DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Effect of menopausal hormone therapy on methylation levels in early and late postmenopausal women.

BACKGROUND: Cardiovascular disease (CVD) remains the leading cause of death among postmenopausal women but standard primary prevention strategies in women are not as effective as in men. By comparison, the Early versus Late Intervention Trial with Estradiol (ELITE) study demonstrated that hormone therapy (HT) was associated with significant reduction in atherosclerosis progression in women who were within six years of menopause compared to those who were 10 or more years from menopause. These findings are consistent with other studies showing significant reductions in all-cause mortality and CVD with HT, particularly when initiated in women younger than 60 years of age or within 10 years since menopause. To explore the biological mechanisms underlying the age-related atheroprotective effects of HT, we investigated changes in methylation of blood cells of postmenopausal women who participated in ELITE. RESULTS: We first validated the epigenetic data generated from blood leukocytes of ELITE participants by replicating previously known associations between smoking and methylation levels at previously identified CpG sites, such as cg05575921 at the AHRR locus. An epigenome-wide association study (EWAS) evaluating changes in methylation through interactions with time-since-menopause and HT revealed two significantly associated CpG sites on chromosomes 12 (cg19552895; p = 1.1 × 10-9) and 19 (cg18515510; p = 2.4 × 10-8). Specifically, HT resulted in modest, but significant, increases in methylation levels at both CpGs but only in women who were 10 or more years since menopause and randomized to HT. Changes in carotid artery intima-media thickness (CIMT) from baseline to 36 months after HT were not significantly correlated with changes in methylation levels at either cg19552895 or cg18515510. Evaluation of other previously identified CpG sites at which methylation levels in either blood or vascular tissue were associated with atherosclerosis also did not reveal any differences in methylation as a function of HT and time-since-menopause or with changes in CIMT. CONCLUSIONS: We identified specific methylation differences in blood in response to HT among women who were 10 or more years since menopause. The functional consequence of these change with respect to atherosclerosis progression and protective effects of HT remains to be determined and will require additional studies.

Epigenetic clock and methylation studies in vervet monkeys.

DNA methylation-based biomarkers of aging have been developed for many mammals but not yet for the vervet monkey (Chlorocebus sabaeus), which is a valuable non-human primate model for biomedical studies. We generated novel DNA methylation data from vervet cerebral cortex, blood, and liver using highly conserved mammalian CpGs represented on a custom array (HorvathMammalMethylChip40). We present six DNA methylation-based estimators of age: vervet multi-tissue epigenetic clock and tissue-specific clocks for brain cortex, blood, and liver. In addition, we developed two dual species clocks (human-vervet clocks) for measuring chronological age and relative age, respectively. Relative age was defined as ratio of chronological age to maximum lifespan to address the species differences in maximum lifespan. The high accuracy of the human-vervet clocks demonstrates that epigenetic aging processes are evolutionary conserved in primates. When applying these vervet clocks to tissue samples from another primate species, rhesus macaque, we observed high age correlations but strong offsets. We characterized CpGs that correlate significantly with age in the vervet. CpG probes that gain methylation with age across tissues were located near the targets of Polycomb proteins SUZ12 and EED and genes possessing the trimethylated H3K27 mark in their promoters. The epigenetic clocks are expected to be useful for anti-aging studies in vervets.

Genetic variation and gene expression across multiple tissues and developmental stages in a nonhuman primate.

By analyzing multitissue gene expression and genome-wide genetic variation data in samples from a vervet monkey pedigree, we generated a transcriptome resource and produced the first catalog of expression quantitative trait loci (eQTLs) in a nonhuman primate model. This catalog contains more genome-wide significant eQTLs per sample than comparable human resources and identifies sex- and age-related expression patterns. Findings include a master regulatory locus that likely has a role in immune function and a locus regulating hippocampal long noncoding RNAs (lncRNAs), whose expression correlates with hippocampal volume. This resource will facilitate genetic investigation of quantitative traits, including brain and behavioral phenotypes relevant to neuropsychiatric disorders.

Genome-wide burden of deleterious coding variants increased in schizophrenia.

Schizophrenia is a common complex disorder with polygenic inheritance. Here we show that by using an approach that compares the individual loads of rare variants in 1,042 schizophrenia cases and 961 controls, schizophrenia cases carry an increased burden of deleterious mutations. At a genome-wide level, our results implicate non-synonymous, splice site as well as stop-altering single-nucleotide variations occurring at minor allele frequency of ≥ 0.01% in the population. In an independent replication sample of 5,585 schizophrenia cases and 8,103 controls of European ancestry we confirm an enrichment in cases of the alleles identified in our study. In addition, the genes implicated by the increased burden of rare coding variants highlight the involvement of neurodevelopment in the aetiology of schizophrenia.

A non-human primate system for large-scale genetic studies of complex traits.

Non-human primates provide genetic model systems biologically intermediate between humans and other mammalian model organisms. Populations of Caribbean vervet monkeys (Chlorocebus aethiops sabaeus) are genetically homogeneous and large enough to permit well-powered genetic mapping studies of quantitative traits relevant to human health, including expression quantitative trait loci (eQTL). Previous transcriptome-wide investigation in an extended vervet pedigree identified 29 heritable transcripts for which levels of expression in peripheral blood correlate strongly with expression levels in the brain. Quantitative trait linkage analysis using 261 microsatellite markers identified significant (n = 8) and suggestive (n = 4) linkages for 12 of these transcripts, including both cis- and trans-eQTL. Seven transcripts, located on different chromosomes, showed maximum linkage to markers in a single region of vervet chromosome 9; this observation suggests the possibility of a master trans-regulator locus in this region. For one cis-eQTL (at B3GALTL, beta-1,3-glucosyltransferase), we conducted follow-up single nucleotide polymorphism genotyping and fine-scale association analysis in a sample of unrelated Caribbean vervets, localizing this eQTL to a region of <200 kb. These results suggest the value of pedigree and population samples of the Caribbean vervet for linkage and association mapping studies of quantitative traits. The imminent whole genome sequencing of many of these vervet samples will enhance the power of such investigations by providing a comprehensive catalog of genetic variation.

Development and implementation of high-throughput SNP genotyping in barley.

BACKGROUND: High density genetic maps of plants have, nearly without exception, made use of marker datasets containing missing or questionable genotype calls derived from a variety of genic and non-genic or anonymous markers, and been presented as a single linear order of genetic loci for each linkage group. The consequences of missing or erroneous data include falsely separated markers, expansion of cM distances and incorrect marker order. These imperfections are amplified in consensus maps and problematic when fine resolution is critical including comparative genome analyses and map-based cloning. Here we provide a new paradigm, a high-density consensus genetic map of barley based only on complete and error-free datasets and genic markers, represented accurately by graphs and approximately by a best-fit linear order, and supported by a readily available SNP genotyping resource. RESULTS: Approximately 22,000 SNPs were identified from barley ESTs and sequenced amplicons; 4,596 of them were tested for performance in three pilot phase Illumina GoldenGate assays. Data from three barley doubled haploid mapping populations supported the production of an initial consensus map. Over 200 germplasm selections, principally European and US breeding material, were used to estimate minor allele frequency (MAF) for each SNP. We selected 3,072 of these tested SNPs based on technical performance, map location, MAF and biological interest to fill two 1536-SNP "production" assays (BOPA1 and BOPA2), which were made available to the barley genetics community. Data were added using BOPA1 from a fourth mapping population to yield a consensus map containing 2,943 SNP loci in 975 marker bins covering a genetic distance of 1099 cM. CONCLUSION: The unprecedented density of genic markers and marker bins enabled a high resolution comparison of the genomes of barley and rice. Low recombination in pericentric regions is evident from bins containing many more than the average number of markers, meaning that a large number of genes are recombinationally locked into the genetic centromeric regions of several barley chromosomes. Examination of US breeding germplasm illustrated the usefulness of BOPA1 and BOPA2 in that they provide excellent marker density and sensitivity for detection of minor alleles in this genetically narrow material.

Identification of brain transcriptional variation reproduced in peripheral blood: an approach for mapping brain expression traits.

Genome-wide gene expression studies may provide substantial insight into gene activities and biological pathways differing between tissues and individuals. We investigated such gene expression variation by analyzing expression profiles in brain tissues derived from eight different brain regions and from blood in 12 monkeys from a biomedically important non-human primate model, the vervet (Chlorocebus aethiops sabaeus). We characterized brain regional differences in gene expression, focusing on transcripts for which inter-individual variation of expression in brain correlates well with variation in blood from the same individuals. Using stringent criteria, we identified 29 transcripts whose expression is measurable, stable, replicable, variable between individuals, relevant to brain function and heritable. Polymorphisms identified in probe regions could, in a minority of transcripts, confound the interpretation of the observed inter-individual variation. The high heritability of levels of these transcripts in a large vervet pedigree validated our approach of focusing on transcripts that showed higher inter-individual compared with intra-individual variation. These selected transcripts are candidate expression Quantitative Trait Loci, differentially regulating transcript levels in the brain among individuals. Given the high degree of conservation of tissue expression profiles between vervets and humans, our findings may facilitate the understanding of regional and individual transcriptional variation and its genetic mechanisms in humans. The approach employed here-utilizing higher quality tissue and more precise dissection of brain regions than is usually possible in humans-may therefore provide a powerful means to investigate variation in gene expression relevant to complex brain related traits, including human neuropsychiatric diseases.

Weighted gene co-expression network analysis of the peripheral blood from Amyotrophic Lateral Sclerosis patients.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is a lethal disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Diagnosis is mainly based on clinical symptoms, and there is currently no therapy to stop the disease or slow its progression. Since access to spinal cord tissue is not possible at disease onset, we investigated changes in gene expression profiles in whole blood of ALS patients. RESULTS: Our transcriptional study showed dramatic changes in blood of ALS patients; 2,300 probes (9.4%) showed significant differential expression in a discovery dataset consisting of 30 ALS patients and 30 healthy controls. Weighted gene co-expression network analysis (WGCNA) was used to find disease-related networks (modules) and disease related hub genes. Two large co-expression modules were found to be associated with ALS. Our findings were replicated in a second (30 patients and 30 controls) and third dataset (63 patients and 63 controls), thereby demonstrating a highly significant and consistent association of two large co-expression modules with ALS disease status. Ingenuity Pathway Analysis of the ALS related module genes implicates enrichment of functional categories related to genetic disorders, neurodegeneration of the nervous system and inflammatory disease. The ALS related modules contain a number of candidate genes possibly involved in pathogenesis of ALS. CONCLUSION: This first large-scale blood gene expression study in ALS observed distinct patterns between cases and controls which may provide opportunities for biomarker development as well as new insights into the molecular mechanisms of the disease.

Polymorphisms in the GRIA1 gene region in psychotic bipolar disorder.

We reported previously a significant linkage signal between psychotic bipolar disorder (BP) and microsatellite markers on chromosome 5q31-34 in the National Institute of Mental Health Bipolar Genetics Initiative (NIMH-BPGI) data set, Wave 1. In an attempt to fine-map this linkage signal we genotyped 1,134 single nucleotide polymorphisms (SNPs) under the linkage peak in 23 informative families (131 individuals) with evidence of linkage. We tested family based association in the presence of linkage with the computer software package FBAT. The most significant association in these families was with a SNP in the second intron of GRIA1 (alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) subunit 1 receptor gene) (rs490922, Z-score = 3.3, P = 0.001). The analysis of 37 additional families with psychotic BP from NIMH-BPGI data sets, Waves 2, 3, and 4 revealed a signal at a SNP in intron 5 of the GRIA1 gene (rs4385264, Z-score = 3.2, P-value = 0.002). A combined analysis of all 60 families continued to support evidence for association of GRIA1 with psychotic BP; however, individual SNPs could not be replicated across datasets. The AMPA1 receptor has been shown to influence cognitive function, such as working memory and reward learning. Our findings suggest that variations in this receptor may contribute to the pathophysiology of BP with psychotic features in some families.

Results of a SNP genome screen in a large Costa Rican pedigree segregating for severe bipolar disorder.

We have ascertained in the Central Valley of Costa Rica a new kindred (CR201) segregating for severe bipolar disorder (BP-I). The family was identified by tracing genealogical connections among eight persons initially independently ascertained for a genome wide association study of BP-I. For the genome screen in CR201, we trimmed the family down to 168 persons (82 of whom are genotyped), containing 25 individuals with a best-estimate diagnosis of BP-I. A total of 4,690 SNP markers were genotyped. Analysis of the data was hampered by the size and complexity of the pedigree, which prohibited using exact multipoint methods on the entire kindred. Two-point parametric linkage analysis, using a conservative model of transmission, produced a maximum LOD score of 2.78 on chromosome 6, and a total of 39 loci with LOD scores >1.0. Multipoint parametric and non-parametric linkage analysis was performed separately on four sections of CR201, and interesting (nominal P-value from either analysis <0.01), although not statistically significant, regions were highlighted on chromosomes 1, 2, 3, 12, 16, 19, and 22, in at least one section of the pedigree, or when considering all sections together. The difficulties of analyzing genome wide SNP data for complex disorders in large, potentially informative, kindreds are discussed.

Magnitude and distribution of linkage disequilibrium in population isolates and implications for genome-wide association studies.

The genome-wide distribution of linkage disequilibrium (LD) determines the strategy for selecting markers for association studies, but it varies between populations. We assayed LD in large samples (200 individuals) from each of 11 well-described population isolates and an outbred European-derived sample, using SNP markers spaced across chromosome 22. Most isolates show substantially higher levels of LD than the outbred sample and many fewer regions of very low LD (termed 'holes'). Young isolates known to have had relatively few founders show particularly extensive LD with very few holes; these populations offer substantial advantages for genome-wide association mapping.

Characterization of mutations in ATP8B1 associated with hereditary cholestasis.

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are clinically distinct hereditary disorders. PFIC patients suffer from chronic cholestasis and develop liver fibrosis. BRIC patients experience intermittent attacks of cholestasis that resolve spontaneously. Mutations in ATP8B1 (previously FIC1) may result in PFIC or BRIC. We report the genomic organization of ATP8B1 and mutation analyses of 180 families with PFIC or BRIC that identified 54 distinct disease mutations, including 10 mutations predicted to disrupt splicing, 6 nonsense mutations, 11 small insertion or deletion mutations predicted to induce frameshifts, 1 large genomic deletion, 2 small inframe deletions, and 24 missense mutations. Most mutations are rare, occurring in 1-3 families, or are limited to specific populations. Many patients are compound heterozygous for 2 mutations. Mutation type or location correlates overall with clinical severity: missense mutations are more common in BRIC (58% vs. 38% in PFIC), while nonsense, frameshifting, and large deletion mutations are more common in PFIC (41% vs. 16% in BRIC). Some mutations, however, lead to a wide range of phenotypes, from PFIC to BRIC or even no clinical disease. ATP8B1 mutations were detected in 30% and 41%, respectively, of the PFIC and BRIC patients screened.

Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene.

OBJECTIVES: There is increasing evidence that polymorphism of the ABCB1 (MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates. The aim of the present study was to (1) identify and describe novel variants in the ABCB1 gene, (2) understand the extent of variation in ABCB1 at the population level, (3) analyze how variation in ABCB1 is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein. METHODS AND RESULTS: Forty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples. These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed. The two most common haplotypes, ABCB1*1 and ABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes. Significant population substructure was detected at both the nucleotide and haplotype level. Linkage disequilibrium was significant across the entire ABCB1 gene, especially between the variant sites found in ABCB1*13, and recombination was inferred. The Ala893Ser change found in the common ABCB1*13 haplotype did not affect P-glycoprotein function. CONCLUSION: This study represents a comprehensive analysis of ABCB1 nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences of ABCB1 polymorphisms.

Natural variation in human membrane transporter genes reveals evolutionary and functional constraints.

Membrane transporters maintain cellular and organismal homeostasis by importing nutrients and exporting toxic compounds. Transporters also play a crucial role in drug response, serving as drug targets and setting drug levels. As part of a pharmacogenetics project, we screened exons and flanking intronic regions for variation in a set of 24 membrane transporter genes (96 kb; 57% coding) in 247 DNA samples from ethnically diverse populations. We identified 680 single nucleotide polymorphisms (SNPs), of which 175 were synonymous and 155 caused amino acid changes, and 29 small insertions and deletions. Amino acid diversity (pi(NS)) in transmembrane domains (TMDs) was significantly lower than in loop domains, suggesting that TMDs have special functional constraints. This difference was especially striking in the ATP-binding cassette superfamily and did not parallel evolutionary conservation: there was little variation in the TMDs, even in evolutionarily unconserved residues. We used allele frequency distribution to evaluate different scoring systems (Grantham, blosum62, SIFT, and evolutionarily conservedevolutionarily unconserved) for their ability to predict which SNPs affect function. Our underlying assumption was that alleles that are functionally deleterious will be selected against and thus under represented at high frequencies and over represented at low frequencies. We found that evolutionary conservation of orthologous sequences, as assessed by evolutionarily conservedevolutionarily unconserved and SIFT, was the best predictor of allele frequency distribution and hence of function. European Americans had an excess of high frequency alleles in comparison to African Americans, consistent with a historic bottleneck. In addition, African Americans exhibited a much higher frequency of population specific medium-frequency alleles than did European Americans.

Evolutionary conservation predicts function of variants of the human organic cation transporter, OCT1.

The organic cation transporter, OCT1, is a major hepatic transporter that mediates the uptake of many organic cations from the blood into the liver where the compounds may be metabolized or secreted into the bile. Because OCT1 interacts with a variety of structurally diverse organic cations, including clinically used drugs as well as toxic substances (e.g., N-methylpyridinium, MPP(+)), it is an important determinant of systemic exposure to many xenobiotics. To understand the genetic basis of extensive interindividual differences in xenobiotic disposition, we functionally characterized 15 protein-altering variants of the human liver organic cation transporter, OCT1, in Xenopus oocytes. All variants that reduced or eliminated function (OCT1-R61C, OCT1-P341L, OCT1-G220V, OCT1-G401S, and OCT1-G465R) altered evolutionarily conserved amino acid residues. In general, variants with decreased function had amino acid substitutions that resulted in more radical chemical changes (higher Grantham values) and were less evolutionarily favorable (lower blosum62 values) than variants that maintained function. A variant with increased function (OCT1-S14F) changed an amino acid residue such that the human protein matched the consensus of the OCT1 mammalian orthologs. Our results indicate that changes at evolutionarily conserved positions of OCT1 are strong predictors of decreased function and suggest that a combination of evolutionary conservation and chemical change might be a stronger predictor of function.

SNP analysis and presentation in the Pharmacogenetics of Membrane Transporters Project.

The multidisciplinary UCSF Pharmacogenetics of Membrane Transporters project seeks to systematically identify sequence variants in transporters and to determine the functional significance of these variants through evaluation of relevant cellular and clinical phenotypes. The project is structured around four interacting cores: genomics, cellular phenotyping, clinical phenotyping, and bioinformatics. The bioinformatics core is responsible for collecting, storing, and analyzing the information obtained by the other cores and for presenting the results, in particular, for the genomic data. Most of this process is automated using locally developed software written in Python, an open source language well suited for rapid, modular development that meets requirements that are themselves constantly evolving. Here we present the details of transforming ABI trace file data into useful information for project investigators and a description of the types of data analysis and display that we have developed.

Polymorphisms in a human kidney xenobiotic transporter, OCT2, exhibit altered function.

The completion of the Human Genome Project and the development of high-throughput polymorphism identification methods have allowed researchers to carry out full genetic analyses of many clinically relevant genes. However, few studies have combined genetic analysis with in vitro phenotyping to better understand the relationship between genetic variation and protein function. Many transporters in the kidney are thought to play key roles in defense against a variety of foreign substances. The goal of this study was to understand the relationship between variation in a gene encoding a major renal xenobiotic transporter, OCT2, and transporter function. We report a comprehensive genetic analysis and functional characterization of variants of OCT2. Twenty-eight variable sites in the OCT2 gene were identified in a collection of 247 ethnically diverse DNA samples. Eight caused non-synonymous amino acid changes, of which four were present at >/= 1% in an ethnic population. All four of these altered transporter function assayed in Xenopus laevis oocytes. Analysis of nucleotide diversity (pi) revealed a higher prevalence of synonymous (pi = 22.4 x 10-4) versus non-synonymous (pi = 2.1 x 10-4) changes in OCT2 than in other genes. In addition, the non-synonymous sites had a significant tendency to exhibit more skewed allele frequencies (more negative Tajima's D-values) compared to synonymous sites. The population-genetic analysis, together with the functional characterization, suggests that selection has acted against amino acid changes in OCT2. This selection may be due to a necessary role of OCT2 in the renal elimination of endogenous amines or xenobiotics, including environmental toxins, neurotoxic amines and therapeutic drugs.