining the overall sequence similarity among the Class I genes. We speculate that the highly conserved sequences on the three duplicated genes in primates have been achieved essentially by maintaining stability of the hetero-dimer formation that might have been related to dietary adaptation in primate evolution.