Quantitative trait loci, G×E and G×G for glycemic traits: response to metformin and placebo in the Diabetes Prevention Program (DPP).

The complex genetic architecture of type-2-diabetes (T2D) includes gene-by-environment (G×E) and gene-by-gene (G×G) interactions. To identify G×E and G×G, we screened markers for patterns indicative of interactions (relationship loci [rQTL] and variance heterogeneity loci [vQTL]). rQTL exist when the correlation between multiple traits varies by genotype and vQTL occur when the variance of a trait differs by genotype (potentially flagging G×G and G×E). In the metformin and placebo arms of the DPP (n = 1762) we screened 280,965 exomic and intergenic SNPs, for rQTL and vQTL patterns in association with year one changes from baseline in glycemia and related traits (insulinogenic index [IGI], insulin sensitivity index [ISI], fasting glucose and fasting insulin). Significant (p < 1.8 × 10-7) rQTL and vQTL generated a priori hypotheses of individual G×E tests for a SNP × metformin treatment interaction and secondarily for G×G screens. Several rQTL and vQTL identified led to 6 nominally significant (p < 0.05) metformin treatment × SNP interactions (4 for IGI, one insulin, and one glucose) and 12G×G interactions (all IGI) that exceeded experiment-wide significance (p < 4.1 × 10-9). Some loci are directly associated with incident diabetes, and others are rQTL and modify a trait's relationship with diabetes (2 diabetes/glucose, 2 diabetes/insulin, 1 diabetes/IGI). rs3197999, an ISI/insulin rQTL, is a possible gene damaging missense mutation in MST1, is associated with ulcerative colitis, sclerosing cholangitis, Crohn's disease, BMI and coronary artery disease. This study demonstrates evidence for context-dependent effects (G×G & G×E) and the complexity of these T2D-related traits.

Genome-wide association study for variants that modulate relationships between cerebrospinal fluid amyloid-beta 42, tau, and p-tau levels.

BACKGROUND: A relationship quantitative trait locus exists when the correlation between multiple traits varies by genotype for that locus. Relationship quantitative trait loci (rQTL) are often involved in gene-by-gene (G×G) interactions or gene-by-environmental interactions, making them a powerful tool for detecting G×G. METHODS: We performed genome-wide association studies to identify rQTL between tau and Aß42 and ptau and Aß42 with over 3000 individuals using age, gender, series, APOE ε2, APOE ε4, and two principal components for population structure as covariates. Each significant rQTL was separately screened for interactions with other loci for each trait in the rQTL model. Parametric bootstrapping was used to assess significance. RESULTS: We found four significant tau/Aß42 rQTL from three unique locations and six ptau/Aß42 rQTL from five unique locations. G×G screens with these rQTL produced four significant G×G interactions (one Aß42, two ptau, and one tau) with four rQTL where each second locus was from a unique location. On follow-up, rs1036819 and rs74025622 were associated with Alzheimer's disease (AD) case/control status; rs15205 and rs79099429 were associated with rate of decline. CONCLUSIONS: The two most significant rQTL (rs8027714 and rs1036819) for ptau/Aß42 are on different chromosomes and both are strong hits for pelvic organ prolapse. While diseases of the nervous system can cause pelvic organ prolapse, it is unlikely related to the ptau/Aß42 relationship but may suggest that these two loci share a pathway. In addition to a ptau/Aß42 rQTL and association with AD case/control status, rs1036819 is a strong rQTL for case/control status/Aß42 and for tau/Aß42. It resides in the ZFAT gene, which is related to autoimmune thyroid disease. For tau, rs9817620 interacts with the tau/Aß42 rQTL rs74025622. It is in the CHL1 gene, which is a neural cell adhesion molecule and may be involved in signal transduction pathways. CHL1 is related to BACE1, which is a ß-secretase enzyme that initiates production of the ß-amyloid peptide involved in AD and is a primary drug target. Overall, there are numerous loci that affect the relationship between these important AD endophenotypes and some are due to interactions with other loci. Some affect the risk of AD and/or rate of progression.

A family-based joint test for mean and variance heterogeneity for quantitative traits.

Traditional quantitative trait locus (QTL) analysis focuses on identifying loci associated with mean heterogeneity. Recent research has discovered loci associated with phenotype variance heterogeneity (vQTL), which is important in studying genetic association with complex traits, especially for identifying gene-gene and gene-environment interactions. While several tests have been proposed to detect vQTL for unrelated individuals, there are no tests for related individuals, commonly seen in family-based genetic studies. Here we introduce a likelihood ratio test (LRT) for identifying mean and variance heterogeneity simultaneously or for either effect alone, adjusting for covariates and family relatedness using a linear mixed effect model approach. The LRT test statistic for normally distributed quantitative traits approximately follows χ(2)-distributions. To correct for inflated Type I error for non-normally distributed quantitative traits, we propose a parametric bootstrap-based LRT that removes the best linear unbiased prediction (BLUP) of family random effect. Simulation studies show that our family-based test controls Type I error and has good power, while Type I error inflation is observed when family relatedness is ignored. We demonstrate the utility and efficiency gains of the proposed method using data from the Framingham Heart Study to detect loci associated with body mass index (BMI) variability.

Mitochondrial genomic variation associated with higher mitochondrial copy number: the Cache County Study on Memory Health and Aging.

BACKGROUND: The mitochondria are essential organelles and are the location of cellular respiration, which is responsible for the majority of ATP production. Each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. The regulation of mitochondrial copy number by nuclear genes has been studied extensively. While mitochondrial variation has been associated with longevity and some of the diseases known to have reduced mitochondrial copy number, the role that the mitochondrial genome itself has in regulating mitochondrial copy number remains poorly understood. RESULTS: We analyzed the complete mitochondrial genomes from 1007 individuals randomly selected from the Cache County Study on Memory Health and Aging utilizing the inferred evolutionary history of the mitochondrial haplotypes present in our dataset to identify sequence variation and mitochondrial haplotypes associated with changes in mitochondrial copy number. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. CONCLUSIONS: We identified three variants associated with higher mitochondrial copy number and suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that causes changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes.

A versatile omnibus test for detecting mean and variance heterogeneity.

Recent research has revealed loci that display variance heterogeneity through various means such as biological disruption, linkage disequilibrium (LD), gene-by-gene (G × G), or gene-by-environment interaction. We propose a versatile likelihood ratio test that allows joint testing for mean and variance heterogeneity (LRT(MV)) or either effect alone (LRT(M) or LRT(V)) in the presence of covariates. Using extensive simulations for our method and others, we found that all parametric tests were sensitive to nonnormality regardless of any trait transformations. Coupling our test with the parametric bootstrap solves this issue. Using simulations and empirical data from a known mean-only functional variant, we demonstrate how LD can produce variance-heterogeneity loci (vQTL) in a predictable fashion based on differential allele frequencies, high D', and relatively low r² values. We propose that a joint test for mean and variance heterogeneity is more powerful than a variance-only test for detecting vQTL. This takes advantage of loci that also have mean effects without sacrificing much power to detect variance only effects. We discuss using vQTL as an approach to detect G × G interactions and also how vQTL are related to relationship loci, and how both can create prior hypothesis for each other and reveal the relationships between traits and possibly between components of a composite trait.

APOE modulates the correlation between triglycerides, cholesterol, and CHD through pleiotropy, and gene-by-gene interactions.

Relationship loci (rQTL) exist when the correlation between multiple traits varies by genotype. rQTL often occur due to gene-by-gene (G × G) or gene-by-environmental interactions, making them a powerful tool for detecting G × G. Here we present an empirical analysis of apolipoprotein E (APOE) with respect to lipid traits and incident CHD leading to the discovery of loci that interact with APOE to affect these traits. We found that the relationship between total cholesterol (TC) and triglycerides (ln TG) varies by APOE isoform genotype in African-American (AA) and European-American (EA) populations. The e2 allele is associated with strong correlation between ln TG and TC while the e4 allele leads to little or no correlation. This led to a priori hypotheses that APOE genotypes affect the relationship of TC and/or ln TG with incident CHD. We found that APOE*TC was significant (P = 0.016) for AA but not EA while APOE*ln TG was significant for EA (P = 0.027) but not AA. In both cases, e2e2 and e2e3 had strong relationships between TC and ln TG with CHD while e2e4 and e4e4 results in little or no relationship between TC and ln TG with CHD. Using ARIC GWAS data, scans for loci that significantly interact with APOE produced four loci for African Americans (one CHD, one TC, and two HDL). These interactions contribute to the rQTL pattern. rQTL are a powerful tool to identify loci that modify the relationship between risk factors and disease and substantially increase statistical power for detecting G × G.

Mitochondrial haplotypes associated with biomarkers for Alzheimer's disease.

Various studies have suggested that the mitochondrial genome plays a role in late-onset Alzheimer's disease, although results are mixed. We used an endophenotype-based approach to further characterize mitochondrial genetic variation and its relationship to risk markers for Alzheimer's disease. We analyzed longitudinal data from non-demented, mild cognitive impairment, and late-onset Alzheimer's disease participants in the Alzheimer's Disease Neuroimaging Initiative with genetic, brain imaging, and behavioral data. We assessed the relationship of structural MRI and cognitive biomarkers with mitochondrial genome variation using TreeScanning, a haplotype-based approach that concentrates statistical power by analyzing evolutionarily meaningful groups (or clades) of haplotypes together for association with a phenotype. Four clades were associated with three different endophenotypes: whole brain volume, percent change in temporal pole thickness, and left hippocampal atrophy over two years. This is the first study of its kind to identify mitochondrial variation associated with brain imaging endophenotypes of Alzheimer's disease. Our results provide additional evidence that the mitochondrial genome plays a role in risk for Alzheimer's disease.

Mitochondrial genomic analysis of late onset Alzheimer's disease reveals protective haplogroups H6A1A/H6A1B: the Cache County Study on Memory in Aging.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia and AD risk clusters within families. Part of the familial aggregation of AD is accounted for by excess maternal vs. paternal inheritance, a pattern consistent with mitochondrial inheritance. The role of specific mitochondrial DNA (mtDNA) variants and haplogroups in AD risk is uncertain. METHODOLOGY/PRINCIPAL FINDINGS: We determined the complete mitochondrial genome sequence of 1007 participants in the Cache County Study on Memory in Aging, a population-based prospective cohort study of dementia in northern Utah. AD diagnoses were made with a multi-stage protocol that included clinical examination and review by a panel of clinical experts. We used TreeScanning, a statistically robust approach based on haplotype networks, to analyze the mtDNA sequence data. Participants with major mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD (p=0.017, corrected for multiple comparisons). The protective haplotypes were defined by three variants: m.3915G>A, m.4727A>G, and m.9380G>A. These three variants characterize two different major haplogroups. Together m.4727A>G and m.9380G>A define H6A1, and it has been suggested m.3915G>A defines H6A. Additional variants differentiate H6A1A and H6A1B; however, none of these variants had a significant relationship with AD case-control status. CONCLUSIONS/SIGNIFICANCE: Our findings provide evidence of a reduced risk of AD for individuals with mtDNA haplotypes H6A1A and H6A1B. These findings are the results of the largest study to date with complete mtDNA genome sequence data, yet the functional significance of the associated haplotypes remains unknown and replication in others studies is necessary.

The importance of context to the genetic architecture of diabetes-related traits is revealed in a genome-wide scan of a LG/J × SM/J murine model.

Variations in diabetic phenotypes are caused by complex interactions of genetic effects, environmental factors, and the interplay between the two. We tease apart these complex interactions by examining genome-wide genetic and epigenetic effects on diabetes-related traits among different sex, diet, and sex-by-diet cohorts in a Mus musculus model. We conducted a genome-wide scan for quantitative trait loci that affect serum glucose and insulin levels and response to glucose stress in an F(16) Advanced Intercross Line of the LG/J and SM/J intercross (Wustl:LG,SM-G16). Half of each sibship was fed a high-fat diet and half was fed a relatively low-fat diet. Context-dependent genetic (additive and dominance) and epigenetic (parent-of-origin imprinting) effects were characterized by partitioning animals into sex, diet, and sex-by-diet cohorts. We found that different cohorts often have unique genetic effects at the same loci, and that genetic signals can be masked or erroneously assigned to specific cohorts if they are not considered individually. Our data demonstrate that the effects of genes on complex trait variation are highly context-dependent and that the same genomic sequence can affect traits differently depending on an individual's sex and/or dietary environment. Our results have important implications for studies of complex traits in humans.

Diet-dependent genetic and genomic imprinting effects on obesity in mice.

Although the current obesity epidemic is of environmental origin, there is substantial genetic variation in individual response to an obesogenic environment. In this study, we perform a genome-wide scan for quantitative trait loci (QTLs) affecting obesity per se, or an obese response to a high-fat diet in mice from the LG/J by SM/J Advanced Intercross (AI) Line (Wustl:LG,SM-G16). A total of 1,002 animals from 78 F16 full sibships were weaned at 3 weeks of age and half of each litter placed on high- and low-fat diets. Animals remained on the diet until 20 weeks of age when they were necropsied and the weights of the reproductive, kidney, mesenteric, and inguinal fat depots were recorded. Effects on these phenotypes, along with total fat depot weight and carcass weight at necropsy, were mapped across the genome using 1,402 autosomal single-nucleotide polymorphism (SNP) markers. Haplotypes were reconstructed and additive, dominance, and imprinting genotype scores were derived every 1 cM along the F16 map. Analysis was performed using a mixed model with additive, dominance, and imprinting genotype scores, their interactions with sex, diet, and with sex-by-diet as fixed effects and with family and its interaction with sex, diet, and sex-by-diet as random effects. We discovered 95 trait-specific QTLs mapping to 40 locations. Most QTLs had additive effects with dominance and imprinting effects occurring at two-thirds of the loci. Nearly every locus interacted with sex and/or diet in important ways demonstrating that gene effects are primarily context dependent, changing depending on sex and/or diet.

Deep resequencing reveals excess rare recent variants consistent with explosive population growth.

Accurately determining the distribution of rare variants is an important goal of human genetics, but resequencing of a sample large enough for this purpose has been unfeasible until now. Here, we applied Sanger sequencing of genomic PCR amplicons to resequence the diabetes-associated genes KCNJ11 and HHEX in 13,715 people (10,422 European Americans and 3,293 African Americans) and validated amplicons potentially harbouring rare variants using 454 pyrosequencing. We observed far more variation (expected variant-site count ∼578) than would have been predicted on the basis of earlier surveys, which could only capture the distribution of common variants. By comparison with earlier estimates based on common variants, our model shows a clear genetic signal of accelerating population growth, suggesting that humanity harbours a myriad of rare, deleterious variants, and that disease risk and the burden of disease in contemporary populations may be heavily influenced by the distribution of rare variants.

Genetic, epigenetic, and gene-by-diet interaction effects underlie variation in serum lipids in a LG/JxSM/J murine model.

Variation in serum cholesterol, free-fatty acids, and triglycerides is associated with cardiovascular disease (CVD) risk factors. There is great interest in characterizing the underlying genetic architecture of these risk factors, because they vary greatly within and among human populations and between the sexes. We present results of a genome-wide scan for quantitative trait loci (QTL) affecting serum cholesterol, free-fatty acids, and triglycerides in an F(16) advanced intercross line of LG/J and SM/J (Wustl:LG,SM-G16). Half of the population was fed a high-fat diet and half was fed a relatively low-fat diet. Context-dependent genetic (additive and dominance) and epigenetic (imprinting) effects were characterized by partitioning animals into sex, diet, and sex-by-diet cohorts. Here we examine genetic, environmental, and genetic-by-environmental interactions of QTL overlapping previously identified loci associated with CVD risk factors, and we add to the serum lipid QTL landscape by identifying new loci.

Genome changes after gene duplication: haploidy vs. diploidy.

Since genome size and the number of duplicate genes observed in genomes increase from haploid to diploid organisms, diploidy might provide more evolutionary probabilities through gene duplication. It is still unclear how diploidy promotes genomic evolution in detail. In this study, we explored the evolution of segmental gene duplication in haploid and diploid populations by analytical and simulation approaches. Results show that (1) under the double null recessive (DNR) selective model, given the same recombination rate, the evolutionary trajectories and consequences are very similar between the same-size gene-pool haploid vs. diploid populations; (2) recombination enlarges the probability of preservation of duplicate genes in either haploid or diploid large populations, and haplo-insufficiency reinforces this effect; and (3) the loss of duplicate genes at the ancestor locus is limited under recombination while under complete linkage the loss of duplicate genes is always random at the ancestor and newly duplicated loci. Therefore, we propose a model to explain the advantage of diploidy: diploidy might facilitate the increase of recombination rate, especially under sexual reproduction; more duplicate genes are preserved under more recombination by originalization (by which duplicate genes are preserved intact at a special quasi-mutation-selection balance under the DNR or haplo-insufficient selective model), so genome sizes and the number of duplicate genes in diploid organisms become larger. Additionally, it is suggested that small genomic rearrangements due to the random loss of duplicate genes might be limited under recombination.

Phylogenetics applied to genotype/phenotype association and selection analyses with sequence data from angptl4 in humans.

Genotype/phenotype association analyses (Treescan) with plasma lipid levels and functional site prediction methods (TreeSAAP and PolyPhen) were performed using sequence data for ANGPTL4 from 3,551 patients in the Dallas Heart Study. Biological assays of rare variants in phenotypic tails and results from a Treescan analysis were used as "known" variants to assess the site prediction abilities of PolyPhen and TreeSAAP. The E40K variant in European Americans and the R278Q variant in African Americans were significantly associated with multiple lipid phenotypes. Combining TreeSAAP and PolyPhen performed well to predict "known" functional variants while reducing noise from false positives.

Estimating population genetic parameters and comparing model goodness-of-fit using DNA sequences with error.

It is known that sequencing error can bias estimation of evolutionary or population genetic parameters. This problem is more prominent in deep resequencing studies because of their large sample size n, and a higher probability of error at each nucleotide site. We propose a new method based on the composite likelihood of the observed SNP configurations to infer population mutation rate theta = 4N(e)micro, population exponential growth rate R, and error rate epsilon, simultaneously. Using simulation, we show the combined effects of the parameters, theta, n, epsilon, and R on the accuracy of parameter estimation. We compared our maximum composite likelihood estimator (MCLE) of theta with other theta estimators that take into account the error. The results show the MCLE performs well when the sample size is large or the error rate is high. Using parametric bootstrap, composite likelihood can also be used as a statistic for testing the model goodness-of-fit of the observed DNA sequences. The MCLE method is applied to sequence data on the ANGPTL4 gene in 1832 African American and 1045 European American individuals.

Inferring population mutation rate and sequencing error rate using the SNP frequency spectrum in a sample of DNA sequences.

One challenge of analyzing samples of DNA sequences is to account for the nonnegligible polymorphisms produced by error when the sequencing error rate is high or the sample size is large. Specifically, those artificial sequence variations will bias the observed single nucleotide polymorphism (SNP) frequency spectrum, which in turn may further bias the estimators of the population mutation rate theta =4N mu for diploids. In this paper, we propose a new approach based on the generalized least squares (GLS) method to estimate theta, given a SNP frequency spectrum in a random sample of DNA sequences from a population. With this approach, error rate epsilon can be either known or unknown. In the latter case, epsilon can be estimated given an estimation of theta. Using coalescent simulation, we compared our estimators with other estimators of theta. The results showed that the GLS estimators are more efficient than other theta estimators with error, and the estimation of epsilon is usable in practice when the theta per bp is small. We demonstrate the application of the estimators with 10-kb noncoding region sequence sampled from a human population and provide suggestions for choosing theta estimators with error.

Replication of long-bone length QTL in the F9-F10 LG,SM advanced intercross.

Quantitative trait locus (QTL) mapping techniques are frequently used to identify genomic regions associated with variation in phenotypes of interest. However, the F(2) intercross and congenic strain populations usually employed have limited genetic resolution resulting in relatively large confidence intervals that greatly inhibit functional confirmation of statistical results. Here we use the increased resolution of the combined F(9) and F(10) generations (n = 1455) of the LG,SM advanced intercross to fine-map previously identified QTL associated with the lengths of the humerus, ulna, femur, and tibia. We detected 81 QTL affecting long-bone lengths. Of these, 49 were previously identified in the combined F(2)-F(3) population of this intercross, while 32 represent novel contributors to trait variance. Pleiotropy analysis suggests that most QTL affect three to four long bones or serially homologous limb segments. We also identified 72 epistatic interactions involving 38 QTL and 88 novel regions. This analysis shows that using later generations of an advanced intercross greatly facilitates fine-mapping of confidence intervals, resolving three F(2)-F(3) QTL into multiple linked loci and narrowing confidence intervals of other loci, as well as allowing identification of additional QTL. Further characterization of the biological bases of these QTL will help provide a better understanding of the genetics of small variations in long-bone length.

Quantitative trait loci associated with susceptibility to therapy-related acute murine promyelocytic leukemia in hCG-PML/RARA transgenic mice.

Therapy-related acute myelogenous leukemia (t-AML) is an important late adverse effect of alkylator chemotherapy. Susceptibility to t-AML has a genetic component, yet specific genetic variants that influence susceptibility are poorly understood. We analyzed an F(2) intercross (n = 282 mice) between mouse strains resistant or susceptible to t-AML induced by the alkylator ethyl-N-nitrosourea (ENU) to identify genes that regulate t-AML susceptibility. Each mouse carried the hCG-PML/RARA transgene, a well-characterized initiator of myeloid leukemia. In the absence of ENU treatment, transgenic F(2) mice developed leukemia with higher incidence (79.4% vs 12.5%) and at earlier time points (108 days vs 234 days) than mice in the resistant background. ENU treatment of F(2) mice further increased incidence (90.4%) and shortened median survival (171 vs 254 days). We genotyped F(2) mice at 384 informative single nucleotide polymorphisms across the genome and performed quantitative trait locus (QTL) analysis. Thirteen QTLs significantly associated with leukemia-free survival, spleen weight, or white blood cell count were identified on 8 chromosomes. These results suggest that susceptibility to ENU-induced leukemia in mice is a complex trait governed by genes at multiple loci. Improved understanding of genetic risk factors should lead to tailored treatment regimens that reduce risk for patients predisposed to t-AML.

DAPK1 variants are associated with Alzheimer's disease and allele-specific expression.

Genetic factors play an important role in the etiology of late-onset Alzheimer's disease (LOAD). We tested gene-centric single nucleotide polymorphisms (SNPs) on chromosome 9 and identified two SNPs in the death-associated protein kinase, DAPK1, that show significant association with LOAD. SNP rs4878104 was significantly associated with LOAD in our discovery case-control sample set (WU) and replicated in each of two initial validation case-control sample sets (P<0.05, UK1, SD). The risk-allele frequency of this SNP showed a similar direction in three other case-control sample sets. A meta-analysis of the six sample sets combined, totaling 2012 cases and 2336 controls, showed an allelic P-value of 0.0016 and an odds ratio (OR) of 0.87 (95%CI: 0.79-0.95). Minor allele homozygotes had a consistently lower risk than major allele homozygotes in the discovery and initial two replication sample sets, which remained significant in the meta-analysis of all six sample sets (OR=0.7, 95%CI: 0.58-0.85), whereas the risk for heterozygous subjects was not significantly different from that of major allele homozygotes. A second SNP, rs4877365, which is in high linkage disequilibrium with rs4878104 (r2=0.64), was also significantly associated with LOAD (meta P=0.0017 in the initial three sample sets). Furthermore, DAPK1 transcripts show differential allelic gene expression, and both rs4878104 and rs4877365 were significantly associated with DAPK1 allele-specific expression (P=0.015 to <0.0001). These data suggest that genetic variation in DAPK1 modulates susceptibility to LOAD.