Patterns of DNA Methylation Across the Leptin Core Promoter in Four Diverse Asian and North American Populations.

DNA methylation is the most widely studied of epigenetic mechanisms, with environmental effects recorded through patterned attachments of methyl groups along the DNA that are capable of modifying gene expression without altering the DNA sequencing. The degree to which these patterns of DNA methylation are heritable, the expected range of normality across populations, and the phenotypic relevance of pattern variation remain unclear. Genes regulating metabolic pathways appear to be vulnerable to ongoing nutritional programming over the life course, as dietary nutrients are significant environmental determinants of DNA methylation, supplying both the methyl groups and energy to generate the methylation process. Here we examine methylation patterns along a region of the metabolic gene leptin (LEP). LEP's putative functions include regulation of energy homeostasis, with its signals affecting energy intake and expenditure, adipogenesis and energy storage, lipid and glucose metabolism, bone metabolism, and reproductive endocrine function. A pattern of differential methylation across CpG sites of the LEP core promoter has been previously identified; however, any consistency of pattern or its phenotypic significance is not fully elucidated among populations. Using DNA extracted from unfractionated white blood cells of peripheral blood samples, our pilot study, divided into two parts, examined the significance of variation in DNA methylation patterns along the leptin core promoter in four populations (phase 1) and used biomarkers reflecting leptin's functional process in two of those populations, western Buryat of Siberia and the Mennonite of central Kansas, to investigate the relevance of the ethnic variation identified in the DNA methylation (phase 2). LEP's core promoter region contains both the binding site for C/EBPα (CCAAT/enhancer binding protein alpha), which tempers the final step in adipocyte maturity and capacity to synthesize leptin, and the TATA motif controlling leptin synthesis. Previous studies report that increased methylation in this region is correlated to decreased gene expression, suggesting tissue-specific methylation variation at this region ( Melzner et al. 2002 ). We hypothesized that evidence of nutritional epigenetic programming would be identified through variation in patterns of DNA methylation and that functional relevance of that variation among populations would be identified through biomarkers that reflect leptin's metabolic signals: serum leptin levels, lipoproteins of the lipid transport system, and anthropometric measures. In phase 1, our combined analyses of 313 individuals documented a distinct and consistent overall pattern of differential DNA methylation across seven CpG sites of LEP core promoter in all ethnicities and both sexes. This pattern replicates those identified in previous studies, suggesting a conserved core promoter region across populations. Phase 2 analyses of two of the four populations (n = 239), correlating methylation at the C/EBPα transcription binding site (TBS) with metabolic and anthropometric biomarkers reflecting LEP roles, showed that stature, which reflects bone growth and remodeling, was significantly and inversely correlated with the percentage of DNA methylation at this site in both sexes. We suggest that variation in DNA methylation along the LEP core promoter plays a substantial role in energy signals affecting both adipogenesis and bone metabolism.

Applications of pooled DNA samples to the assessment of population affinities: short tandem repeats.

Pooled DNA samples have been used in association studies of Mendelian disease genes. This method involves combining equal quantities of DNA from patients and control subjects into separate pools and comparing the pools for distributions of genetic markers. In this study identical quantities of DNA from 300 individuals representing 6 populations were pooled and amplified for 296 loci using the touchdown polymerase chain reaction (PCR) method. The purpose of this study is to test the efficacy of pooled DNA markers in the reconstruction of the genetic structure of human populations. The populations sampled included Chuvash, Buryats, Kizhi, Native Americans, South Africans, and New York City whites. To test the accuracy of the allele-frequency distributions, we genotyped the Buryats and New York samples individually for six microsatellite markers and compared their frequencies to the allele frequencies derived from the electropherogram peak heights for the pooled DNA, producing a correlation of 0.9811 with a variance of less than 0.04. Two-dimensional scaling of genetic distances among the six populations produced clusters that reflected known historical relationships. A distance matrix was created using all 296 loci, and matrices based on individual chromosomes were correlated against the total matrix. As expected, the largest chromosomes had the highest correlations with the total matrix, whereas one of the smallest chromosomes, chromosome 22, had the lowest correlation and differed most from the combined STR distance matrix.

Phylogenetic relationships of human populations in sub-Saharan Africa.

This study utilizes the GM/KM immunoglobulin allotype system to elucidate the phylogenetic relationships of sub-Saharan Africans. The importance of understanding the relatedness of these peoples stems from the sub-Saharan region being the possible birthplace of humans. Haplotype distributions were determined for 19 populations and compared using chi-square analysis. Published data of other sub-Saharan Africans and representative populations worldwide were also added for comparison. Genetic distances between populations were calculated based on haplotype frequencies, and genetic relationships were observed through principal components analysis. Data from the GM/KM system showed a genetic homogeneity of the Bantu populations, with some exceptions, supporting the possibility of a common origin of these peoples. The Malagasy appeared as a divergent population, most likely due to Southeast Asian/Austronesian admixture, as indicated by the presence of the GM*AF B haplotype. The Cape Coloured also showed a divergence, with their genetic structures containing Caucasoid and Khoisan contributions. Finally, the Mbuti Pygmies appeared genetically isolated and had the highest frequency of the GM*A B haplotype out of all studied populations.

Distribution of the 3' VNTR polymorphism in the human dopamine transporter gene in world populations.

A polymorphism with a variable number of tandem repeats (VNTR) found in the 3' untranslated region of the human dopamine transporter gene (DAT1) was scored in unrelated individuals drawn from 10 geographically widely dispersed populations in order to assess this marker's usefulness in human population genetics. The populations that were analyzed in this study included 4 indigenous groups of Siberia, natives of North and South America, as well as Caucasian and Oceanic groups, most of which represented small-scale societies. A total of 5 DAT1 alleles were seen overall, but only in one Siberian population, the Altai-Kizhi, were all 5 present, and in the Native Americans of Colombia the locus was monomorphic. The most common allele, DAT1*10, ranged in frequency from 52% in Greeks to 100% in South Americans. The high frequency of the DAT1*10 allele (approximately 90%) among Mongoloid groups of north and east Asia distinguishes them from most Caucasian groups. The presence of the rare DAT1*7 allele in relatively high frequency (approximately 5%) among all Siberian groups suggests a close affinity with north Asian groups, especially Mongolians. The presence of the even rarer DAT1*13 allele in one Siberian population, the Altai-Kizhi, reflects this group's long historical contact with Mongolians. The results demonstrated that the DAT1 VNTR polymorphism is useful in investigating population relationships, and that rare alleles at this locus may be particularly valuable in understanding the extent of genetic affinity between neighboring groups and in situations where admixture is suspected. However, because of both the association and linkage of this VNTR locus with attention-deficit hyperactivity disorder (ADHD) in children, and its highly restricted polymorphism (usually 3 alleles) in most human groups, the possibility of selection constraints on the DAT1 gene cannot be ignored.

Application of forensic DNA testing in the legal system.

DNA technology has taken an irreplaceable position in the field of the forensic sciences. Since 1985, when Peter Gill and Alex Jeffreys first applied DNA technology to forensic problems, to the present, more than 50,000 cases worldwide have been solved through the use of DNA based technology. Although the development of DNA typing in forensic science has been extremely rapid, today we are witnessing a new era of DNA technology including automation and miniaturization. In forensic science, DNA analysis has become "the new form of scientific evidence" and has come under public scrutiny and the demand to show competence. More and more courts admit the DNA based evidence. We believe that in the near future this technology will be generally accepted in the legal system. There are two main applications of DNA analysis in forensic medicine: criminal investigation and paternity testing. In this article we present background information on DNA, human genetics, and the application of DNA analysis to legal problems, as well as the commonly applied respective mathematics.

Deletion polymorphism in the human COL1A2 gene: genetic evidence of a non-African population whose descendants spread to all continents.

We report the frequencies of a deletion polymorphism at the alpha 2 (1) collagen gene (COL1A2) and argue that this distribution has major implications for understanding the evolution of modern humans immediately after their exodus from sub-Saharan Africa as well as their subsequent spread to all continents. The high frequency of the deletion in non-African populations and its complete absence in sub-Saharan African groups suggest that the deletion event occurred just before or shortly after modern humans left Africa. The deletion probably arose shortly after the African exodus in a group whose descendants were among the ancestors of all contemporary populations, except for sub-Saharan Africans. This, of course, does not imply that there was a single migration out of Africa. The GM immunoglobulin haplotype GM*A,X G displays a similar distribution to that for the COL1A2 deletion, and these 2 polymorphisms suggest that the exodus from Africa may not have been a rapid dispersion to all other regions of the world. Instead, it may have involved a period of time for the savanna-derived gene pool to adapt to novel selective agents, such as bacteria, viruses, and/or environmental xenobiotics found in both animal and plant foods in their new environment. In this context these polymorphisms are indicators of the evolution that occurred before the diaspora of these populations to the current distribution of modern peoples.

Premature termination codon in the aggrecan gene of nanomelia and its influence on mRNA transport and stability.

AIM: To analyze the influence of the premature termination codon on mRNA transport and stability METHODS: Chondrocyte mRNA was isolated from homozygous and heterozygous nanomelic 17-days old embryos and examined by RT-PCR analysis. To analyze aggrecan mRNA stability, mRNA synthesis was inhibited with DRB [5,6 dichloro-1-(-D-ribofuranosyl benzimidazole)], a specific inhibitor of RNA polymerase II. Visualization of the aggrecan alleles was performed by in situ hybridization. RESULTS: The level of mutant aggrecan mRNA within the nucleus was equal to that of the control, but no mutant mRNA was observed in the cytoplasm. RT-PCR revealed that the mutant transcript was only detectable in the nucleus, compared with house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or collagen type II. A restriction site induced by premature termination codon TAA allowed the distinction of normal and mutant transcripts in chondrocytes derived from embryos heterozygous for the nanomelic mutation. After the treatment with DRB, identical decay rates were demonstrated for both transcripts within the heterozygous nucleus. In situ hybridization showed no abnormal mRNA accumulation. CONCLUSION: This is the first evidence suggesting that the transcript of the mRNA with the premature termination codon within an exon does exit the nucleus.

Allele frequencies of six highly polymorphic DNA loci in the Croatian population.

The allele frequency distributions in a series of Croats were analyzed for six unlinked polymorphic DNA loci: THO1, FESFPS, VWA01, APOB, D1S80, and D17S5. The allele frequencies were determined for 100 unrelated genomic DNA samples. The observed heterozygote frequencies of the loci ranged from 0.63 to 0.76; however, the the expected heterozygosity ranged from 0.68 to 0.82, with only D17S5 having a significant excess of homozygous phenotypes (p < 0.001). The excess homozygosity seen in the D17S5 system may be due to allelic drop-out and warrants further technical analysis of that system, given the uniform lack of significant deviation in the other five systems. The forensic usefulness of these systems can be measured using two different statistics: the power of discrimination and the likelihood of a coincidental match. The power of discrimination ranged from 0.85 to 0.94 for the 6 systems with the combined likelihood of a coincidental match based on these 6 systems of 1 in 3.6 million, or slightly less than the population of Croatia. A second, more conservative estimator of the likelihood of a match is based on the most common phenotype for each system. If someone had the most common phenotype for each of the 6 systems, the chance of a coincidental match would be approximately 1 in 64,000. For paternity testing the usefulness of a system is measured by the average power of exclusion or (1-power of exclusion), the random man not excluded. The average power of exclusion, based on observed heterozygosity, ranged from 0.33 to 0.53, and the average power of exclusion based on the expected heterozygosity ranged from 0.39 to 0.64. The combined average power of exclusion was 99.2% for these 6 systems, using the expected heterozygosity. Based on the results of testing these six systems, there is no significant substructuring within the southern Croatian populations, and these systems provide a useful tool for forensic, paternity, and anthropological applications.

Genetic admixture and gallbladder disease in Mexican Americans.

Gallbladder disease is a common source of morbidity in the Mexican American population. Genetic heritage has been proposed as a possible contributor, but evidence for this is limited. Because gallbladder disease has been associated with Native American heritage, genetic admixture may serve as a useful proxy for genetic susceptibility to the disease in epidemiologic studies. The objective of our study was to examine the possibility that gallbladder disease is associated with greater Native American admixture in Mexican Americans. This study used data from the Hispanic Health and Nutrition Examination Survey and was based on 1,145 Mexican Americans who underwent gallbladder ultrasonography and provided usable phenotypic information. We used the GM and KM immunoglobulin antigen system to generate estimates of admixture proportions and compared these for individuals with and without gallbladder disease. Overall, the proportionate genetic contributions from European, Native American, and African ancestries in our sample were 0.575, 0.390, and 0.035, respectively. Admixture proportions did not differ between cases and noncases: Estimates of Native American admixture for the two groups were 0.359 and 0.396, respectively, but confidence intervals for estimates overlapped. This study found no evidence for the hypothesis that greater Native American admixture proportion is associated with higher prevalence of gallbladder disease in Mexican Americans. Reasons for the finding that Native American admixture proportions did not differ between cases and noncases are discussed. Improving our understanding of the measurement, use, and limitations of genetic admixture may increase its usefulness as an epidemiologic tool as well as its potential for contributing to our understanding of disease distributions across populations.

Anti-D in a D-positive renal transplant patient.

BACKGROUND: The detection of anti-D in a D-positive renal transplant recipient is unusual and may arise by several potential mechanisms. These include passive transfer of alloantibody and the presence of autoanti-D or alloanti-D that is due to microchimerism when the allograft is from a D-negative donor. In the latter case, overt hemolysis has been seen or suspected. The occurrence of anti-D in a D-positive renal transplant recipient without hemolysis, which is most likely attributable to microchimerism, is reported. CASE REPORT: A 51-year-old group O, D-positive woman, who was serologically HLA type A1, A2; B8, B44; DR3, DR6, DR52; DQ1, DQ2, underwent the transplantation of a kidney from a cadaveric donor who was serologically HLA type A1, A2; B8, B44; DR13, DR17, DR52; DQ1, DQ2. The donor was known to be D-negative and immunized to D. No blood components or derivatives were administered at the time of organ graft. Ten weeks after the transplant, the direct antiglobulin test was positive in the recipient, and anti-D was eluted. Polymerase chain reaction amplification using primers to distinguish DR13 (donor) from DR14 alleles (recipient split of DR6) in the peripheral blood showed the recipient to be DR14. No DR13 could be detected, and thus microchimerism could not be confirmed. However, in the peripheral blood, GM and KM allotyping of the serum (GM A,F,X B,G and KM 1,3) and eluate (G1M F, KM 3) showed a pattern of allotypes most consistent with an alloantibody. Eleven months after transplantation, the graft continued to function; the direct antiglobulin test was still positive, and elution of anti-D persisted. CONCLUSION: This case of anti-D in a D-positive renal transplant recipient is attributed to microchimerism, despite the lack of confirmation by genotypic analysis of the peripheral blood. It raises the possibility that microchimerism may be a more common phenomenon in solid allograft recipients than is realized.

Analysis of human specificity in AFLP systems APOB, PAH, and D1S80.

We have previously characterized and databased three human amplified fragment length polymorphism (AFLP) loci: the hypervariable regions 3' to apolipoprotein B (APOB), phenylalanine hydroxylase (PAH) and at locus D1S80. The analysis utilized polymerase chain reaction (PCR) technology for human identification in forensic and paternity testing. This study extended that work by assessment of specificity of amplicons produced with non-human and human control DNAs for APOB, PAH and D1S80 under high and low stringency PCR conditions. It was seen that primate and other animal templates (with the exception of chimpanzee) yielded products below the human allele range under high stringency PCR parameters. Under reduced stringency PCR with animal and primate samples, reproducible genetic fingerprints were generated spanning the human allele range. The patterns were produced with defined human AFLP primer pairs under specifically relaxed PCR reaction and thermalcycling parameters. They showed genetic relationships between species at the DNA level. Amplicon patterns were compared for band size and intensity matches within the PCR synthesis range defined by the conditions used. This technique could become a useful tool in species identification and molecular evolutionary studies.

Immunoglobulin allotypes and estimation of genetic admixture among populations of Kinnaur District, Himachal Pradesh, India.

Four regional populations of the Kanet (Puh, Kalpa, Sangla, and Nachar) and an endogamous group of Koli from Kinnaur District, Himachal Pradesh, India, were studied to determine the extent of genetic variation of immunoglobulin allotypes (GM, KM, and AM) and the genetic contribution from ancestral populations of Tibet and northwest India. Haplotype GM*A G showed a higher frequency in the Kanet (40-60%)-a frequency that is more comparable to Asian populations-whereas in the Koli a lower frequency was observed, which is nearer the values for populations from northwest India. The IG haplotype data suggest that the Kanet population of Kinnaur District and the northeastern population of Nepal have different European origins than the more central population of India, represented by a sample from Delhi. The present results suggest that the populations of Kinnaur District are of admixed origin with contributions of Tibetan genes of 87.3%, 51.3%, 49.9%, 40.0%, and 9.5% in the Puh, Kalpa, Sangla, and Nachar Kanet and the Koli, respectively. The genetic distance obtained from 19 loci (9 blood groups, 8 biochemical markers, GM, and KM) showed an inverse relationship between the distance of the hybrid population from the parental gene pool. The Puh Kanet, nearest the Tibetan border, had the highest proportion of Tibetan genes but showed the lowest genetic distance with Tibetans. As the geographic distance of the other regional populations of the Kanet increases from the border of Tibet, genetic distance compared with the parental Tibetan population increases and the proportion of Tibetan admixture decreases. In the Kinnaur District admixture seems to contribute largely to the present-day observed high level of genetic differentiation.

Identification of war victims from mass graves in Croatia, Bosnia, and Herzegovina by use of standard forensic methods and DNA typing.

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.

Immunoglobulin haplotype frequencies in Anabaptist population samples: Kansas and Nebraska Mennonites and Indiana Amish.

Anabaptist history is a chronicle of repeated migrations, fissions, and fusions of various subgroups. The effects of these events should be evident in the population biology of the Anabaptist groups. No prior genetic studies have included the polymorphic and highly informative immunoglobulin markers. Here, 685 serum samples representing 1 Amish and 3 Mennonite community samples (7 congregations) were studied for immunoglobulin allotypes. The haplotypes IGHG*F B, IGHG*A,Z G, and IGHG*A,X,Z G range in frequency from 0.542 to 0.765, 0.123 to 0.290, and 0.075 to 0.170, respectively. IGK*1 frequencies range from 0.035 to 0.077. All frequencies are within expected ranges for central and western European population samples. There was considerable intergroup variability among the Anabaptist samples that was statistically significant (chi29 = 22.63, 0.005 < p < 0.01). Principal component analyses, including the immunoglobulin allotype frequencies and published data on ABO, MN, and Rhesus (Dd) markers, demonstrate that the Mennonite congregation samples with close historical ties group together and are distinct from the Amish and Meridian congregation samples.

The distribution of F13A subtypes in four populations using agarose isoelectric focusing and Western Blot detection.

Using an isoelectric focusing gel containing agarose, urea, a separator and a narrow range ampholyte of pH 5-6, a system was designed to split the F13A*1 and F13A*2 alleles into the subtypes 1A, 1B, 2A and 2B. Four population groups (European-Americans, African-Americans, Mexican-Americans and Native-Americans) were data based. Subtyping F13A increased the information content significantly over the previous F13A typing system by doubling the number of alleles and increasing the number of phenotypes from three to ten. The new system has proved to be of value in parentage testing by increasing exclusionary power in cases of non-paternity and increasing the paternity index in non-exclusionary cases. Though there does not appear to be any significant variation among U.S. populations, published data on Japanese populations suggests that significant differences among populations may exist, leading to an anthropological usefulness as well.

DNA interpopulational variation in Siberian indigenous populations: The mountain Altai.

Using mtDNA and classical markers, previous studies have found that the Altai are genetically divergent from the rest of Siberia. This study uses five variable number tandem repeat (VNTR) loci to examine the relationship of the Altai to other indigenous Siberian populations. Frequencies of VNTR fragments have been obtained from the DNA of 95 individuals living in the Altai village of Mendur-Sokkon. A Kolmogorov-Smirnov test shows that the Altai are significantly different from the Evenki village of Surinda at loci D11S129 and D20S15. In addition, the Altai are also statistically different from the Evenki village of Poligus at locus D11S129. The test reveals no differences between the Ket village of Sulamai and Mendur-Sokkon. The GST value obtained for Siberia is significant and is almost equal to that found for the GST of American ethnic groups. The significance of the GST values was verified through random resampling of the data. The GST value is an effect of the relative isolation of the Evenki as well as gene flow into the Kets and Altai; this is shown in a plot of rn versus mean heterozygosity. Although genetic differentiation between the Siberian groups is significant, an R matrix analysis, which uses American and Siberian ethnic groups, shows that the Siberians form a tight cluster. When the R matrix, GST , and the Kolmogorov-Smirnov results are combined, the Altai appear to be genetically different among Siberian populations, yet they are not as genetically divergent as previous studies have shown. © 1996 Wiley-Liss, Inc.

VNTR DNA variation in Siberian indigenous populations.

The VNTR loci D7S104, D11S129, D18S17, D20S15, and D21S112 in three indigenous Siberian populations were analyzed to determine the populations' genetic structure. Using the Kolmogorov-Smirnov test, we found that the Siberian indigenous populations of Surinda and Sulamai are separated at the D11S129 locus (p < 0.05). However, the population of Poligus is genetically homogeneous compared with the villages of Sulamai and Surinda. Principal component plots for the sets of VNTR loci cluster the Siberian groups together, reflecting the homogeneity of these populations. An analysis of mean per locus heterozygosity versus the distance from the centroid of distribution suggests gene flow into Sulamai but little genetic exchange with Surinda and Poligus. Ultimately, the VNTR data reflect the genetic distinctiveness of the Kets and the Evenki.

Characterization of human AFLP systems apolipoprotein B, phenylalanine hydroxylase, and D1S80.

Methodology is presented for amplified fragment length polymorphism (AFLP) typing using a nonisotopic, PCR protocol. Human variable number tandem repeat (VNTR) loci used for identification in forensic and paternity testing were optimized for reaction and thermal-cycling parameters. Loci analyzed were the apolipoprotein B (APOB) 3' hypervariable region (HVR), phenylalanine hydroxylase 3' HVR (PAH), and D1S80. Coamplification of a monomorphic beta-globin fragment serves as an amplification control. Biotin is integrated into PCR amplicon through primer incorporation. AFLP products undergo agarose gel electrophoresis and Southern transfer to a nylon membrane. Amplicons were detected using a streptavidin-enzyme conjugate. Either colorimetric- or chemiluminescent-developed bands are genotyped using locus-specific allele ladders with known VNTR repeat numbers. Using this methodology, we have successfully typed > 500 individuals from three population groups for each locus during data basing and casework.

Simultaneous subtyping of group specific component and esterase D by isoelectric focusing on agarose gels.

A quick, sensitive and economical technique has been developed to subtype GC and ESD simultaneously on the same agarose IEF gel. This method could be a useful tool for forensic application.

Immunoglobulin allotypes (GM and KM) indicate multiple founding populations of Native Americans: evidence of at least four migrations to the New World.

On the basis of GM and KM typing and language, approximately 28,000 Amerindians were divided into 4 groups of populations: non-Nadene South American (8 groups), non-Nadene North American (7 groups), Nadene (4 groups), and Eskaleuts (6 groups). These groups were compared to four groups of Asian populations. The distribution of GM haplotypes differed significantly among and within these groups as measured by chi-square analysis. Furthermore, as reflected in a maximum linkage cluster analysis, Amerindian populations in general cluster along geographic divisions, with Eskaleuts and Nadenes clustering with the Asian populations and non-Nadene North American and non-Nadene South American populations forming two additional clusters. Based on GM haplotype data and other genetic polymorphisms, the divisions appear to reflect populations that entered the New World at different times. It appears that the South American non-Nadene populations are the oldest, characterized by the haplotypes GM*A G and GM*X G, whereas later North American non-Nadene populations are characterized by high frequencies of GM*A G and low frequencies of GM*X G and GM*A T. In contrast, Eskaleuts appear to have only GM*A G and GM*A T. The Nadene speakers have GM*X G and GM*A T in higher and approximately equal frequencies. Maximum linkage cluster analysis places the Alaskan Athapaskans closest to northwestern Siberian populations and the Eskaleuts closest to the Chukchi, their closest Asian neighbor. These analyses, when combined with other data, suggest that, in the peopling of the New World, at least four separate migrant groups crossed Beringia at various times. It appears likely that the South American non-Nadene entered the New World before 17,000 years B.P. and that the North American non-Nadene entered in the immediate postglacial period, with the Eskaleut and Nadene arriving at a later date.