Evidence for three genetic loci involved in both anorexia nervosa risk and variation of body mass index.

The maintenance of normal body weight is disrupted in patients with anorexia nervosa (AN) for prolonged periods of time. Prior to the onset of AN, premorbid body mass index (BMI) spans the entire range from underweight to obese. After recovery, patients have reduced rates of overweight and obesity. As such, loci involved in body weight regulation may also be relevant for AN and vice versa. Our primary analysis comprised a cross-trait analysis of the 1000 single-nucleotide polymorphisms (SNPs) with the lowest P-values in a genome-wide association meta-analysis (GWAMA) of AN (GCAN) for evidence of association in the largest published GWAMA for BMI (GIANT). Subsequently we performed sex-stratified analyses for these 1000 SNPs. Functional ex vivo studies on four genes ensued. Lastly, a look-up of GWAMA-derived BMI-related loci was performed in the AN GWAMA. We detected significant associations (P-values <5 × 10-5, Bonferroni-corrected P<0.05) for nine SNP alleles at three independent loci. Interestingly, all AN susceptibility alleles were consistently associated with increased BMI. None of the genes (chr. 10: CTBP2, chr. 19: CCNE1, chr. 2: CARF and NBEAL1; the latter is a region with high linkage disequilibrium) nearest to these SNPs has previously been associated with AN or obesity. Sex-stratified analyses revealed that the strongest BMI signal originated predominantly from females (chr. 10 rs1561589; Poverall: 2.47 × 10-06/Pfemales: 3.45 × 10-07/Pmales: 0.043). Functional ex vivo studies in mice revealed reduced hypothalamic expression of Ctbp2 and Nbeal1 after fasting. Hypothalamic expression of Ctbp2 was increased in diet-induced obese (DIO) mice as compared with age-matched lean controls. We observed no evidence for associations for the look-up of BMI-related loci in the AN GWAMA. A cross-trait analysis of AN and BMI loci revealed variants at three chromosomal loci with potential joint impact. The chromosome 10 locus is particularly promising given that the association with obesity was primarily driven by females. In addition, the detected altered hypothalamic expression patterns of Ctbp2 and Nbeal1 as a result of fasting and DIO implicate these genes in weight regulation.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa.

Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

Gender difference in interactions between MAOA promoter uVNTR polymorphism and negative familial stressors on body mass index among Chinese adolescents.

OBJECTIVES: Monoamine oxidase A (MAOA) modulates metabolism of serotonin and dopamine metabolism, neurotransmitters involved in regulation of appetite and food intake. The gene coding for MAOA contains a 30-bp tandem repeat (uVNTR) polymorphism in its promoter region that has been previously identified to be associated with obesity with mixed findings in the literature. Our goals were to replicate the population effects of this functional polymorphism on obesity risk, and to further explore gender differences and interaction effects with negative stressors. METHODS: Analyses were conducted with data on genotypes, measured weight and height, and self-reported behavioural characteristics among 1101 Chinese adolescents 11-15 years old living in Wuhan, China. RESULTS: Girls with the high-activity allele had significantly lower body mass index (BMI; ß = -0.25 ± 0.98, P = 0.011) compared to those with the low activity allele. Experience of negative familial stressors (e.g., death or illness of family members, hit or scolded by parents and increased quarrelling with parents, parents argued frequently) significantly weakened this protective genetic effect on BMI (P for interaction = 0.043). Stratified analyses showed a significant protective genetic effect on BMI only within the stratum of low stress level (ß = -0.44 ± 0.14, P = 0.002). No similar effect was observed among boys. CONCLUSIONS: Our findings confirm the genetic effects of MAOA uVNTR polymorphism on BMI in a Chinese adolescent population and suggest potential genetic interactions with negative familial stressors.

Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition.

Genome-wide meta-analyses of smoking behaviors in African Americans.

The identification and exploration of genetic loci that influence smoking behaviors have been conducted primarily in populations of the European ancestry. Here we report results of the first genome-wide association study meta-analysis of smoking behavior in African Americans in the Study of Tobacco in Minority Populations Genetics Consortium (n = 32,389). We identified one non-coding single-nucleotide polymorphism (SNP; rs2036527[A]) on chromosome 15q25.1 associated with smoking quantity (cigarettes per day), which exceeded genome-wide significance (ß = 0.040, s.e. = 0.007, P = 1.84 × 10(-8)). This variant is present in the 5'-distal enhancer region of the CHRNA5 gene and defines the primary index signal reported in studies of the European ancestry. No other SNP reached genome-wide significance for smoking initiation (SI, ever vs never smoking), age of SI, or smoking cessation (SC, former vs current smoking). Informative associations that approached genome-wide significance included three modestly correlated variants, at 15q25.1 within PSMA4, CHRNA5 and CHRNA3 for smoking quantity, which are associated with a second signal previously reported in studies in European ancestry populations, and a signal represented by three SNPs in the SPOCK2 gene on chr10q22.1. The association at 15q25.1 confirms this region as an important susceptibility locus for smoking quantity in men and women of African ancestry. Larger studies will be needed to validate the suggestive loci that did not reach genome-wide significance and further elucidate the contribution of genetic variation to disparities in cigarette consumption, SC and smoking-attributable disease between African Americans and European Americans.

Gender-stratified gene and gene-treatment interactions in smoking cessation.

We conducted gender-stratified analyses on a systems-based candidate gene study of 53 regions involved in nicotinic response and the brain-reward pathway in two randomized clinical trials of smoking cessation treatments (placebo, bupropion, transdermal and nasal spray nicotine replacement therapy). We adjusted P-values for multiple correlated tests, and used a Bonferroni-corrected α-level of 5 × 10(-4) to determine system-wide significance. Four single-nucleotide polymorphisms (rs12021667, rs12027267, rs6702335, rs12039988; r2 > 0.98) in erythrocyte membrane protein band 4.1 (EPB41) had a significant male-specific marginal association with smoking abstinence (odds ratio (OR) = 0.5; 95% confidence interval (CI): 0.3-0.6) at end of treatment (adjusted P < 6 × 10(-5)). rs806365 in cannabinoid receptor 1 (CNR1) had a significant male-specific gene-treatment interaction at 6-month follow-up (adjusted P = 3.9 × 10(-5)); within males using nasal spray, rs806365 was associated with a decrease in odds of abstinence (OR = 0.04; 95% CI: 0.01-0.2). While the role of CNR1 in substance abuse has been well studied, we report EPB41 for the first time in the nicotine literature.

Varenicline for smoking cessation: nausea severity and variation in nicotinic receptor genes.

This study evaluated association between common and rare sequence variants in 10 nicotinic acetylcholine receptor subunit genes and the severity of nausea 21 days after initiating the standard, Food and Drug Administration-approved varenicline regimen for smoking cessation. A total of 397 participants from a randomized clinical effectiveness trial with complete clinical and DNA resequencing data were included in the analysis (mean age=49.2 years; 68.0% female). Evidence for significant association between common sequence variants in CHRNB2 and nausea severity was obtained after adjusting for age, gender and correlated tests (all P(ACT)<0.05). Individuals with the minor allele of CHRNB2 variants experienced less nausea than did those without the minor allele, consistent with previously reported findings for CHRNB2 and the occurrence of nausea and dizziness as a consequence of first smoking attempt in adolescents, and with the known neurophysiology of nausea. As nausea is the most common reason for discontinuance of varenicline, further pharmacogenetic investigations are warranted.

Epstein-Barr virus microRNAs and lung cancer.

BACKGROUND: We conducted the first analysis of viral microRNAs (miRNAs) in lung cancer, with a focus on Epstein-Barr virus (EBV). METHODS: We evaluated viral miRs with a two-channel oligo-array targeting mature, anti-sense miRNAs in 290 cases. In 48 cases, we compared microarray and real-time quantitative PCR (qPCR) expression for three EBV miRNAs. We tested for EBV DNA, RNA, and protein in tumour tissue from six cases with and six cases without strong qPCR-based evidence of EBV miRNAs. RESULTS: The EBV miRNAs strongly differentiated between adenocarcinoma and squamous cell carcinoma using the microarray (P<0.01 for 9 out of 16 EBV miRNAs). However, microarray and qPCR measurements of BART1, BART2, and BHRF1-3 expression were not significantly correlated (P=0.53, 0.94, and 0.47, respectively). Although qPCR provided substantial evidence of EBV miRNAs in 7 out of 48 cases, only 1 of these 7 cases had detectable EBV DNA in tumour tissue. None had detectable EBV RNA or protein by histochemical stains. CONCLUSION: In a comprehensive evaluation of EBV miRNA, DNA, RNA, and protein in lung cancer, we found little evidence of EBV in lung tumour tissue. Discrepancies between microarray- and qPCR-based strategies highlight the difficulty of validating molecular markers of disease. Our results do not support a role of EBV in lung cancer.

Inherited polymorphisms in the RNA-mediated interference machinery affect microRNA expression and lung cancer survival.

BACKGROUND: MicroRNAs (miRs) have an important role in lung carcinogenesis and progression. Single-nucleotide polymorphisms (SNPs) in genes involved in miR biogenesis may affect miR expression in lung tissue and be associated with lung carcinogenesis and progression. METHODS: we analysed 12 SNPs in POLR2A, RNASEN and DICER1 genes in 1984 cases and 2073 controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We investigated miR expression profiles in 165 lung adenocarcinoma (AD) and 125 squamous cell carcinoma tissue samples from the same population. We used logistic and Cox regression models to examine the association of individual genotypes and haplotypes with lung cancer risk and with lung cancer-specific survival, respectively. SNPs-miR expression associations in cases were assessed using two-sample t-tests and global permutation tests. RESULTS: a haplotype in RNASEN (Drosha) was significantly associated with shorter lung cancer survival (hazard ratio=1.86, 95% CI=1.19-2.92, P=0.007). In AD cases, a SNP within the same haplotype was associated with reduced RNASEN mRNA expression (P=0.013) and with miR expression changes (global P=0.007) of miRs known to be associated with cancer (e.g., let-7 family, miR-21, miR-25, miR-126 and miR15a). CONCLUSION: inherited variation in the miR-processing machinery can affect miR expression levels and lung cancer-specific survival.

Correlation of population parameters leading to power differences in association studies with population stratification.

The power of statistical tests to measure effect sizes in the presence of population stratification is an important issue for the design and analysis of population-based association studies. Comparisons of statistical tests have shown that the power of different statistical approaches varies in different genetic scenarios. However, the impact of stratified population parameters on statistical power is not yet understood in a general statistical framework, particularly the impact of correlated population parameters. To investigate such impact in detail, we implemented a genetic model for population-based association studies with stratified samples and evaluated the impact on power with different genetic scenarios. The investigation shows that correlation between disease prevalence and risk allele frequency among subpopulations impacts statistical power. In a model with five subpopulations and moderate population divergence (Fst= 0.01), the correlation accounts for more than 85% of power difference. Our results also show that the estimation of genetic effect for candidate loci is biased by population divergence. Beneficial alleles could be wrongly characterized as risk alleles when prevalence differences and divergences of risk loci are large among subpopulations.

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.

Candidate genes for anorexia nervosa in the 1p33-36 linkage region: serotonin 1D and delta opioid receptor loci exhibit significant association to anorexia nervosa.

Serotonergic and opioidergic neurotransmitter system alterations have been observed in people with eating disorders; the genes for the serotonin 1D receptor (HTR1D) and the opioid delta receptor (OPRD1) are found on chr1p36.3-34.3, a region identified by our group in a linkage analysis of anorexia nervosa (AN). These candidate genes were evaluated for sequence variation and for linkage and association of this sequence variation to AN in family and case : control data sets. Resequencing of the HTR1D locus and a portion of the OPRD1 locus identified novel SNPs and confirmed existing SNPs. Genotype assay development and genotyping of nine SNPs (four at HTR1D and five at OPRD1) was performed on 191 unrelated individuals fulfilling DSM-IV criteria (w/o amenorrhea criterion) for AN, 442 relatives of AN probands and 98 psychiatrically screened controls. Linkage analysis of these candidate gene SNPs with 33 microsatellite markers in families including relative pairs concordantly affected with restricting AN (N=37) substantially increased the evidence for linkage of this region to restricting AN to an NPL score of 3.91. Statistically significant genotypic, allelic, and haplotypic association to AN in the case : control design was observed at HTR1D and OPRD1 with effect sizes for individual SNPs of 2.63 (95% CI=1.21-5.75) for HTR1D and 1.61 (95% CI=1.11-2.44) for OPRD1. Using genotype data on parents and AN probands, three SNPs at HTR1D were found to exhibit significant transmission disequilibrium (P&<0.05). The combined statistical genetic evidence suggests that HTR1D and OPRD1 or linked genes may be involved in the etiology of AN.

Candidate gene analysis of the Price Foundation anorexia nervosa affected relative pair dataset.

The eating disorders are severe psychiatric illnesses with significant morbidity and mortality that exhibit statistically significant familial risk and heritability, providing support for a molecular genetic approach toward defining etiological factors. An emerging candidate gene literature has concentrated on serotinergic and dopaminergic candidates. With the financial support of the Price Foundation, a group of investigators initiated an international multi-center collaboration (Price Foundation Collaborative Group) in 1995 to study the genetics of anorexia and bulimia nervosa by collecting and analyzing phenotypes and genotypes of individuals and their relatives affected with eating disorders. The first sample of families collected by this collaborative group, known as the Price Foundation Anorexia Nervosa Affected Relative Pair (AN-ARP) dataset, was ascertained on an proband affected with Anorexia Nervosa (AN), with relative pairs affected with the eating disorders AN, Bulimia Nervosa or Eating Disorders Not Otherwise Specified [1]. Biognosis U.S., Inc. was founded to identify and characterize candidate susceptibility genes for anorexia and bulimia nervosa phenotypes in the Price Foundation eating disorder datasets. During 2000-2001, Biognosis U.S., Inc. developed and implemented a research program with a focus on the analysis of candidate genes nominated by neurochemical characteristics of eating disorder patients [2], serotonergic and dopaminergic candidate gene polymorphisms [3], neuroendocrine regulation of appetite [4], and by a positional hypothesis from a linkage analysis of the AN-ARP dataset [5]. This report reviews the anorexia nervosa candidate gene literature through 2001, the candidate gene research program implemented at Biognosis U.S., Inc. and selected candidate gene findings in the AN-ARP dataset derived from that research program.

A genome-wide search for loci contributing to smoking and alcoholism.

Using the Collaborative Study on the Genetics of Alcoholism (COGA) data, we performed a sib-pair linkage analysis of two smoking-related traits and one alcoholism phenotype. The first trait, EVRNVR, was a dichotomous one we constructed based on epidemiological definitions of smoking. The second trait, PKYRS, used the quantitative pack-year history provided, and the third trait was the COGA alcoholism classification, ALDX1. There was some evidence for linkage of the EVRNVR trait to regions-on chromosomes 6, 9, and 14. Smaller numbers of loci provided nominal evidence for linkage to PKYRS, although some candidate gene regions were identified. The number of loci identified using EVRNVR suggests that a threshold-based phenotype may better identify loci affecting smoking history. Approximately one-third of the loci that showed evidence for linkage to EVRNVR at a nominal significance level (p < 0.01) also showed evidence for linkage to ALDX1. Some of these regions may represent loci increasing vulnerability to both smoking and alcoholism.

Sib-pair linkage analyses of alcoholism: dichotomous and quantitative measures.

We hypothesized that a quantitative alcoholism trait would have greater power than the Collaborative Study on the Genetics of Alcoholism (COGA) dichotomous alcoholism traits, ALDX1 and ALDX2, to detect putative alcoholism loci. To test this, we performed nonparametric sib-pair linkage analysis to screen 285 polymorphic autosomal markers for evidence of linkage to ALDX1, ALDX2, and a quantitative trait, QUANT, defined from the 11 COGA latent class variables. We also examined the effects on the analyses of including covariates (sex, age, and pack-years of smoking) and of transforming QUANT (log and square root). ALDX1 and ALDX2 showed the greatest evidence for linkage to markers on chromosome 1, by both the affected sib-pair and the Haseman-Elston tests. Regions of interest were also identified on chromosomes 4, 8, 16, and 17. QUANT showed little evidence for linkage to any chromosomal region, having no more significant results than were expected by chance. Including covariates or transforming QUANT had little effect on the analyses. A quantitative trait based on all 37 latent class variables, with each variable appropriately weighted, may have had more power than QUANT to detect genomic regions of relevance to alcoholism.

Cigarette smoking.

Cigarette smoking is the largest preventable risk factor for morbidity and mortality in developed countries. Dramatic changes in the prevalence of cigarette smoking in the second half of this century in the United States (i.e., a reduction among men and an increase among women) have reduced current smoking levels to approximately one quarter of the adult population and have reduced differences in smoking prevalence and smoking-attributable diseases between the sexes. Current smoking in the United States is positively associated with younger age, lower income, reduced educational achievement, and disadvantaged neighborhood environment. Daily smokers smoke cigarettes to maintain nicotine levels in the brain, primarily to avoid the negative effects of nicotine withdrawal, but also to modulate mood. Regular smokers exhibit higher and lower levels of stress and arousal, respectively, than nonsmokers, as well as higher impulsivity and neuroticism trait values. Nicotine dependence is the single most common psychiatric diagnosis in the United States, and substance abuse, major depression, and anxiety disorders are the most prevalent psychiatric comorbid conditions associated with nicotine dependence. Studies in twins have implicated genetic factors that explain most of the variability in vulnerability to smoking and in persistence of the smoking phenotype. Future research into the causes of smoking must take into account these associated demographics, social factors, comorbid psychiatric conditions, and genetic factors to understand this complex human behavior.

Autosomal, mitochondrial, and Y chromosome DNA variation in Finland: evidence for a male-specific bottleneck.

The high prevalence of rare genetic diseases in Finland has been attributed to a founder effect some 2,000 years ago. However, this hypothesis has not been supported from mtDNA sequence and autosomal microsatellite data which indicate high levels of gene diversity. Here we have identified genetic evidence for a population bottleneck by examining variable microsatellite loci on the nonrecombining portion of Y chromosomes from Finland and four populations from Europe and the Americas. Sequence data from segment I of the control region (HVS-1) of mtDNA (360 bases) and 20 autosomal dinucleotide repeat markers were also analyzed. Partitions of genetic variance within and between populations revealed significant levels of Y-chromosome differentiation between populations. Phylogenetic and diversity analyses revealed divergent Finnish Y-haplotype clades and significantly lower Y-haplotype diversity among Finns as compared to other populations. Surprisingly, Finnish Y-haplotype diversity was even lower than the Native American populations. These results provide support for the Finnish bottleneck hypothesis. Evidence for two separate founding Finnish Y-chromosome lineages was also observed from the Y-chromosome phylogeny. A limited number of closely related founding males may have contributed to the low number of paternal lineages in the Finnish population. In contrast, high levels of genetic diversity for mtDNA and autosomal STRs may be the result of sex-biased gene flow and recent immigration to urban areas from established internal isolates within Finland.

Cladistic association analysis of Y chromosome effects on alcohol dependence and related personality traits.

Association between Y chromosome haplotype variation and alcohol dependence and related personality traits was investigated in a large sample of psychiatrically diagnosed Finnish males. Haplotypes were constructed for 359 individuals using alleles at eight loci (seven microsatellite loci and a nucleotide substitution in the DYZ3 alphoid satellite locus). A cladogram linking the 102 observed haplotype configurations was constructed by using parsimony with a single-step mutation model. Then, a series of contingency tables nested according to the cladogram hierarchy were used to test for association between Y haplotype and alcohol dependence. Finally, using only alcohol-dependent subjects, we tested for association between Y haplotype and personality variables postulated to define subtypes of alcoholism-antisocial personality disorder, novelty seeking, harm avoidance, and reward dependence. Significant association with alcohol dependence was observed at three Y haplotype clades, with significance levels of P = 0.002, P = 0.020, and P = 0.010. Within alcohol-dependent subjects, no relationship was revealed between Y haplotype and antisocial personality disorder, novelty seeking, harm avoidance, or reward dependence. These results demonstrate, by using a fully objective association design, that differences among Y chromosomes contribute to variation in vulnerability to alcohol dependence. However, they do not demonstrate an association between Y haplotype and the personality variables thought to underlie the subtypes of alcoholism.

An Asian-Native American paternal lineage identified by RPS4Y resequencing and by microsatellite haplotyping.

Human paternal population history was studied in 9 populations [three Native American, three Asian, two Caucasian and one African-derived sample(s)] using sequence and short tandem repeat haplotype diversity within the non-pseudoautosegmal region of the Y chromosome. Complete coding and additional flanking sequences (949 base pairs) of the RPS4Y locus were determined in 59 individuals from three of the populations, revealing a nucleotide diversity of 0.0147%, consistent with previous estimates from Y chromosome resequencing studies. One RPS4Y sequence variant, 711C > T, was polymorphic in Asian and Native American populations, but not in African and Caucasian population samples. The RPS4Y 711C > T variant, a second unique sequence variant at DYS287 and nine Y chromosome short tandem repeat (YSTR) loci were used to analyze the evolution of Y chromosome lineages. Three unambiguous lineages were defined in Asian, Native American and Jamaican populations using sequence variants at RPS4Y and DYS287. These lineages were independently supported by the haplotypes defined solely by YSTR alleles, demonstrating the haplotypes constructed from YSTRs can evaluate population diversity, admixture and phylogeny.