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The epigenome of stem cells occupies a critical interface between genes and environment, serving to regulate expression through modification by intrinsic and extrinsic factors. We hypothesized that aging and obesity, which represent major risk factors for a variety of diseases, synergistically modify the epigenome of adult adipose stem cells (ASCs). Using integrated RNA- and targeted bisulfite-sequencing in murine ASCs from lean and obese mice at 5- and 12- months of age, we identified global DNA hypomethylation with either aging or obesity, and a synergistic effect of aging combined with obesity. The transcriptome of ASCs in lean mice was relatively stable to the effects of age, but this was not true in obese mice. Functional pathway analyses identified a subset of genes with critical roles in progenitors and in diseases of obesity and aging. Specifically, Mapt, Nr3c2, App, and Ctnnb1 emerged as potential hypomethylated upstream regulators in both aging and obesity (AL vs YL and AO vs YO), and App, Ctnnb1, Hipk2, Id2, and Tp53 exhibited additional effects of aging in obese animals. Further, Foxo3 and Ccnd1 were potential hypermethylated upstream regulators of healthy aging (AL vs YL), and of the effects of obesity in young animals (YO vs YL), suggesting that these factors could play a role in accelerated aging with obesity. Finally, we identified candidate driver genes that appeared recurrently in all analyses and comparisons undertaken. Further mechanistic studies are needed to validate the roles of these genes capable of priming ASCs for dysfunction in aging- and obesity-associated pathologies.

During pandemics, individuals exhibit differences in risk and clinical outcomes. Here, we developed single-cell high-throughput human in vitro susceptibility testing (scHi-HOST), a method for rapidly identifying genetic variants that confer resistance and susceptibility. We applied this method to influenza A virus (IAV), the cause of four pandemics since the start of the 20th century. scHi-HOST leverages single-cell RNA sequencing (scRNA-seq) to simultaneously assign genetic identity to cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral burden, and identify expression quantitative trait loci. Integration of scHi-HOST with human challenge and experimental validation demonstrated that a missense variant in endoplasmic reticulum aminopeptidase 1 (ERAP1; rs27895) increased IAV burden in cells and human volunteers. rs27895 exhibits population differentiation, likely contributing to greater permissivity of cells from African populations to IAV. scHi-HOST is a broadly applicable method and resource for decoding infectious-disease genetics.

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.

Interleukin 7 receptor α-chain is crucial for the development and maintenance of T cells and is genetically associated with autoimmune disorders including multiple sclerosis (MS), a demyelinating disease of the CNS. Exon 6 of IL7R encodes for the transmembrane domain of the receptor and is regulated by alternative splicing: inclusion or skipping of IL7R exon 6 results in membrane-bound or soluble IL7R isoforms, respectively. We previously identified a SNP (rs6897932) in IL7R exon 6, strongly associated with MS risk and showed that the risk allele (C) increases skipping of the exon, resulting in elevated levels of sIL7R. This has important pathological consequences as elevated levels of sIL7R has been shown to exacerbate the disease in the experimental autoimmune encephalomyelitis mouse model of MS. Understanding the regulation of exon 6 splicing provides important mechanistic insights into the pathogenesis of MS. Here we report two mechanisms by which IL7R exon 6 is controlled. First, a competition between PTBP1 and U2AF2 at the polypyrimidine tract (PPT) of intron 5, and second, an unexpected U2AF2-mediated assembly of spicing factors in the exon. We noted the presence of a branchpoint sequence (BPS) (TACTAAT or TACTAAC) within exon 6, which is stronger with the C allele. We also noted that the BPS is followed by a PPT and conjectured that silencing could be mediated by the binding of U2AF2 to that tract. In support of this model, we show that evolutionary conservation of the exonic PPT correlates well with the degree of alternative splicing of exon 6 in two non-human primate species and that U2AF2 binding to this PPT recruits U2 snRNP components to the exon. These observations provide the first explanation for the stronger silencing of IL7R exon 6 with the disease associated C allele at rs6897932.

The genomic regulatory networks underlying the pathogenesis of non-ST-segment elevation acute coronary syndrome (NSTE-ACS) are incompletely understood. As intermediate traits, protein biomarkers report on underlying disease severity and prognosis in NSTE-ACS. We hypothesized that integration of dense microRNA (miRNA) profiling with biomarker measurements would highlight potential regulatory pathways that underlie the relationships between prognostic biomarkers, miRNAs, and cardiovascular phenotypes. We performed miRNA sequencing using whole blood from 186 patients from the TRILOGY-ACS trial. Seven circulating prognostic biomarkers were measured: NH2-terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein, osteopontin (OPN), myeloperoxidase, growth differentiation factor 15, monocyte chemoattractant protein, and neopterin. We tested miRNAs for association with each biomarker with generalized linear models and controlled the false discovery rate at 0.05. Ten miRNAs, including known cardiac-related miRNAs 25-3p and 423-3p, were associated with NT-proBNP levels (min. P = 7.5 × 10−4) and 48 miRNAs, including cardiac-related miRNAs 378a-3p, 20b-5p and 320a, -b, and -d, were associated with OPN levels (min. P = 1.6 × 10−6). NT-proBNP and OPN were also associated with time to cardiovascular death, myocardial infarction (MI), or stroke in the sample. By integrating large-scale miRNA profiling with circulating biomarkers as intermediate traits, we identified associations of known cardiac-related and novel miRNAs with two prognostic biomarkers and identified potential genomic networks regulating these biomarkers. These results, highlighting plausible biological pathways connecting miRNAs with biomarkers and outcomes, may inform future studies seeking to delineate genomic pathways underlying NSTE-ACS outcomes.

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.

Background The neuropeptide oxytocin regulates mammalian social behavior. Disruptions in oxytocin signaling are a feature of many psychopathologies. One commonly studied biomarker for oxytocin involvement in psychiatric diseases is DNA methylation at the oxytocin receptor gene (OXTR). Such studies focus on DNA methylation in two regions of OXTR, exon 3 and a region termed MT2 which overlaps exon 1 and intron 1. However, the relative contribution of exon 3 and MT2 in regulating OXTR gene expression in the brain is currently unknown. Results Here, we use the prairie vole as a translational animal model to investigate genetic, epigenetic, and environmental factors affecting Oxtr gene expression in a region of the brain that has been shown to drive Oxtr related behavior in the vole, the nucleus accumbens. We show that the genetic structure of Oxtr in prairie voles resembles human OXTR. We then studied the effects of early life experience on DNA methylation in two regions of a CpG island surrounding the Oxtr promoter: MT2 and exon 3. We show that early nurture in the form of parental care results in DNA hypomethylation of Oxtr in both MT2 and exon 3, but only DNA methylation in MT2 is associated with Oxtr gene expression. Network analyses indicate that CpG sites in the 3′ portion of MT2 are most highly associated with Oxtr gene expression. We also identify two novel SNPs in exon 3 of Oxtr in prairie voles and a novel alternative transcript originating from the third intron of the gene. Expression of the novel alternative transcript is associated with genotype at SNP KLW2. Conclusions These results identify putative regulatory features of Oxtr in prairie voles which inform future studies examining OXTR in human social behaviors and disorders. These studies indicate that in prairie voles, DNA methylation in MT2, particularly in the 3′ portion, is more predictive of Oxtr gene expression than DNA methylation in exon 3. Similarly, in human temporal cortex, we find that DNA methylation in the 3′ portion of MT2 is associated with OXTR expression. Together, these results suggest that among the CpG sites studied, DNA methylation of MT2 may be the most reliable indicator of OXTR gene expression. We also identify novel features of prairie vole Oxtr, including SNPs and an alternative transcript, which further develop the prairie vole as a translational model for studies of OXTR.

Lymphoblastoid cell lines (LCLs) are generated by transforming primary B cells with Epstein–Barr virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing (scRNA-seq) revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, activation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host–pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

Lymphoblastoid Cell Lines (LCLs) are generated by transforming primary B cells with Epstein-Barr Virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, proliferation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

Background DNA methylation is implicated in many chronic diseases and may contribute to mortality. Therefore, we conducted an epigenome‐wide association study (EWAS) for all‐cause mortality with whole‐transcriptome data in a cardiovascular cohort (CATHGEN [Catheterization Genetics]). Methods and Results Cases were participants with mortality≥7 days postcatheterization whereas controls were alive with≥2 years of follow‐up. The Illumina Human Methylation 450K and EPIC arrays (Illumina, San Diego, CA) were used for the discovery and validation sets, respectively. A linear model approach with empirical Bayes estimators adjusted for confounders was used to assess difference in methylation (Δβ). In the discovery set (55 cases, 49 controls), 25 629 (6.5%) probes were differently methylated (P0.05); the SLC4A9 transcript did not pass quality control. The cg17944110 probe is located within a potential regulatory element; expression of predicted targets (using GeneHancer) of the regulatory element, UBIAD1 (P=0.01) and CLSTN1 (P=0.03), were lower in cases. Conclusions We identified 6 novel methylation sites associated with all‐cause mortality. Methylation in CASZ1 may serve as a regulatory element associated with mortality in cardiovascular patients. Larger studies are necessary to confirm these observations.

The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (P, Fuchs endothelial corneal dystrophy (FECD) is one of the most common reasons for corneal transplantation, and is known to cluster in families. Here, the authors discover new genetic loci associated with FECD with sex-specific effects and implications for disease mechanism.

Circulating tumor cell (CTC) and cell-free (cf) DNA-based genomic alterations are increasingly being used for clinical decision-making in oncology. However, the concordance and discordance between paired CTC and cfDNA genomic profiles remain largely unknown. We performed comparative genomic hybridization (CGH) on CTCs and cfDNA, and low-pass whole genome sequencing (lpWGS) on cfDNA to characterize genomic alterations (CNA) and tumor content in two independent prospective studies of 93 men with mCRPC treated with enzalutamide/abiraterone, or radium-223. Comprehensive analysis of 69 patient CTCs and 72 cfDNA samples from 93 men with mCRPC, including 64 paired samples, identified common concordant gains in FOXA1, AR, and MYC, and losses in BRCA1, PTEN, and RB1 between CTCs and cfDNA. Concordant PTEN loss and discordant BRCA2 gain were associated with significantly worse outcomes in Epic AR-V7 negative men with mCRPC treated with abiraterone/enzalutamide. We identified and externally validated CTC-specific genomic alternations that were discordant in paired cfDNA, even in samples with high tumor content. These CTC/cfDNA-discordant regions included key genomic regulators of lineage plasticity, osteomimicry, and cellular differentiation, including MYCN gain in CTCs (31%) that was rarely detected in cfDNA. CTC MYCN gain was associated with poor clinical outcomes in AR-V7 negative men and small cell transformation. In conclusion, we demonstrated concordance of multiple genomic alterations across CTC and cfDNA platforms; however, some genomic alterations displayed substantial discordance between CTC DNA and cfDNA despite the use of identical copy number analysis methods, suggesting tumor heterogeneity and divergent evolution associated with poor clinical outcomes.

Centromeres are defined by a specialized chromatin organization that includes nucleosomes that contain the centromeric histone variant centromere protein A (CENP-A) instead of canonical histone H3. Studies in various organisms have shown that centromeric chromatin (i.e., CENP-A chromatin or centrochromatin) exhibits plasticity, in that it can assemble on different types of DNA sequences. However, once established on a chromosome, the centromere is maintained at the same position. In humans, this location is the highly homogeneous repetitive DNA alpha satellite. Mislocalization of centromeric chromatin to atypical locations can lead to genome instability, indicating that restriction of centromeres to a distinct genomic position is important for cell and organism viability. Here, we describe a rearrangement of Homo sapiens chromosome 17 (HSA17) that has placed alpha satellite DNA next to euchromatin. We show that on this mutant chromosome, CENP-A chromatin has spread from the alpha satellite into the short arm of HSA17, establishing a ∼700 kb hybrid centromeric domain that spans both repetitive and unique sequences and changes the expression of at least one gene over which it spreads. Our results illustrate the plasticity of human centromeric chromatin and suggest that heterochromatin normally constrains CENP-A chromatin onto alpha satellite DNA. This work highlights that chromosome rearrangements, particularly those that remove the pericentromere, create opportunities for centromeric nucleosomes to move into non-traditional genomic locations, potentially changing the surrounding chromatin environment and altering gene expression.

Summary Multiple sclerosis (MS) is an autoimmune disorder where T cells attack neurons in the central nervous system (CNS) leading to demyelination and neurological deficits. A driver of increased MS risk is the soluble form of the interleukin-7 receptor alpha chain gene (sIL7R) produced by alternative splicing of IL7R exon 6. Here, we identified the RNA helicase DDX39B as a potent activator of this exon and consequently a repressor of sIL7R, and we found strong genetic association of DDX39B with MS risk. Indeed, we showed that a genetic variant in the 5′ UTR of DDX39B reduces translation of DDX39B mRNAs and increases MS risk. Importantly, this DDX39B variant showed strong genetic and functional epistasis with allelic variants in IL7R exon 6. This study establishes the occurrence of biological epistasis in humans and provides mechanistic insight into the regulation of IL7R exon 6 splicing and its impact on MS risk.

Air pollution is a worldwide contributor to cardiovascular disease mortality and morbidity. Traffic-related air pollution is a widespread environmental exposure and is associated with multiple cardiovascular outcomes such as coronary atherosclerosis, peripheral arterial disease, and myocardial infarction. Despite the recognition of the importance of both genetic and environmental exposures to the pathogenesis of cardiovascular disease, studies of how these two contributors operate jointly are rare. We performed a genome-wide interaction study (GWIS) to examine gene-traffic exposure interactions associated with coronary atherosclerosis. Using race-stratified cohorts of 538 African-Americans (AA) and 1562 European-Americans (EA) from a cardiac catheterization cohort (CATHGEN), we identify gene-by-traffic exposure interactions associated with the number of significantly diseased coronary vessels as a measure of chronic atherosclerosis. We found five suggestive (P

Background Neural tube defects (NTD) have a strong genetic component, with up to 70% of variance in human prevalence determined by heritable factors. Although the identification of causal DNA variants by sequencing candidate genes from functionally relevant pathways and model organisms has provided some success, alternative approaches are demanded.

DNA methylation age (DNAm age) has become a widely utilized epigenetic biomarker for the aging process. The Horvath method for determining DNAm age is perhaps the most widely utilized and validated DNA methylation age assessment measure. Horvath DNAm age is calculated based on methylation measurements at 353 loci, present on Illumina's 450k and 27k DNA methylation microarrays. With increasing use of the more recently developed Illumina MethylationEPIC (850k) microarray, it is worth revisiting this aging measure to evaluate estimation differences due to array design. Of the requisite 353 loci, 17 are missing from the 850k microarray. Similarly, an alternate, 71 loci DNA methylation age assessment measure created by Hannum et al. is missing 6 requisite loci. Using 17 datasets with 27k, 450k, and/or 850k methylation data, we compared each sample's epigenetic age estimated from all 353 loci required by the Horvath DNAm age calculator, and using only the 336 loci available on the 850k array. In 450k/27k data, removing loci not on the 850k array resulted in underestimation of Horvath's DNAm age. Underestimation of Horvath DNAm age increased from ages 0 to ~20, remaining stable thereafter (mean deviation = -3.46 y, SD = 1.13 for individuals ≥20 years). Underestimation of Horvath's DNAm age by the reduced 450k/27k data was similar to the underestimation observed in the 850k data indicating it is driven by missing probes. In analogous examination of Hannum's DNAm age, the magnitude and direction of epigenetic age misestimation varied with chronological age. In conclusion, inter-array deviations in DNAm age estimations may be largely driven by missing probes between arrays, despite default probe imputation procedures. Though correlations and associations based on Horvath's DNAm age may be unaffected, researchers should exercise caution when interpreting results based on absolute differences in DNAm age or when mixing samples assayed on different arrays.

Neural tube defects (NTDs) are caused by improper neural tube closure during the early stages of embryonic development. NTDs are hypothesized to have a complex genetic origin and numerous candidate genes have been proposed. The nitric oxide synthase 3 (NOS3) G594T polymorphism has been implicated in risk for spina bifida, and interactions between that single nucleotide polymorphism (SNP) and the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism have also been observed. To evaluate other genetic variation in the NO pathway in the development of NTDs, we examined all three NOS genes: NOS1, NOS2, and NOS3. Using 3109 Caucasian samples in 745 families, we evaluated association in the overall dataset and within specific phenotypic subsets. Haplotype tagging SNPs in the NOS genes were tested for genetic association with NTD subtypes, both for main effects as well as for the presence of interactions with the MTHFR C677T polymorphism. Nominal main effect associations were found with all subtypes, across all three NOS genes, and interactions were observed between SNPs in all three NOS genes and MTHFR C677T. Unlike the previous report, the most significant associations in our dataset were with cranial subtypes and the AG genotype of rs4795067 in NOS2 (p = 0.0014) and the interaction between the rs9658490 G allele in NOS1 and MTHFR 677TT genotype (p = 0.0014). Our data extend the previous findings by implicating a role for all three NOS genes, independently and through interactions with MTHFR, in risk not only for spina bifida, but all NTD subtypes.

We performed a gene–smoking interaction analysis using families from an early-onset coronary artery disease cohort (GENECARD). This analysis was focused on validating and expanding results from previous studies implicating single nucleotide polymorphisms (SNPs) on chromosome 3 in smoking-mediated coronary artery disease. We analyzed 430 SNPs on chromosome 3 and identified 16 SNPs that showed a gene–smoking interaction at P < 0.05 using association in the presence of linkage—ordered subset analysis, a method that uses permutations of the data to empirically estimate the strength of the association signal. Seven of the 16 SNPs were in the Rho-GTPase pathway indicating a 1.87-fold enrichment for this pathway. A meta-analysis of gene–smoking interactions in three independent studies revealed that rs9289231 in KALRN had a Fisher’s combined P value of 0.0017 for the interaction with smoking. In a gene-based meta-analysis KALRN had a P value of 0.026. Finally, a pathway-based analysis of the association results using WebGestalt revealed several enriched pathways including the regulation of the actin cytoskeleton pathway as defined by the Kyoto Encyclopedia of Genes and Genomes.

Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P Only two genome-wide significant loci associated with longevity have been identified so far, probably because of insufficient sample sizes of centenarians, whose genomes may harbor genetic variants associated with health and longevity. Here we report a genome-wide association study (GWAS) of Han Chinese with a sample size 2.7 times the largest previously published GWAS on centenarians. We identified 11 independent loci associated with longevity replicated in Southern-Northern regions of China, including two novel loci (rs2069837-IL6; rs2440012-ANKRD20A9P) with genome-wide significance and the rest with suggestive significance (P

There is a growing literature indicating that genetic variants modify many of the associations between environmental exposures and clinical outcomes, potentially by increasing susceptibility to these exposures. However, genome-scale investigations of these interactions have been rarely performed particularly in the case of air pollution exposures. We performed race-stratified genome-wide gene-environment interaction association studies on European-American (EA, N = 1623) and African-American (AA, N = 554) cohorts to investigate the joint influence of common single nucleotide polymorphisms (SNPs) and residential exposure to traffic ("traffic exposure")-a recognized vascular disease risk factor-on peripheral arterial disease (BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10(-6)) and rs1180341 (tibial artery, eQTL P = 5.3x10(-6)). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.PAD). Traffic exposure was estimated via the distance from the primary residence to the nearest major roadway, defined as the nearest limited access highways or major arterial. The rs755249-traffic exposure interaction was associated with PAD at a genome-wide significant level (P = 2.29x10(-8)) in European-Americans. Rs755249 is located in the 3' untranslated region of BMP8A, a member of the bone morphogenic protein (BMP) gene family. Further investigation revealed several variants in BMP genes associated with PAD via an interaction with traffic exposure in both the EA and AA cohorts; this included interactions with non-synonymous variants in BMP2, which is regulated by air pollution exposure. The BMP family of genes is linked to vascular growth and calcification and is a novel gene family for the study of PAD pathophysiology. Further investigation of BMP8A using the Genotype Tissue Expression Database revealed multiple variants with nominally significant (P < 0.05) interaction P-values in our EA cohort were significant BMP8A eQTLs in tissue types highlight relevant for PAD such as rs755249 (tibial nerve, eQTL P = 3.6x10(-6)) and rs1180341 (tibial artery, eQTL P = 5.3x10(-6)). Together these results reveal a novel gene, and possibly gene family, associated with PAD via an interaction with traffic air pollution exposure. These results also highlight the potential for interactions studies, particularly at the genome scale, to reveal novel biology linking environmental exposures to clinical outcomes.

On the basis of the genotypic/phenotypic data from Chinese Longitudinal Healthy Longevity Survey (CLHLS) and Cox proportional hazard model, the present study demonstrates that interactions between carrying FOXO1A-209 genotypes and tea drinking are significantly associated with lower risk of mortality at advanced ages. Such a significant association is replicated in two independent Han Chinese CLHLS cohorts (p = 0.028-0.048 in the discovery and replication cohorts, and p = 0.003-0.016 in the combined dataset). We found the associations between tea drinking and reduced mortality are much stronger among carriers of the FOXO1A-209 genotype compared to non-carriers, and drinking tea is associated with a reversal of the negative effects of carrying FOXO1A-209 minor alleles, that is, from a substantially increased mortality risk to substantially reduced mortality risk at advanced ages. The impacts are considerably stronger among those who carry two copies of the FOXO1A minor allele than those who carry one copy. On the basis of previously reported experiments on human cell models concerning FOXO1A-by-tea-compounds interactions, we speculate that results in the present study indicate that tea drinking may inhibit FOXO1A-209 gene expression and its biological functions, which reduces the negative impacts of FOXO1A-209 gene on longevity (as reported in the literature) and offers protection against mortality risk at oldest-old ages. Our empirical findings imply that the health outcomes of particular nutritional interventions, including tea drinking, may, in part, depend upon individual genetic profiles, and the research on the effects of nutrigenomics interactions could potentially be useful for rejuvenation therapies in the clinic or associated healthy aging intervention programs.

Received: June 10, 2010 , Accepted: July 29, 2010 , Published: August 8, 2010 // The glioblastoma genome displays remarkable chromosomal aberrations, which harbor critical glioblastoma-specific genes contributing to several oncogenetic pathways. To identify glioblastoma-targeted genes, we completed a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in glioblastomas. We performed copy number analysis of 111 glioblastomas by Digital Karyotyping and Illumina BeadChip assays and validated our findings using data from the TCGA (The Cancer Genome Atlas) glioblastoma project. From this study, we identified recurrent focal copy number alterations in 1p36.23 and 4p16.3. Expression analyses of genes located in the two regions revealed genes which are dysregulated in glioblastomas. Specifically, we identify EGFR negative regulator, ERRFI1, within the minimal region of deletion in 1p36.23. In glioblastoma cells with a focal deletion of the ERRFI1 locus, restoration of ERRFI1 expression slowed cell migration. Furthermore, we demonstrate that TACC3, an Aurora-A kinase substrate, on 4p16.3, displays gain of copy number, is overexpressed in a glioma-grade-specific pattern, and correlates with Aurora kinase overexpression in glioblastomas. Our multifaceted genomic evaluation of glioblastoma establishes ERRFI1 as a potential candidate tumor suppressor gene and TACC3 as a potential oncogene, and provides insight on targets for oncogenic pathway-based therapy.

Alternative splicing is a general mechanism for regulating gene expression that affects the RNA products of more than 90% of human genes. Not surprisingly, alternative splicing is observed among gene products of metazoan immune systems, which have evolved to efficiently recognize pathogens and discriminate between “self” and “non-self”, and thus need to be both diverse and flexible. In this review we focus on the specific interface between alternative splicing and autoimmune diseases, which result from a malfunctioning of the immune system and are characterized by the inappropriate reaction to self-antigens. Despite the widespread recognition of alternative splicing as one of the major regulators of gene expression, the connections between alternative splicing and autoimmunity have not been apparent. We summarize recent findings connecting splicing and autoimmune disease, and attempt to find common patterns of splicing regulation that may advance our understanding of autoimmune diseases and open new avenues for therapy.

Objective The genetic contributions to the multifactorial disorder osteoarthritis (OA) have been increasingly recognized. The goal of the current study was to use OA-related biomarkers of severity and disease burden as quantitative traits to identify genetic susceptibility loci for OA. Methods In a large multigenerational extended family (n = 350), we measured 5 OA-related biomarkers: hyaluronan (HA), cartilage oligomeric matrix protein (COMP), N-propeptide of type IIA collagen (PIIANP), C-propeptide of type II procollagen (CPII), and type II collagen neoepitope (C2C). Single-nucleotide polymorphism markers (n = 6,090) covering the whole genome were genotyped using the Illumina HumanLinkage-12 BeadChip. Variance components analysis, as implemented in the Sequential Oligogenic Linkage Analysis Routines, was used to estimate heritabilities of the quantitative traits and to calculate 2-point and multipoint logarithm of odds (LOD) scores using a polygenic model. Results After adjusting for age and sex, we found that 4 of the 5 biomarkers exhibited significant heritability (PIIANP 0.57, HA 0.49, COMP 0.43, C2C 0.30; P ≤ 0.01 for all). Fourteen of the 19 loci that had multipoint LOD scores of >1.5 were near to or overlapped with previously reported OA susceptibility loci. Four of these loci were identified by more than 1 biomarker. The maximum multipoint LOD scores for the heritable quantitative biomarker traits were 4.3 for PIIANP (chromosome 8p23.2), 3.2 for COMP (chromosome 8q11.1), 2.0 for HA (chromosome 6q16.3), and 2.0 for C2C (chromosome 5q31.2). Conclusion Herein, we report the first evidence of genetic susceptibility loci identified by OA-related biomarkers in an extended family. Our results demonstrate that serum concentrations of PIIANP, HA, COMP, and C2C have substantial heritable components, and using these biomarkers, several genetic loci potentially contributing to the genetic diversity of OA were identified.

Leukotrienes are arachidonic acid derivatives long known for their inflammatory properties and their involvement with a number of human diseases, most particularly asthma. Recently, leukotriene-based inflammation has also been shown to play an important role in atherosclerosis: ALOX5AP and LTA4H, both genes in the leukotriene biosynthesis pathway, have individually been shown to be associated with various cardiovascular disease (CVD) phenotypes. To assess the role of the leukotriene pathway in CVD pathogenesis, we performed genetic association studies of ALOX5AP and LTA4H in a family based study of early onset coronary artery disease (EOCAD) (GENECARD, 1,101 families) and in a non-familial dataset of EOCAD (CATHGEN, 656 cases and 405 controls). We found weak to moderate association between single nucleotide polymorphisms (SNPs) in ALOX5AP and LTA4H with EOCAD. The previously reported four-SNP haplotype (HapA) in ALOX5AP showed association with EOCAD in CATHGEN (P = 0.02), while controlling for age, race and CVD risk factors. HapK, the previously reported ten-SNP haplotype in LTA4H was associated with EOCAD in CATHGEN (P = 0.04). Another previously reported four-SNP haplotype in ALOX5AP (HapB) was not significant in our sample (P = 0.39). The overall lack of (or weak) association of single SNPs as compared with the haplotype results demonstrates the need for analyzing multiple SNPs within each gene in such studies. Interestingly, we detected an association of SNPs in ALOX5 (P < 0.05), the target of ALOX5AP, with CVD. Using a pathway-based approach, we also detected statistical evidence for interactions among ALOX5, ALOX5AP and LTA4H using RNA expression data from a collection of freshly harvested human aortas with varying degrees of atherosclerosis. The GENECARD families did not demonstrate evidence for linkage or association with ALOX5, ALOX5AP or LTA4H. Our results support a modest role for the leukotriene pathway in atherosclerosis pathogenesis, reveal important genomic interactions within the pathway, and suggest the importance of using pathway-based modeling for evaluating the genomics of atherosclerosis susceptibility.

Background Neural tube defects (NTDs), including spina bifida and anencephaly, are the second most common birth defect with an incidence of 1/1000. Genetic factors are believed to contribute to NTD risk and family-based studies can be useful for identifying such risk factors. Methods We ascertained 1066 NTD families (1467 affected patients), including 307 multiplex NTD families. We performed pedigree analysis to describe the inheritance patterns, pregnancy outcomes, and recurrence risks to relatives of various types. Results Myelomeningocele or spina bifida (66.9%) and cranial defects (17.7%) were the most common NTD subtypes observed. The overall male:female ratio for affected individuals was 0.82, and there were even fewer males among individuals with an upper level NTD (0.62). Among twins, 2 of the 5 monozygotic twins and only 3 of 35 dizygotic twins were concordant, while 27% of the same sex twins were concordant, but none of the different sex twins. The estimated 6.3% recurrence risk to siblings (CI 0.04-0.08) is consistent with previous reports. Families with two or more affected individuals show a higher proportion of female transmitters (p = 0.0002). Additionally, the number of affected relatives in maternal compared to paternal lineages was more than double (p = 0.006). There were significantly more miscarriages, infant deaths, and stillborn pregnancies of the maternal aunts and uncles (p Conclusions Our data provide several lines of evidence consistent with a maternal effect, as well as a sex-influenced effect, in the etiology of NTDs.

Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor α chain ( IL7R ) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 × 10−7). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.

A susceptibility locus for coronary artery disease (CAD) has been mapped to chromosome 3q13-21 in a linkage study of early-onset CAD. We completed an association-mapping study across the 1-LOD–unit-down supporting interval, using two independent white case-control data sets (CATHGEN, initial and validation) to evaluate association under the peak. Single-nucleotide polymorphisms (SNPs) evenly spaced at 100-kb intervals were screened in the initial data set (N=468). Promising SNPs (P

Autoimmune diseases are thought to result from imbalances in normal immune physiology and regulation. Here, we show that autoimmune disease susceptibility and resistance alleles on mouse chromosome 3 (Idd3) correlate with differential expression of the key immunoregulatory cytokine interleukin-2 (IL-2). In order to test directly that an approximately two-fold reduction in IL-2 underpins the Idd3-linked destabilization of immune homeostasis, we demonstrate that engineered haplodeficiency of IL-2 gene expression not only reduces T cell IL-2 production by two-fold but also mimics the autoimmune dysregulatory effects of the naturally-occurring susceptibility alleles of IL-2. Reduced IL-2 production achieved by both genetic mechanisms correlates with fewer and less functional CD4+CD25+ regulatory T cells, which are critical for maintaining immune homeostasis.

It is well established that the major histocompatibility complex (MHC) on chromosome 6p21 contains at least one gene that influences susceptibility to multiple sclerosis.1–6 Although this association was first identified more than 30 years ago1 through the study of class I human leukocyte antigens (HLAs), it was quickly realized that this signal was predominantly, if not exclusively, the result of linkage disequilibrium (LD) with class II HLA genes, and that these exert the primary effect on susceptibility.7, 8 The complex nature of the MHC, especially its high gene content, extreme polymorphism, and extensive LD,9 has confounded efforts to resolve the nature of the MHC association in multiple sclerosis, although progress and useful clarifications have been made, especially in recent years. In virtually every population studied, multiple sclerosis is found to be associated with the DRB1*1501 allele.10 The only exceptions are those populations where this allele has a low frequency, and analysis is therefore underpowered; but even in these situations, DRB1*1501 is generally overrepresented in cases.11 The DRB1*1501 allele is carried on a particularly extensive haplotype,12 the most common DR15 haplotype found in white Europeans. As a result, many variants from flanking genes, even some located quite a distance from DRB1, have sufficient LD with DRB1*1501 that they invariably also show evidence for association with the disease in any population where association with DRB1*1501 can be demonstrated.11, 13–17 This extensive LD has made it difficult to establish which of the variants making up this haplotype is primarily responsible for the association. The distinction between DRB1*1501 and DQB1*0602 has been particularly taxing because the LD between these closely mapped genes is especially tight in those populations where the disease is frequent, that is, white Europeans and their migrant descendants. However, recent studies in the admixed African American population indicate the supremacy of the DRB1*1501 allele.18 In the presence of one susceptibility allele it is difficult to identify effects attributable to a second allele,19 especially if the second allele exerts a more modest effect or has a low frequency, or both. However, by analyzing populations where DR15 haplotypes are less common, and by using large cohorts, it has been possible to demonstrate that the DRB1*0301 allele also confers susceptibility to multiple sclerosis, thereby confirming allelic heterogeneity at the DRB1 locus.4, 6, 18, 20 Furthermore available evidence suggests that the susceptibility effects of the DRB1*1501 allele may be modulated by other DRB1 alleles.6, 20 The relation between the MHC and multiple sclerosis is further complicated by the accumulating evidence suggesting that MHC loci mapping outside DRB1 also influence susceptibility to the disease.11, 13–16 Work in animal models suggests that clustering of susceptibility loci is a common phenomenon in complex disease,21 and it therefore appears reasonable to expect that other genes from the MHC region may influence susceptibility to multiple sclerosis. The observation of positive logarithm of odds scores in the MHC region in linkage studies stratified for the effects of DRB1 supports the existence of secondary loci,22–24 although none of these data reaches a level providing statistical confidence. In considering these linkage data, it is important to remember that early linkage studies in multiple sclerosis25–27 were significantly underpowered,28 to the point that they could not even convincingly demonstrate evidence for linkage resulting from the effects of DRB1. Confirmation of linkage in this region has been established only in more recent studies involving many hundreds of families.23, 24, 29 Given the inherently limited resolution of linkage-based studies,28 the absence of statistically significant linkage in the MHC region after exclusion of primary effects attributable to DRB1 does not exclude the presence of secondary loci. Several authors have attempted to identify secondary loci using more powerful association-based methods. Two groups have typed dense microsatellite maps of the region and both found evidence for a secondary locus maximal in a region close to HLA-A: one group identifying the marker D6S1683 just telomeric of HLA-A,11 and the second group implicating a region including HLA-A extending from MOGCA to D6S265 marker.14 Follow-up studies in Norway also found evidence implicating the D6S265 marker.16 In another smaller study, a microsatellite marker close to HLA-C (marker C1_3_2) also showed evidence for an independent effect.15 In contrast, a systematic effort to screen the MHC and flanking regions using single nucleotide polymorphisms (SNPs) found no evidence for association beyond that attributable to DRB1*1501, although this study was limited by a high genotyping failure rate (40%) and, more importantly, a distribution of markers leaving regions close to the classical loci essentially unexplored.17 In all of these studies, statistical power has inevitably been reduced by the processes required to filter out the primary effect attributable to DRB1 and the large correction required for multiple testing. Unfortunately, none of the published studies has used sufficient samples to compensate for these statistical penalties, and thus none is able to provide unequivocal evidence supporting any particular secondary locus.

TMEM231, a functional component of the MKS complex at the ciliary transition zone, is mutated in orofaciodigital syndrome type 3 and Meckel syndrome., The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.

Levels of certain circulating short-chain dicarboxylacylcarnitine (SCDA), long-chain dicarboxylacylcarnitine (LCDA) and medium chain acylcarnitine (MCA) metabolites are heritable and predict cardiovascular disease (CVD) events. Little is known about the biological pathways that influence levels of most of these metabolites. Here, we analyzed genetics, epigenetics, and transcriptomics with metabolomics in samples from a large CVD cohort to identify novel genetic markers for CVD and to better understand the role of metabolites in CVD pathogenesis. Using genomewide association in the CATHGEN cohort (N = 1490), we observed associations of several metabolites with genetic loci. Our strongest findings were for SCDA metabolite levels with variants in genes that regulate components of endoplasmic reticulum (ER) stress (USP3, HERC1, STIM1, SEL1L, FBXO25, SUGT1) These findings were validated in a second cohort of CATHGEN subjects (N = 2022, combined p = 8.4x10-6–2.3x10-10). Importantly, variants in these genes independently predicted CVD events. Association of genomewide methylation profiles with SCDA metabolites identified two ER stress genes as differentially methylated (BRSK2 and HOOK2). Expression quantitative trait loci (eQTL) pathway analyses driven by gene variants and SCDA metabolites corroborated perturbations in ER stress and highlighted the ubiquitin proteasome system (UPS) arm. Moreover, culture of human kidney cells in the presence of levels of fatty acids found in individuals with cardiometabolic disease, induced accumulation of SCDA metabolites in parallel with increases in the ER stress marker BiP. Thus, our integrative strategy implicates the UPS arm of the ER stress pathway in CVD pathogenesis, and identifies novel genetic loci associated with CVD event risk., Author Summary Cardiovascular disease is a strongly heritable trait. Despite application of the latest genomic technologies, the genetic architecture of disease risk remains poorly defined, and mechanisms underlying this susceptibility are incompletely understood. In this study, we performed genome-wide mapping of heart disease-related metabolites measured in the blood as the genetic traits of interest (instead of the disease itself), in a large cohort of 3512 patients at risk of heart disease from the CATHGEN study. Our goal was to discover new cardiovascular disease genes and thereby mechanisms of disease pathogenesis by understanding the genes that regulate levels of these metabolites. These analyses identified novel genetic variants associated with metabolite levels and with cardiovascular disease itself. Importantly, by utilizing an unbiased systems-based approach integrating genetics, gene expression, epigenetics and metabolomics, we uncovered a novel pathway of heart disease pathogenesis, that of endoplasmic reticulum (ER) stress, represented by elevated levels of circulating short-chain dicarboxylacylcarnitine (SCDA) metabolites.

The proteins encoded by the classical HLA class I and class II genes in the major histocompatibility complex (MHC) are highly polymorphic and play an essential role in self/non-self immune recognition. HLA variation is a crucial determinant of transplant rejection and susceptibility to a large number of infectious and autoimmune disease1. Yet identification of causal variants is problematic due to linkage disequilibrium (LD) that extends across multiple HLA and non-HLA genes in the MHC2,3. We therefore set out to characterize the LD patterns between the highly polymorphic HLA genes and background variation by typing the classical HLA genes and >7,500 common single nucleotide polymorphisms (SNPs) and deletion/insertion polymorphisms (DIPs) across four population samples. The analysis provides informative tag SNPs that capture some of the variation in the MHC region and that could be used in initial disease association studies, and provides new insight into the evolutionary dynamics and ancestral origins of the HLA loci and their haplotypes.

The complex inheritance involved in multiple sclerosis (MS) risk has been extensively investigated, but our understanding of MS genetics remains rudimentary. In this study, we explore 51 single nucleotide polymorphisms (SNPs) in 36 candidate genes from the inflammatory pathway and test for gene-gene interactions using complementary case-control, discordant sibling pair, and trio family study designs. We used a sample of 421 carefully diagnosed MS cases and 96 unrelated, healthy controls; discordant sibling pairs from 146 multiplex families; and 275 trio families. We used multifactor dimensionality reduction to explore gene-gene interactions. Based on our analyses, we have identified several statistically significant models including both main effect models and two-locus, three-locus, and four-locus epistasis models that predict MS disease risk with between approximately 61% and 85% accuracy. These results suggest that significant epistasis, or gene-gene interactions, may exist even in the absence of statistically significant individual main effects.

Multiple sclerosis (MS) is a common disease of the central nervous system characterized by inflammation, myelin loss, gliosis, varying degrees of axonal pathology, and progressive neurological dysfunction. Multiple sclerosis exhibits many of the characteristics that distinguish complex genetic disorders including polygenic inheritance and environmental exposure risks. Here, we used a highly efficient multilocus genotyping assay representing variation in 34 genes associated with inflammatory pathways to explore gene–gene interactions and disease susceptibility in a well-characterized African-American case–control MS data set. We applied the multifactor dimensionality reduction (MDR) test to detect epistasis, and identified single-IL4R(Q576R)- and three-IL4R(Q576R), IL5RA(-80), CD14(-260)- locus association models that predict MS risk with 75–76% accuracy (P < 0.01). These results demonstrate the importance of exploring both main effects and gene–gene interactions in the study of complex diseases.

Summary: Single nucleotide polymorphisms (SNPs) are commonly used for association studies to find genes responsible for complex genetic diseases. With the recent advance of SNP technology, researchers are able to assay thousands of SNPs in a single experiment. But the process of manually choosing thousands of genotyping SNPs for tens or hundreds of genes is time consuming. We have developed a web-based program, SNPselector, to automate the process. SNPselector takes a list of gene names or a list of genomic regions as input and searches the Ensembl genes or genomic regions for available SNPs. It prioritizes these SNPs on their tagging for linkage disequilibrium, SNP allele frequencies and source, function, regulatory potential and repeat status. SNPselector outputs result in compressed Excel spreadsheet files for review by the user. Availability: SNPselector is freely available at http://primer.duhs.duke.edu/ Contact: mike.hauser@duke.edu

Meningiomas are common neoplasms of the meninges lining of the central nervous system. Deletions of 1p have been established as important for the initiation and/or progression of meningioma. The rationale of this array-CGH study was to characterize copy number imbalances of chromosome 1 in meningioma, using a full-coverage genomic microarray containing 2,118 distinct measurement points. In total, 82 meningiomas were analyzed, making this the most detailed analysis of chromosome 1 in a comprehensive series of tumors. We detected a broad range of aberrations, such as deletions and/or gains of various sizes. Deletions were the predominant finding and ranged from monosomy to a 3.5-Mb terminal 1p homozygous deletion. Although multiple aberrations were observed across chromosome 1, every meningioma in which imbalances were detected harbored 1p deletions. Tumor heterogeneity was also observed in three recurrent meningiomas, which most likely reflects a progressive loss of chromosomal segments at different stages of tumor development. The distribution of aberrations supports the existence of at least four candidate loci on chromosome 1, which are important for meningioma tumorigenesis. In one of these regions, our results already allow the analysis of a number of candidate genes. In a large series of cases, we observed an association between the presence of segmental duplications and deletion breakpoints, which suggests their role in the generation of these tumor-specific aberrations. As 1p is the site of the genome most frequently affected by tumor-specific aberrations, our results indicate loci of general importance for cancer development and progression.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

The Del(13)Svea36H mutation (referred to hereafter as Del36H) is a microscopically visible deletion of ∼20% of mouse chromosome 13 (Arkell et al. 2001). Mice that are heterozygous for Del36H display a phenotype that varies with genetic background and that can involve reduced size, craniofacial malformation, eyes open at birth, and a mild tail kink. These mice may model some aspects of human genetic disease, because the Del36H region shows conserved synteny with regions of human chromosome 6p22.1-6p22.3 and 6p25 that are lost in some deletion syndromes (Davies et al. 1999). Furthermore, several disease loci map to this region in humans: two eye defects (iridogoniodysgenesis and Axenfeld-Rieger anomaly; Mears et al. 1998; Nishimura et al. 1998), haemochromatosis (Feder et al. 1996), dyslexia (Grigorenko et al. 2003), and schizophrenia susceptibility (Straub et al. 2002). Mice with interstitial chromosome deletions like Del36H are potent experimental tools for functional genomics. In particular, they can be used to reveal recessive phenotypes due to mutations that map to a specific chromosomal region. However, the positional candidate approach to identifying mutations in genes underlying mutant phenotypes remains nontrivial, especially for point mutations such as those induced by ENU (Brown and Hardisty 2003). A prerequisite for effective mutation detection using this approach is a comprehensive gene list, with exhaustive annotation of exons and regulatory elements. A limited catalog of the genes deleted in Del36H can be found in genetic and radiation hybrid maps (Arkell et al. 2001; Avner et al. 2001; Hudson et al. 2001), and an automatically annotated genomic sequence is available (Waterston et al. 2002), but the current public mouse genome assembly is a mixture of draft and finished sequence and, by definition, draft genomic sequence contains gaps and regions of lower sequence quality. These artefacts can influence gene annotation and, therefore, the subsequent design of mutation detection assays. Manual annotation, in contrast, should provide a gold standard reference set. As well as being an invaluable resource for functional genomics, a large genomic region of this kind provides an opportunity to investigate the organization and evolution of a significant piece of the mouse genome. Such studies also rely on high-quality sequence and manual gene annotation to avoid errors in sequence alignment, identification of coding and pseudogenes, classification of repetitive elements, and so on. The accumulating information on genome sequences from a number of species raises many questions about genome evolution. Important among these are the relative roles of whole-genome, segmental, and individual gene duplication, and the mechanisms underlying these processes (Lynch and Conery 2000; Dehal et al. 2001; Eichler and Sankoff 2003; Friedman and Hughes 2004); the usefulness of inter-genome comparisons for identifying selectively conserved regions in genomes, including not only genes, but regulatory regions and functional RNA genes (Mallon et al. 2000; Dehal et al. 2001; Dermitzakis et al. 2002; Kondrashov and Shabalina 2002; Margulies et al. 2003; Frazer et al. 2004); the roles of repeated (transposable element-like) and repetitive (satellites, microsatellites, and minisatellites) sequences in genome evolution (Toth et al. 2000; Hancock 2002; Babcock et al. 2003; Alba and Guigo 2004; Han et al. 2004; Kazazian Jr. 2004); and the characteristics of sites of evolutionary chromosome breakpoints (Puttagunta et al. 2000; Dehal et al. 2001; Pevzner and Tesler 2003). Here, we describe the genomic architecture of Del36H based on 12.66 Mb of finished DNA sequence, annotated using a combination of manual annotation with synteny and comparative sequence analysis. We find that the region is gene rich, primarily as the result of high gene densities in regions containing gene families that are smaller or absent in the orthologous human regions, and which appear to contribute to the special requirements of the lifestyle of the mouse. We consider forces and processes that may have contributed to the expansion of these gene families during evolution. We also identify a segment of Del36H containing two nearby evolutionary breakpoints, and show that these lie in a gene desert, a potentially optimal site for chromosome breakage. Finally, we consider the evolutionary dynamics of Evolutionarily Conserved Regions (ECRs; Mallon et al. 2000) within Del36H and their potential application to the identification of regulatory, and potentially other functional sequences within noncoding regions of the mouse genome.

At least two loci that determine susceptibility to type 1 diabetes in the NOD mouse have been mapped to chromosome 1, Idd5.1 (insulin-dependent diabetes 5.1) and Idd5.2. In this study, using a series of novel NOD.B10 congenic strains, Idd5.1 has been defined to a 2.1-Mb region containing only four genes, Ctla4, Icos, Als2cr19, and Nrp2 (neuropilin-2), thereby excluding a major candidate gene, Cd28. Genomic sequence comparison of the two functional candidate genes, Ctla4 and Icos, from the B6 (resistant at Idd5.1) and the NOD (susceptible at Idd5.1) strains revealed 62 single nucleotide polymorphisms (SNPs), only two of which were in coding regions. One of these coding SNPs, base 77 of Ctla4 exon 2, is a synonymous SNP and has been correlated previously with type 1 diabetes susceptibility and differential expression of a CTLA-4 isoform. Additional expression studies in this work support the hypothesis that this SNP in exon 2 is the genetic variation causing the biological effects of Idd5.1. Analysis of additional congenic strains has also localized Idd5.2 to a small region (1.52 Mb) of chromosome 1, but in contrast to the Idd5.1 interval, Idd5.2 contains at least 45 genes. Notably, the Idd5.2 region still includes the functionally polymorphic Nramp1 gene. Future experiments to test the identity of Idd5.1 and Idd5.2 as Ctla4 and Nramp1, respectively, can now be justified using approaches to specifically alter or mimic the candidate causative SNPs.

Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)--which encodes a vital negative regulatory molecule of the immune system--as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1 kb 3' region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.

Familial cold autoinflammatory syndrome (FCAS) is an autosomal dominant inflammatory disease with a high degree of penetrance that is characterized by episodes of rash, arthralgia, fever, conjunctivitis, and leukocytosis after generalized exposure to cold. FCAS was previously mapped to a 10-cM region on chromosome 1q44, and subsequently the gene ( CIAS1) responsible for FCAS was identified. In this paper, we describe the physical and genetic mapping of the FCAS locus, and we report a large ancestral haplotype and a new disease-causing mutation. A BAC contig of approximately 3 Mb was developed and subsequently used for high throughput sequencing. We identified a critical region of 4 cM using rare crossover events in four large North American FCAS families. An unusually large shared haplotype (40 cM) was identified in three of the four families. We found a single heterozygous missense mutation (T1058C=L353P) in exon 3 of CIAS1 in all four families that is responsible for the large majority of FCAS cases described in the literature. We also report a comprehensive list of intragenic single nucleotide polymorphisms. The data provided here will assist others researching the 1q44 region and will aid clinicians in the diagnosis of FCAS.

The syndrome of congenital hypoparathyroidism, mental retardation, facial dysmorphism and extreme growth failure (HRD or Sanjad-Sakati syndrome; OMIM 241410) is an autosomal recessive disorder reported almost exclusively in Middle Eastern populations. A similar syndrome with the additional features of osteosclerosis and recurrent bacterial infections has been classified as autosomal recessive Kenny-Caffey syndrome (AR-KCS; OMIM 244460). Both traits have previously been mapped to chromosome 1q43-44 (refs 5,6) and, despite the observed clinical variability, share an ancestral haplotype, suggesting a common founder mutation. We describe refinement of the critical region to an interval of roughly 230 kb and identification of deletion and truncation mutations of TBCE in affected individuals. The gene TBCE encodes one of several chaperone proteins required for the proper folding of alpha-tubulin subunits and the formation of alpha-beta-tubulin heterodimers. Analysis of diseased fibroblasts and lymphoblastoid cells showed lower microtubule density at the microtubule-organizing center (MTOC) and perturbed microtubule polarity in diseased cells. Immunofluorescence and ultrastructural studies showed disturbances in subcellular organelles that require microtubules for membrane trafficking, such as the Golgi and late endosomal compartments. These findings demonstrate that HRD and AR-KCS are chaperone diseases caused by a genetic defect in the tubulin assembly pathway, and establish a potential connection between tubulin physiology and the development of the parathyroid.

Purpose We investigated whether mitochondrial DNA (mtDNA) variants affect the susceptibility of Fuchs endothelial corneal dystrophy (FECD). Methods Ten mtDNA variants defining European haplogroups were genotyped in a discovery dataset consisting of 530 cases and 498 controls of European descent from the Duke FECD cohort. Association tests for mtDNA markers and haplogroups were performed using logistic regression models with adjustment of age and sex. Subset analyses included controlling for additional effects of either the TCF4 SNP rs613872 or cigarette smoking. Our replication dataset was derived from the genome-wide association study (GWAS) of the FECD Genetics Consortium, where genotypes for three of 10 mtDNA markers were available. Replication analyses were performed to compare non-Duke cases to all GWAS controls (GWAS1, N = 3200), and to non-Duke controls (GWAS2, N = 3043). Results The variant A10398G was significantly associated with FECD (odds ratio [OR] = 0.72; 95% confidence interval [CI] = [0.53, 0.98]; P = 0.034), and remains significant after adjusting for smoking status (min P = 0.012). This variant was replicated in GWAS1 (P = 0.019) and GWAS2 (P = 0.036). Haplogroup I was significantly associated with FECD (OR = 0.46; 95% CI = [0.22, 0.97]; P = 0.041) and remains significant after adjusting for the effect of smoking (min P = 0.008) or rs613872 (P = 0.034). Conclusions The 10398G allele and Haplogroup I appear to confer significant protective effects for FECD. The effect of A10398G and Haplogroup I to FECD is likely independent of the known TCF4 variant. More data are needed to decipher the interaction between smoking and mtDNA haplogroups.

Background Chiari Type I Malformation (CMI) is characterized by herniation of the cerebellar tonsils through the foramen magnum at the base of the skull, resulting in significant neurologic morbidity. As CMI patients display a high degree of clinical variability and multiple mechanisms have been proposed for tonsillar herniation, it is hypothesized that this heterogeneous disorder is due to multiple genetic and environmental factors. The purpose of the present study was to gain a better understanding of what factors contribute to this heterogeneity by using an unsupervised statistical approach to define disease subtypes within a case-only pediatric population. Methods A collection of forty-four pediatric CMI patients were ascertained to identify disease subtypes using whole genome expression profiles generated from patient blood and dura mater tissue samples, and radiological data consisting of posterior fossa (PF) morphometrics. Sparse k-means clustering and an extension to accommodate multiple data sources were used to cluster patients into more homogeneous groups using biological and radiological data both individually and collectively. Results All clustering analyses resulted in the significant identification of patient classes, with the pure biological classes derived from patient blood and dura mater samples demonstrating the strongest evidence. Those patient classes were further characterized by identifying enriched biological pathways, as well as correlated cranial base morphological and clinical traits. Conclusions Our results implicate several strong biological candidates warranting further investigation from the dura expression analysis and also identified a blood gene expression profile corresponding to a global down-regulation in protein synthesis.

The homozygous loop-tail ( Lp ) mouse has a severe neural tube closure defect, analogous to the craniorachischisis phenotype seen in humans. Linkage analysis and physical mapping have previously localized the Lp locus to a region on mouse chromosome 1 defined by the markers D1Mit113–Tagln2. Here we report the construction of sequence-ready bacterial clone contigs encompassing the Lp critical region in both mouse and the orthologous human region (1q22–q23). Twenty-two genes, one EST, and one pseudogene have been identified using a combination of EST database screening, exon amplification, and genomic sequence analysis. The preliminary gene map is Cen– Estm33 –AA693056– Ly9–Cd48–Slam–Cd84–Kiaa1215–Nhlh1–Kiaa0253–Copa–Pxf–H326–Pea15–Casq1–Atp1a4–Atp1a2–Estm34–Kcnj9–Kcnj10–Kiaa1355–Tagln2–Nesg1–Crp –Tel. The genes between Slam and Kiaa1355 are positional candidates for Lp. The comparative gene content and order are identical between mouse and human, indicating a high degree of conservation between the two species in this region. Together, the physical and transcript maps described here serve as resources for the identification of the Lp mutation and further define the conservation of this genomic region between mouse and human.