Pharmacogenetic Variation at CYP2D6, CYP2C9, and CYP2C19: Population Genetic and Forensic Aspects

Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would cover the most important genetic variants altering the enzyme activity, and, for the first time, to describe the distribution of genetic variation at these loci on global and microgeographic scales. In addition, pharmacogenetics was applied to a postmortem forensic setting to elucidate the role of genetic variation in drug intoxications, focusing mainly on cases related to tricyclic antidepressants, which are commonly involved in fatal drug poisonings in Finland. Genetic variability data were obtained by genotyping new population samples by the methods developed based on PCR and multiplex single-nucleotide primer extension reaction, as well as by collecting data from the literature. Data consisted of 138, 129, and 146 population samples for CYP2D6, CYP2C9, and CYP2C19, respectively. In addition, over 200 postmortem forensic cases were examined with respect to drug and metabolite concentrations and genotypic variation at CYP2D6 and CYP2C19. The distribution of genetic variation within and among human populations was analyzed by descriptive statistics and variance analysis and by correlating the genetic and geographic distances using Mantel tests and spatial autocorrelation. The correlation between phenotypic and genotypic variation in drug metabolism observed in postmortem cases was also analyzed statistically. The genotyping methods developed proved to be informative, technically feasible, and cost-effective. Detailed molecular analysis of CYP2D6 genetic variation in a global survey of human populations revealed that the pattern of variation was similar to those of neutral genomic markers. Most of the CYP2D6 diversity was observed within populations, and the spatial pattern of variation was best described as clinal. On the other hand, genetic variants of CYP2D6, CYP2C9, and CYP2C19 associated with altered enzymatic activity could reach extremely high frequencies in certain geographic regions. Pharmacogenetic variation may also be significantly affected by population-specific demographic histories, as seen within the Finnish population. When pharmacogenetics was applied to a postmortem forensic setting, a correlation between amitriptyline metabolic ratios and genetic variation at CYP2D6 and CYP2C19 was observed in the sample material, even in the presence of confounding factors typical for these cases. In addition, a case of doxepin-related fatal poisoning was shown to be associated with a genetic defect at CYP2D6. Each of the genes studied showed a distinct variation pattern in human populations and high frequencies of altered activity variants, which may reflect the neutral evolution and/or selective pressures caused by dietary or environmental exposure. The results are relevant also from the clinical point of view since the genetic variation at CYP2D6, CYP2C9, and CYP2C19 already has a range of clinical applications, e.g. in cancer treatment and oral anticoagulation therapy. This study revealed that pharmacogenetics may also contribute valuable information to the medicolegal investigation of sudden, unexpected deaths.

Genome-wide association meta-analysis identifies new endometriosis risk loci

We conducted a genome-wide association meta-analysis of 4,604 endometriosis cases and 9,393 controls of Japanese and European ancestry. We show that rs12700667 on chromosome 7p15.2, previously found to associate with disease in Europeans, replicates in Japanese (P = 3.6 x 10(-3)), and we confirm association of rs7521902 at 1p36.12 near WNT4. In addition, we establish an association of rs13394619 in GREB1 at 2p25.1 with endometriosis and identify a newly associated locus at 12q22 near VEZT (rs10859871). Excluding cases of European ancestry of minimal or unknown severity, we identified additional previously unknown loci at 2p14 (rs4141819), 6p22.3 (rs7739264) and 9p21.3 (rs1537377). All seven SNP effects were replicated in an independent cohort and associated at P <5 x 10(-8) in a combined analysis. Finally, we found a significant overlap in polygenic risk for endometriosis between the genome-wide association cohorts of European and Japanese descent (P = 8.8 x 10(-11)), indicating that many weakly associated SNPs represent true endometriosis risk loci and that risk prediction and future targeted disease therapy may be transferred across these populations.

Using genomic data to make indirect (and unauthorized) estimates of disease risk

The number of genetic factors associated with common human traits and disease is increasing rapidly, and the general public is utilizing affordable, direct-to-consumer genetic tests. The results of these tests are often in the public domain. A combination of factors has increased the potential for the indirect estimation of an individual's risk for a particular trait. Here we explain the basic principals underlying risk estimation which allowed us to test the ability to make an indirect risk estimation from genetic data by imputing Dr. James Watson's redacted apolipoprotein E gene (APOE) information. The principles underlying risk prediction from genetic data have been well known and applied for many decades, however, the recent increase in genomic knowledge, and advances in mathematical and statistical techniques and computational power, make it relatively easy to make an accurate but indirect estimation of risk. There is a current hazard for indirect risk estimation that is relevant not only to the subject but also to individuals related to the subject; this risk will likely increase as more detailed genomic data and better computational tools become available.

Six novel susceptibility Loci for early-onset androgenetic alopecia and their unexpected association with common diseases

Androgenetic alopecia (AGA) is a highly heritable condition and the most common form of hair loss in humans. Susceptibility loci have been described on the X chromosome and chromosome 20, but these loci explain a minority of its heritable variance. We conducted a large-scale meta-analysis of seven genome-wide association studies for early-onset AGA in 12,806 individuals of European ancestry. While replicating the two AGA loci on the X chromosome and chromosome 20, six novel susceptibility loci reached genome-wide significance (p = 2.62x10(-)(9)-1.01x10(-)(1)(2)). Unexpectedly, we identified a risk allele at 17q21.31 that was recently associated with Parkinson's disease (PD) at a genome-wide significant level. We then tested the association between early-onset AGA and the risk of PD in a cross-sectional analysis of 568 PD cases and 7,664 controls. Early-onset AGA cases had significantly increased odds of subsequent PD (OR = 1.28, 95% confidence interval: 1.06-1.55, p = 8.9x10(-)(3)). Further, the AGA susceptibility alleles at the 17q21.31 locus are on the H1 haplotype, which is under negative selection in Europeans and has been linked to decreased fertility. Combining the risk alleles of six novel and two established susceptibility loci, we created a genotype risk score and tested its association with AGA in an additional sample. Individuals in the highest risk quartile of a genotype score had an approximately six-fold increased risk of early-onset AGA [odds ratio (OR) = 5.78, p = 1.4x10(-)(8)(8)]. Our results highlight unexpected associations between early-onset AGA, Parkinson's disease, and decreased fertility, providing important insights into the pathophysiology of these conditions.

Genome-wide association analysis identifies susceptibility loci for migraine without aura

Migraine without aura is the most common form of migraine, characterized by recurrent disabling headache and associated autonomic symptoms. To identify common genetic variants associated with this migraine type, we analyzed genome-wide association data of 2,326 clinic-based German and Dutch individuals with migraine without aura and 4,580 population-matched controls. We selected SNPs from 12 loci with 2 or more SNPs associated with P values of <1 x 10(-5) for replication testing in 2,508 individuals with migraine without aura and 2,652 controls. SNPs at two of these loci showed convincing replication: at 1q22 (in MEF2D; replication P = 4.9 x 10(-4); combined P = 7.06 x 10(-11)) and at 3p24 (near TGFBR2; replication P = 1.0 x 10(-4); combined P = 1.17 x 10(-9)). In addition, SNPs at the PHACTR1 and ASTN2 loci showed suggestive evidence of replication (P = 0.01; combined P = 3.20 x 10(-8) and P = 0.02; combined P = 3.86 x 10(-8), respectively). We also replicated associations at two previously reported migraine loci in or near TRPM8 and LRP1. This study identifies the first susceptibility loci for migraine without aura, thereby expanding our knowledge of this debilitating neurological disorder.

Genome-wide linkage and association analyses implicate FASN in predisposition to Uterine Leiomyomata

Uterine leiomyomata (UL), the most prevalent pelvic tumors in women of reproductive age, pose a major public health problem given their high frequency, associated morbidities, and most common indication for hysterectomies. A genetic component to UL predisposition is supported by analyses of ethnic predisposition, twin studies, and familial aggregation. A genome-wide SNP linkage panel was genotyped and analyzed in 261 white UL-affected sister-pair families from the Finding Genes for Fibroids study. Two significant linkage regions were detected in 10p11 (LOD = 4.15) and 3p21 (LOD = 3.73), and five additional linkage regions were identified with LOD scores > 2.00 in 2q37, 5p13, 11p15, 12q14, and 17q25. Genome-wide association studies were performed in two independent cohorts of white women, and a meta-analysis was conducted. One SNP (rs4247357) was identified with a p value (p = 3.05 x 10(-8)) that reached genome-wide significance (odds ratio = 1.299). The candidate SNP is under a linkage peak and in a block of linkage disequilibrium in 17q25.3, which spans fatty acid synthase (FASN), coiled-coil-domain-containing 57 (CCDC57), and solute-carrier family 16, member 3 (SLC16A3). By tissue microarray immunohistochemistry, we found elevated (3-fold) FAS levels in UL-affected tissue compared to matched myometrial tissue. FAS transcripts and/or protein levels are upregulated in various neoplasms and implicated in tumor cell survival. FASN represents the initial UL risk allele identified in white women by a genome-wide, unbiased approach and opens a path to management and potential therapeutic intervention.

A genome-wide association study of caffeine-related sleep disturbance: Confirmation of a role for a common variant in the adenosine receptor

OBJECTIVES To identify common genetic variants that predispose to caffeine-induced insomnia and to test whether genes whose expression changes in the presence of caffeine are enriched for association with caffeine-induced insomnia. DESIGN A hypothesis-free, genome-wide association study. SETTING Community-based sample of Australian twins from the Australian Twin Registry. PARTICIPANTS After removal of individuals who said that they do not drink coffee, a total of 2,402 individuals from 1,470 families in the Australian Twin Registry provided both phenotype and genotype information. MEASUREMENTS AND RESULTS A dichotomized scale based on whether participants reported ever or never experiencing caffeine-induced insomnia. A factor score based on responses to a number of questions regarding normal sleep habits was included as a covariate in the analysis. More than 2 million common single nucleotide polymorphisms (SNPs) were tested for association with caffeine-induced insomnia. No SNPs reached the genome-wide significance threshold. In the analysis that did not include the insomnia factor score as a covariate, the most significant SNP identified was an intronic SNP in the PRIMA1 gene (P = 1.4 x 10(-)(6), odds ratio = 0.68 [0.53 - 0.89]). An intergenic SNP near the GBP4 gene on chromosome 1 was the most significant upon inclusion of the insomnia factor score into the model (P = 1.9 x 10(-)(6), odds ratio = 0.70 [0.62 - 0.78]). A previously identified association with a polymorphism in the ADORA2A gene was replicated. CONCLUSIONS Several genes have been identified in the study as potentially influencing caffeine-induced insomnia. They will require replication in another sample. The results may have implications for understanding the biologic mechanisms underlying insomnia.

Genome-wide meta-analysis of common variant differences between men and women

The male-to-female sex ratio at birth is constant across world populations with an average of 1.06 (106 male to 100 female live births) for populations of European descent. The sex ratio is considered to be affected by numerous biological and environmental factors and to have a heritable component. The aim of this study was to investigate the presence of common allele modest effects at autosomal and chromosome X variants that could explain the observed sex ratio at birth. We conducted a large-scale genome-wide association scan (GWAS) meta-analysis across 51 studies, comprising overall 114 863 individuals (61 094 women and 53 769 men) of European ancestry and 2 623 828 common (minor allele frequency >0.05) single-nucleotide polymorphisms (SNPs). Allele frequencies were compared between men and women for directly-typed and imputed variants within each study. Forward-time simulations for unlinked, neutral, autosomal, common loci were performed under the demographic model for European populations with a fixed sex ratio and a random mating scheme to assess the probability of detecting significant allele frequency differences. We do not detect any genome-wide significant (P < 5 x 10(-8)) common SNP differences between men and women in this well-powered meta-analysis. The simulated data provided results entirely consistent with these findings. This large-scale investigation across ~115 000 individuals shows no detectable contribution from common genetic variants to the observed skew in the sex ratio. The absence of sex-specific differences is useful in guiding genetic association study design, for example when using mixed controls for sex-biased traits.

Evaluation of polymorphisms in predicted target sites for micro RNAs differentially expressed in endometriosis

Previous microarray analyses identified 22 microRNAs (miRNAs) differentially expressed in paired ectopic and eutopic endometrium of women with and without endometriosis. To investigate further the role of these miRNAs in women with endometriosis, we conducted an association study aiming to explore the relationship between endometriosis risk and single-nucleotide polymorphisms (SNPs) in miRNA target sites for these differentially expressed miRNAs. A panel of 102 SNPs in the predicted miRNA binding sites were evaluated for an endometriosis association study and an ingenuity pathway analysis was performed. Fourteen rare variants were identified in this study. We found SNP rs14647 in the Wolf-Hirschhorn syndrome candidate gene1 (WHSC1) 3'UTR (untranslated region) was associated with endometriosis-related infertility presenting an odds ratio of 12.2 (95% confidence interval = 2.4-60.7, P = 9.03 x 10(-5)). SNP haplotype AGG in the solute carrier family 22, member 23 (SLC22A23) 3'UTR was associated with endometriosis-related infertility and more severe disease. With the individual genotyping data, ingenuity pathways analysis identified the tumour necrosis factor and cyclin-dependant kinase inhibitor as major factors in the molecular pathways. Significant associations between WHSC1 alleles and endometriosis-related infertility and SLC22A23 haplotypes and the disease severe stage were identified. These findings may help focus future research on subphenotypes of this disease. Replication studies in independent large sample sets to confirm and characterize the involvement of the gene variation in the pathogenesis of endometriosis are needed.

Genomic inflation factors under polygenic inheritance

Population structure, including population stratification and cryptic relatedness, can cause spurious associations in genome-wide association studies (GWAS). Usually, the scaled median or mean test statistic for association calculated from multiple single-nucleotide-polymorphisms across the genome is used to assess such effects, and 'genomic control' can be applied subsequently to adjust test statistics at individual loci by a genomic inflation factor. Published GWAS have clearly shown that there are many loci underlying genetic variation for a wide range of complex diseases and traits, implying that a substantial proportion of the genome should show inflation of the test statistic. Here, we show by theory, simulation and analysis of data that in the absence of population structure and other technical artefacts, but in the presence of polygenic inheritance, substantial genomic inflation is expected. Its magnitude depends on sample size, heritability, linkage disequilibrium structure and the number of causal variants. Our predictions are consistent with empirical observations on height in independent samples of ~4000 and ~133,000 individuals.

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption

Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of approximately 2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 x 10(-8) to P = 4 x 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.

High-density fine-mapping of a chromosome 10q26 linkage peak suggests association between endometriosis and variants close to CYP2C19

OBJECTIVE To refine a previously reported linkage peak for endometriosis on chromosome 10q26, and conduct follow-up analyses and a fine-mapping association study across the region to identify new candidate genes for endometriosis. DESIGN Case-control study. SETTING Academic research. PATIENT(S) Cases=3,223 women with surgically confirmed endometriosis; controls=1,190 women without endometriosis and 7,060 population samples. INTERVENTION(S) Analysis of 11,984 single nucleotide polymorphisms on chromosome 10. MAIN OUTCOME MEASURE(S) Allele frequency differences between cases and controls. RESULT(S) Linkage analyses on families grouped by endometriosis symptoms (primarily subfertility) provided increased evidence for linkage (logarithm of odds score=3.62) near a previously reported linkage peak. Three independent association signals were found at 96.59 Mb (rs11592737), 105.63 Mb (rs1253130), and 124.25 Mb (rs2250804). Analyses including only samples from linkage families supported the association at all three regions. However, only rs11592737 in the cytochrome P450 subfamily C (CYP2C19) gene was replicated in an independent sample of 2,079 cases and 7,060 population controls. CONCLUSION(S) The role of the CYP2C19 gene in conferring risk for endometriosis warrants further investigation.

Educational attainment: A genome wide association study in 9538 Australians

BACKGROUND Correlations between Educational Attainment (EA) and measures of cognitive performance are as high as 0.8. This makes EA an attractive alternative phenotype for studies wishing to map genes affecting cognition due to the ease of collecting EA data compared to other cognitive phenotypes such as IQ. METHODOLOGY In an Australian family sample of 9538 individuals we performed a genome-wide association scan (GWAS) using the imputed genotypes of approximately 2.4 million single nucleotide polymorphisms (SNP) for a 6-point scale measure of EA. Top hits were checked for replication in an independent sample of 968 individuals. A gene-based test of association was then applied to the GWAS results. Additionally we performed prediction analyses using the GWAS results from our discovery sample to assess the percentage of EA and full scale IQ variance explained by the predicted scores. RESULTS The best SNP fell short of having a genome-wide significant p-value (p = 9.77x10(-7)). In our independent replication sample six SNPs among the top 50 hits pruned for linkage disequilibrium (r(2)<0.8) had a p-value<0.05 but only one of these SNPs survived correction for multiple testing--rs7106258 (p = 9.7*10(-4)) located in an intergenic region of chromosome 11q14.1. The gene based test results were non-significant and our prediction analyses show that the predicted scores explained little variance in EA in our replication sample. CONCLUSION While we have identified a polymorphism chromosome 11q14.1 associated with EA, further replication is warranted. Overall, the absence of genome-wide significant p-values in our large discovery sample confirmed the high polygenic architecture of EA. Only the assembly of large samples or meta-analytic efforts will be able to assess the implication of common DNA polymorphisms in the etiology of EA.

Genome-wide association study identifies a new melanoma susceptibility locus at 1q21.3

We performed a genome-wide association study of melanoma in a discovery cohort of 2,168 Australian individuals with melanoma and 4,387 control individuals. In this discovery phase, we confirm several previously characterized melanoma-associated loci at MC1R, ASIP and MTAP-CDKN2A. We selected variants at nine loci for replication in three independent case-control studies (Europe: 2,804 subjects with melanoma, 7,618 control subjects; United States 1: 1,804 subjects with melanoma, 1,026 control subjects; United States 2: 585 subjects with melanoma, 6,500 control subjects). The combined meta-analysis of all case-control studies identified a new susceptibility locus at 1q21.3 (rs7412746, P = 9.0 x 10(-11), OR in combined replication cohorts of 0.89 (95% CI 0.85-0.95)). We also show evidence suggesting that melanoma associates with 1q42.12 (rs3219090, P = 9.3 x 10(-8)). The associated variants at the 1q21.3 locus span a region with ten genes, and plausible candidate genes for melanoma susceptibility include ARNT and SETDB1. Variants at the 1q21.3 locus do not seem to be associated with human pigmentation or measures of nevus density.

Meta-analysis of genome-wide association for migraine in six population-based European cohorts

Migraine is a common neurological disorder with a genetically complex background. This paper describes a meta-analysis of genome-wide association (GWA) studies on migraine, performed by the Dutch-Icelandic migraine genetics (DICE) consortium, which brings together six population-based European migraine cohorts with a total sample size of 10,980 individuals (2446 cases and 8534 controls). A total of 32 SNPs showed marginal evidence for association at a P-value<10(-5). The best result was obtained for SNP rs9908234, which had a P-value of 8.00 x 10(-8). This top SNP is located in the nerve growth factor receptor (NGFR) gene. However, this SNP did not replicate in three cohorts from the Netherlands and Australia. Of the other 31 SNPs, 18 SNPs were tested in two replication cohorts, but none replicated. In addition, we explored previously identified candidate genes in the meta-analysis data set. This revealed a modest gene-based significant association between migraine and the metadherin (MTDH) gene, previously identified in the first clinic-based GWA study (GWAS) for migraine (Bonferroni-corrected gene-based P-value=0.026). This finding is consistent with the involvement of the glutamate pathway in migraine. Additional research is necessary to further confirm the involvement of glutamate.