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1.  The Kinetics of Urinary Fumonisin B1 Excretion in Humans Consuming Maize-Based Diets 
Molecular nutrition & food research  2012;56(9):10.1002/mnfr.201200166.
Fumonisins (FB) are mycotoxins found in maize. The purpose of this study was to 1) determine the relationship between FB1, FB2 and FB3 intake and urinary excretion in humans, 2) validate a method to isolate urinary FB on C18-SPE cartridges for international shipment, and 3) test the method using samples from Guatemala. Volunteers (n=10) consumed 206 grams/day of tortillas and biscuits prepared from masa flour and a product containing maize flour. Volunteers estimated their daily urine output and samples were analyzed for FB1, FB2 and FB3 and hydrolyzed FB1. Only FB1 was detected in urine suggesting lower absorption of FB2 and FB3. Excretion was highly variable peaking soon after consumption began and decreasing rapidly after consumption stopped. Within five days after consumption ended FB1 was not detected in urine. In a study with eight volunteers, the average total urinary FB1 was 0.5% of the intake. FB1 was detected in 61% (107/177) of the samples collected in Guatemala. The results support the use of urinary FB1 to assess ongoing exposure in population based studies. However, relating the FB1 concentration in urine to dietary intake of FB by individual subjects will be complicated due to inter-individual variability and the rapidity of clearance.
doi:10.1002/mnfr.201200166
PMCID: PMC3820424  PMID: 22815244
Fumonisin; Fusarium verticillioides; Urinary fumonisin B1
2.  Genome-Wide Linkage Analysis of Cardiovascular Disease Biomarkers in a Large, Multigenerational Family 
PLoS ONE  2013;8(8):e71779.
Given the importance of cardiovascular disease (CVD) to public health and the demonstrated heritability of both disease status and its related risk factors, identifying the genetic variation underlying these susceptibilities is a critical step in understanding the pathogenesis of CVD and informing prevention and treatment strategies. Although one can look for genetic variation underlying susceptibility to CVD per se, it can be difficult to define the disease phenotype for such a qualitative analysis and CVD itself represents a convergence of diverse etiologic pathways. Alternatively, one can study the genetics of intermediate traits that are known risk factors for CVD, which can be measured quantitatively. Using the latter strategy, we have measured 21 cardiovascular-related biomarkers in an extended multigenerational pedigree, the CARRIAGE family (Carolinas Region Interaction of Aging, Genes, and Environment). These biomarkers belong to inflammatory and immune, connective tissue, lipid, and hemostasis pathways. Of these, 18 met our quality control standards. Using the pedigree and biomarker data, we have estimated the broad sense heritability (H2) of each biomarker (ranging from 0.09–0.56). A genome-wide panel of 6,015 SNPs was used subsequently to map these biomarkers as quantitative traits. Four showed noteworthy evidence for linkage in multipoint analysis (LOD score ≥ 2.6): paraoxonase (chromosome 8p11, 21), the chemokine RANTES (22q13.33), matrix metalloproteinase 3 (MMP3, 17p13.3), and granulocyte colony stimulating factor (GCSF, 8q22.1). Identifying the causal variation underlying each linkage score will help to unravel the genetic architecture of these quantitative traits and, by extension, the genetic architecture of cardiovascular risk.
doi:10.1371/journal.pone.0071779
PMCID: PMC3732259  PMID: 23936524
3.  Genome-Wide Linkage Analysis of Quantitative Biomarker Traits of Osteoarthritis in a Large Multigenerational Extended Family 
Arthritis and rheumatism  2010;62(3):781-790.
Objective
The genetic contributions to the multifactorial disorder osteoarthritis (OA) have been increasingly recognized. Our goal 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 (CARRIAGE family, n=350), we measured five OA-related biomarkers: HA (hyaluronan), COMP (cartilage oligomeric matrix protein), PIIANP (type IIA collagen N-propeptide), CPII (type II procollagen carboxy-propeptide), and C2C (type II collagen cleavage neoepitope). SNP markers (6,090) covering the whole genome were genotyped using the Illumina HumanLinkage-12 BeadChip. Variance components analysis as implemented in SOLAR was used to estimate heritabilities of the quantitative traits, and to calculate two-point and multi-point LOD scores using a polygenic model.
Results
Four of the five biomarkers showed significant heritability (p<0.01 age and sex adjusted h2r: PIIANP 0.57, HA 0.49, COMP 0.43, C2C 0.30). Fourteen of the 19 loci with multi-point LOD scores >1.5 were near or overlapped previously reported OA susceptibility loci. Four of these loci were identified by more than one biomarker. The maximum multi-point LOD scores for the heritable quantitative biomarker traits were LOD 4.3 for PIIANP (chromosome 8p23.2); LOD 3.2 for COMP (chromosome 8q11.1); LOD 2.0 for HA (chromosome 6q16.3); LOD 2.0 for C2C (chromosome 5q31.2).
Conclusions
We report the first evidence of genetic susceptibility loci identified by OA-related biomarkers in an extended family. Serum concentrations of PIIANP, HA, COMP and C2C have substantial heritable components and identified several genetic loci potentially contributing to the genetic diversity of OA.
doi:10.1002/art.27288
PMCID: PMC3684272  PMID: 20187133
whole-genome scan; osteoarthritis; biomarkers; quantitative trait; linkage; heritability
4.  Clinical, radiological, and genetic similarities between patients with Chiari Type I and Type 0 malformations 
Object
Although Chiari Type I (CM-I) and Type 0 (CM-0) malformations have been previously characterized clinically and radiologically, there have been no studies focusing on the possible genetic link between these disorders. The goal of this study was to identify families in whom CM-0 and CM-I co-occurred and to further assess the similarities between these disorders.
Methods
Families were ascertained through a proband with CM-I. Detailed family histories were obtained to identify first-degree relatives diagnosed with CM-0. Several criteria were used to exclude individuals with acquired forms of CM-I and/or syringomyelia. Individuals were excluded with syndromic, traumatic, infectious, or tumor-related syringomyelia, as well as CM-I due to a supratentorial mass, hydrocephalus, history of cervical or cranial surgery unrelated to CM-I, or development of symptoms following placement of a lumbar shunt. Medical records and MR images were used to characterize CM-I and CM-0 individuals clinically and radiologically.
Results
Five families were identified in which the CM-I proband had a first-degree relative with CM-0. Further assessment of affected individuals showed similar clinical and radiological features between CM-0 and CM-I individuals, although CM-I patients in general had more severe symptoms and skull base abnormalities than their CM-0 relatives. Overall, both groups showed improvement in symptoms and/or syrinx size following craniocervical decompression surgery.
Conclusions
There is accumulating evidence suggesting that CM-0 and CM-I may be caused by a common underlying developmental mechanism. The data in this study are consistent with this hypothesis, showing similar clinical and radiological features between CM-0 and CM-I individuals, as well as the occurrence of both disorders within families. Familial clustering of CM-0 and CM-I suggests that these disorders may share an underlying genetic basis, although additional epigenetic and/or environmental factors are likely to play an important role in the development of CM-0 versus CM-I.
doi:10.3171/2011.12.PEDS11113
PMCID: PMC3678957  PMID: 22462700
Chiari malformation; idiopathic syringomyelia; genetics
5.  A stress response pathway regulates DNA damage through β2-adrenoreceptors and β-arrestin-1 
Nature  2011;477(7364):349-353.
The human mind and body respond to stress1, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the ‘fight-or-flight’ response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term2. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders3, and epidemiological studies strongly indicate that chronic stress leads to DNA damage4,5. This stress-induced DNA damage may promote ageing6, tumorigenesis4,7, neuropsychiatric conditions8,9 and miscarriages10. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β2-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos11. Activated β2-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right12. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β2-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1−/−) mice, which show pre served p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress1, and in the testes, in which paternal stress may affect the offspring’s genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.
doi:10.1038/nature10368
PMCID: PMC3628753  PMID: 21857681
6.  The S1103Y Cardiac Sodium Channel Variant Is Associated with ICD Events in African Americans with Heart Failure and Reduced Ejection Fraction 
Background
Risk stratifying heart failure patients for primary prevention implantable cardioverter-defibrillators (ICDs) remains a challenge, especially for African Americans, who have an increased incidence of sudden cardiac death but have been underrepresented in clinical trials. We hypothesized that the S1103Y cardiac sodium channel SCN5A variant influences the propensity for ventricular arrhythmias in African American patients with heart failure and reduced ejection fraction.
Methods and Results
112 African Americans with ejection fractions (EF) <35% receiving primary prevention ICDs were identified from the Duke Electrophysiology Genetic and Genomic Studies (EPGEN) biorepository and followed for appropriate ICD therapy (either antitachycardia pacing or shock) for documented sustained ventricular tachycardia or fibrillation. The S1103Y variant was over-represented in patients receiving appropriate ICD therapy compared to subjects who did not (35% vs 13%, p=0.03). Controlling for baseline characteristics, the adjusted hazard ratio using a Cox Proportional Hazard Model for ICD therapy in Y1103 allele carriers was 4.33 (95% CI 1.60–11.73, p=<0.01). There was no difference in mortality between carriers and non-carriers.
Conclusion
This is the first report that the S1103Y variant is associated with a higher incidence of ventricular arrhythmias in African Americans with heart failure and reduced ejection fraction.
doi:10.1161/CIRCGENETICS.110.958652
PMCID: PMC3086077  PMID: 21498565
heart failure; death; sudden; genetics; ion channels; race/ethnicity
7.  HDMX regulates p53 activity and confers chemoresistance to 3-Bis(2-chloroethyl)-1-nitrosourea 
Neuro-Oncology  2010;12(9):956-966.
Glioblastoma multiforme (GBM) is one of the deadliest tumors afflicting humans, and the mechanisms of its onset and progression remain largely undefined. Our attempts to elucidate its molecular pathogenesis through DNA copy-number analysis by genome-wide digital karyotyping and single nucleotide polymorphism arrays identified a dramatic focal amplification on chromosome 1q32 in 4 of 57 GBM tumors. Quantitative real-time PCR measurements revealed that HDMX is the most commonly amplified and overexpressed gene in the 1q32 locus. Further genetic screening of 284 low- and high-grade gliomas revealed that HDMX amplifications occur solely in pediatric and adult GBMs and that they are mutually exclusive of TP53 mutations and MDM2 amplifications. Here, we demonstrate that HDMX regulates p53 to promote GBM growth and attenuates tumor response to chemotherapy. In GBM cells, HDMX overexpression inhibits p53-mediated transcriptional activation of p21, releases cells from G0 to G1 phase, and enhances cellular proliferation. HDMX overexpression does not affect the expression of PUMA and BAX proapoptotic genes. While in GBM cells treated with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), HDMX appears to stabilize p53 and promote phosphorylation of the DNA double-stranded break repair protein H2AX, up-regulate the DNA repair gene VPX, stimulate DNA repair, and confer resistance to BCNU. In summary, HDMX exhibits bona fide oncogenic properties and offers a promising molecular target for GBM therapeutic intervention.
doi:10.1093/neuonc/noq045
PMCID: PMC2940701  PMID: 20472715
chemoresistance; GBM; HDMX; oncogene; p53
8.  Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies 
Human Molecular Genetics  2010;19(14):2754-2766.
Aging is believed to be among the most important contributors to atherosclerosis, through mechanisms that remain largely obscure. Serum levels of tumor necrosis factor (TNF) rise with aging and have been correlated with the incidence of myocardial infarction. We therefore sought to determine whether genetic variation in the TNF receptor-1 gene (TNFR1) contributes to aging-related atherosclerosis in humans and whether Tnfr1 expression aggravates aging-related atherosclerosis in mice. With 1330 subjects from a coronary angiography database, we performed a case–control association study of coronary artery disease (CAD) with 16 TNFR1 single-nucleotide polymorphisms (SNPs). Two TNFR1 SNPs significantly associated with CAD in subjects >55 years old, and this association was supported by analysis of a set of 759 independent CAD cases. In multiple linear regression analysis, accounting for TNFR1 SNP rs4149573 significantly altered the relationship between aging and CAD index among 1811 subjects from the coronary angiography database. To confirm that TNFR1 contributes to aging-dependent atherosclerosis, we grafted carotid arteries from 18- and 2-month-old wild-type (WT) and Tnfr1−/− mice into congenic apolipoprotein E-deficient (Apoe−/−) mice and harvested grafts from 1 to 7 weeks post-operatively. Aged WT arteries developed accelerated atherosclerosis associated with enhanced TNFR1 expression, enhanced macrophage recruitment, reduced smooth muscle cell proliferation and collagen content, augmented apoptosis and plaque hemorrhage. In contrast, aged Tnfr1−/− arteries developed atherosclerosis that was indistinguishable from that in young Tnfr1−/− arteries and significantly less than that observed in aged WT arteries. We conclude that TNFR1 polymorphisms associate with aging-related CAD in humans, and TNFR1 contributes to aging-dependent atherosclerosis in mice.
doi:10.1093/hmg/ddq172
PMCID: PMC2893804  PMID: 20421368
9.  Distinct patterns of 1p and 19q alterations identify subtypes of human gliomas that have different prognoses† 
Neuro-Oncology  2010;12(7):664-678.
We studied the status of chromosomes 1 and 19 in 363 astrocytic and oligodendroglial tumors. Whereas the predominant pattern of copy number abnormality was a concurrent loss of the entire 1p and 19q regions (total 1p/19q loss) among oligodendroglial tumors and partial deletions of 1p and/or 19q in astrocytic tumors, a subset of apparently astrocytic tumors also had total 1p/19q loss. The presence of total 1p/19q loss was associated with longer survival of patients with all types of adult gliomas independent of age and diagnosis (P = .041). The most commonly deleted region on 19q in astrocytic tumors spans 885 kb in 19q13.33–q13.41, which is telomeric to the previously proposed region. Novel regions of homozygous deletion, including a part of DPYD (1p21.3) or the KLK cluster (19q13.33), were observed in anaplastic oligodendrogliomas. Amplifications encompassing AKT2 (19q13.2) or CCNE1 (19q12) were identified in some glioblastomas. Deletion mapping of the centromeric regions of 1p and 19q in the tumors that had total 1p/19q loss, indicating that the breakpoints lie centromeric to NOTCH2 within the pericentromeric regions of 1p and 19q. Thus, we show that the copy number abnormalities of 1p and 19q in human gliomas are complex and have distinct patterns that are prognostically predictive independent of age and pathological diagnosis. An accurate identification of total 1p/19q loss and discriminating this from other 1p/19q changes is, however, critical when the 1p/19q copy number status is used to stratify patients in clinical trials.
doi:10.1093/neuonc/nop075
PMCID: PMC2940668  PMID: 20164239
array-CGH; astrocytoma; centromere; deletion; microarray; oligodendroglioma; translocation
10.  Integrated genomic analyses identify ERRFI1 and TACC3 as glioblastoma-targeted genes 
Oncotarget  2010;1(4):265-277.
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.
PMCID: PMC2992381  PMID: 21113414
glioblastoma; genomics; copy number; 1p36; 4p16; ERRFI1; TACC3
11.  Integrated genomic analyses identify ERRFI1 and TACC3 as glioblastoma-targeted genes 
Oncotarget  2010;1(4):265-277.
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.
PMCID: PMC2992381  PMID: 21113414
glioblastoma; genomics; copy number; 1p36; 4p16; ERRFI1; TACC3
12.  Alternative splicing in multiple sclerosis and other autoimmune diseases 
RNA Biology  2010;7(4):462-473.
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.
doi:10.4161/rna.7.4.12301
PMCID: PMC3070910  PMID: 20639696
alternative splicing; autoimmune disease; SNP; multiple sclerosis; interleukin 7 receptor alpha chain
13.  Follow-up examination of linkage and association to chromosome 1q43 in multiple sclerosis 
Genes and immunity  2009;10(7):624-630.
Multiple sclerosis is a debilitating neuroimmunological and neurodegenerative disease affecting more than 400,000 individuals in the United States. Population and family-based studies have suggested that there is a strong genetic component. Numerous genomic linkage screens have identified regions of interest for MS loci. Our own second-generation genome-wide linkage study identified a handful of non-MHC regions with suggestive linkage. Several of these regions were further examined using single-nucleotide polymorphisms (SNPs) with average spacing between SNPs of approximately 1.0 Mb in a dataset of 173 multiplex families. The results of that study provided further evidence for the involvement of the chromosome 1q43 region. This region is of particular interest given linkage evidence in studies of other autoimmune and inflammatory diseases including rheumatoid arthritis and systemic lupus erythematosus. In this follow-up study, we saturated the region with ~700 SNPs (average spacing of 10kb per SNP) in search of disease associated variation within this region. We found preliminary evidence to suggest that common variation within the RGS7 locus may be involved in disease susceptibility.
doi:10.1038/gene.2009.53
PMCID: PMC2765552  PMID: 19626040
multiple sclerosis; linkage; association; 1q43; RGS7
14.  A general integrative genomic feature transcription factor binding site prediction method applied to analysis of USF1 binding in cardiovascular disease 
Human genomics  2009;3(3):221-235.
Transcription factors are key mediators of human complex disease processes. Identifying the target genes of transcription factors will increase our understanding of the biological network leading to disease risk. The prediction of transcription factor binding sites (TFBSs) is one method to identify these target genes; however, current prediction methods need improvement. We chose the transcription factor upstream stimulatory factor 1 (USF1) to evaluate the performance of our novel TFBS prediction method because of its known genetic association with coronary artery disease (CAD) and the recent availability of USF1 chromatin immunoprecipitation microarray (ChIP-chip) results. The specific goals of our study were to develop a novel and accurate genome-scale method for predicting USF1 binding sites and associated target genes to aid in the study of CAD. Previously published USF1 ChIP-chip data for 1 per cent of the genome were used to develop and evaluate several kernel logistic regression prediction models. A combination of genomic features (phylogenetic conservation, regulatory potential, presence of a CpG island and DNaseI hypersensitivity), as well as position weight matrix (PWM) scores, were used as variables for these models. Our most accurate predictor achieved an area under the receiver operator characteristic curve of 0.827 during cross-validation experiments, significantly outperforming standard PWM-based prediction methods. When applied to the whole human genome, we predicted 24,010 USF1 binding sites within 5 kilobases upstream of the transcription start site of 9,721 genes. These predictions included 16 of 20 genes with strong evidence of USF1 regulation. Finally, in the spirit of genomic convergence, we integrated independent experimental CAD data with these USF1 binding site prediction results to develop a prioritised set of candidate genes for future CAD studies. We have shown that our novel prediction method, which employs genomic features related to the presence of regulatory elements, enables more accurate and efficient prediction of USF1 binding sites. This method can be extended to other transcription factors identified in human disease studies to help further our understanding of the biology of complex disease.
PMCID: PMC2742312  PMID: 19403457
transcription factors; cardiovascular disease; human genetics; binding site prediction
15.  Genomic and epigenetic evidence for oxytocin receptor deficiency in autism 
BMC Medicine  2009;7:62.
Background
Autism comprises a spectrum of behavioral and cognitive disturbances of childhood development and is known to be highly heritable. Although numerous approaches have been used to identify genes implicated in the development of autism, less than 10% of autism cases have been attributed to single gene disorders.
Methods
We describe the use of high-resolution genome-wide tilepath microarrays and comparative genomic hybridization to identify copy number variants within 119 probands from multiplex autism families. We next carried out DNA methylation analysis by bisulfite sequencing in a proband and his family, expanding this analysis to methylation analysis of peripheral blood and temporal cortex DNA of autism cases and matched controls from independent datasets. We also assessed oxytocin receptor (OXTR) gene expression within the temporal cortex tissue by quantitative real-time polymerase chain reaction (PCR).
Results
Our analysis revealed a genomic deletion containing the oxytocin receptor gene, OXTR (MIM accession no.: 167055), previously implicated in autism, was present in an autism proband and his mother who exhibits symptoms of obsessive-compulsive disorder. The proband's affected sibling did not harbor this deletion but instead may exhibit epigenetic misregulation of this gene through aberrant gene silencing by DNA methylation. Further DNA methylation analysis of the CpG island known to regulate OXTR expression identified several CpG dinucleotides that show independent statistically significant increases in the DNA methylation status in the peripheral blood cells and temporal cortex in independent datasets of individuals with autism as compared to control samples. Associated with the increase in methylation of these CpG dinucleotides is our finding that OXTR mRNA showed decreased expression in the temporal cortex tissue of autism cases matched for age and sex compared to controls.
Conclusion
Together, these data provide further evidence for the role of OXTR and the oxytocin signaling pathway in the etiology of autism and, for the first time, implicate the epigenetic regulation of OXTR in the development of the disorder.
See the related commentary by Gurrieri and Neri:
doi:10.1186/1741-7015-7-62
PMCID: PMC2774338  PMID: 19845972
16.  A general integrative genomic feature transcription factor binding site prediction method applied to analysis of USF1 binding in cardiovascular disease 
Human Genomics  2009;3(3):221-235.
Transcription factors are key mediators of human complex disease processes. Identifying the target genes of transcription factors will increase our understanding of the biological network leading to disease risk. The prediction of transcription factor binding sites (TFBSs) is one method to identify these target genes; however, current prediction methods need improvement. We chose the transcription factor upstream stimulatory factor l (USF1) to evaluate the performance of our novel TFBS prediction method because of its known genetic association with coronary artery disease (CAD) and the recent availability of USF1 chromatin immunoprecipitation microarray (ChIP-chip) results. The specific goals of our study were to develop a novel and accurate genome-scale method for predicting USF1 binding sites and associated target genes to aid in the study of CAD. Previously published USF1 ChIP-chip data for 1 per cent of the genome were used to develop and evaluate several kernel logistic regression prediction models. A combination of genomic features (phylogenetic conservation, regulatory potential, presence of a CpG island and DNaseI hypersensitivity), as well as position weight matrix (PWM) scores, were used as variables for these models. Our most accurate predictor achieved an area under the receiver operator characteristic curve of 0.827 during cross-validation experiments, significantly outperforming standard PWM-based prediction methods. When applied to the whole human genome, we predicted 24,010 USF1 binding sites within 5 kilobases upstream of the transcription start site of 9,721 genes. These predictions included 16 of 20 genes with strong evidence of USF1 regulation. Finally, in the spirit of genomic convergence, we integrated independent experimental CAD data with these USF1 binding site prediction results to develop a prioritised set of candidate genes for future CAD studies. We have shown that our novel prediction method, which employs genomic features related to the presence of regulatory elements, enables more accurate and efficient prediction of USF1 binding sites. This method can be extended to other transcription factors identified in human disease studies to help further our understanding of the biology of complex disease.
doi:10.1186/1479-7364-3-3-221
PMCID: PMC2742312  PMID: 19403457
transcription factors; cardiovascular disease; human genetics; binding site prediction
17.  Comprehensive genetic analysis of the platelet activating factor acetylhydrolase (PLA2G7) gene and cardiovascular disease in case–control and family datasets 
Human molecular genetics  2008;17(9):1318-1328.
Platelet-activating factor acetylhydrolase (PLA2G7) is a potent pro- and anti-inflammatory molecule that has been implicated in multiple inflammatory disease processes, including cardiovascular disease. The goal of this study was to investigate the genetic effects of PLA2G7 on coronary artery disease (CAD) risk in two large, independent datasets with CAD. Using a haplotype tagging (ht) approach, 19 ht single nucleotide polymorphisms (SNPs) were genotyped in CATHGEN case–control samples (cases = 806 and controls = 267) and in the GENECARD Family Study (n = 1101 families, 2954 individuals). Single SNP analysis using logistic regression revealed nine SNPs with significant association in all CATHGEN subjects (P = 0.0004–0.02). CATHGEN cases were further stratified into subgroups based on age of CAD onset (AOO) and severity of disease; 599 young affecteds (YA, AOO <56) and 207 old affected (OA, AOO >56). Significant genetic effects were observed in both OA and YA (P = 0.0001–0.02). The GENECARD probands demonstrated results similar to those seen in the YA CATHGEN cases (P = 0.002–0.05). Of the 19 SNPs genotyped, 3 SNPs result in non-synonymous coding changes (I198T, A379V and R92H). Two of the coding SNPs, R92H and A379V, constitute two of the most significantly associated SNPs, even after Bonferroni correction and appear to represent independent associations (r2 = 0.09). Multiple additional polymorphisms in low linkage disequilibrium with these coding SNPs were also strongly associated. In summary, PLA2G7 represents an important, potentially functional candidate in the pathophysiology of CAD based on replicated associations using two independent datasets and multiple statistical approaches. Further functional studies involving a combination of risk alleles are warranted.
doi:10.1093/hmg/ddn020
PMCID: PMC2652668  PMID: 18204052
18.  SNPs in Multi-Species Conserved Sequences (MCS) as useful markers in association studies: a practical approach 
BMC Genomics  2007;8:266.
Background
Although genes play a key role in many complex diseases, the specific genes involved in most complex diseases remain largely unidentified. Their discovery will hinge on the identification of key sequence variants that are conclusively associated with disease. While much attention has been focused on variants in protein-coding DNA, variants in noncoding regions may also play many important roles in complex disease by altering gene regulation. Since the vast majority of noncoding genomic sequence is of unknown function, this increases the challenge of identifying "functional" variants that cause disease. However, evolutionary conservation can be used as a guide to indicate regions of noncoding or coding DNA that are likely to have biological function, and thus may be more likely to harbor SNP variants with functional consequences. To help bias marker selection in favor of such variants, we devised a process that prioritizes annotated SNPs for genotyping studies based on their location within Multi-species Conserved Sequences (MCSs) and used this process to select SNPs in a region of linkage to a complex disease. This allowed us to evaluate the utility of the chosen SNPs for further association studies. Previously, a region of chromosome 1q43 was linked to Multiple Sclerosis (MS) in a genome-wide screen. We chose annotated SNPs in the region based on location within MCSs (termed MCS-SNPs). We then obtained genotypes for 478 MCS-SNPs in 989 individuals from MS families.
Results
Analysis of our MCS-SNP genotypes from the 1q43 region and comparison to HapMap data confirmed that annotated SNPs in MCS regions are frequently polymorphic and show subtle signatures of selective pressure, consistent with previous reports of genome-wide variation in conserved regions. We also present an online tool that allows MCS data to be directly exported to the UCSC genome browser so that MCS-SNPs can be easily identified within genomic regions of interest.
Conclusion
Our results showed that MCS can easily be used to prioritize markers for follow-up and candidate gene association studies. We believe that this novel approach demonstrates a paradigm for expediting the search for genes contributing to complex diseases.
doi:10.1186/1471-2164-8-266
PMCID: PMC1959193  PMID: 17683615
19.  SNPselector: a web tool for selecting SNPs for genetic association studies 
Bioinformatics (Oxford, England)  2005;21(22):4181-4186.
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: hong.xu@duke.edu, mike.hauser@duke.edu
doi:10.1093/bioinformatics/bti682
PMCID: PMC1361283  PMID: 16179360
20.  Mapping and characterization of the amplicon near APOA2 in 1q23 in human sarcomas by FISH and array CGH 
Molecular Cancer  2005;4:39.
Background
Amplification of the q21-q23 region on chromosome 1 is frequently found in sarcomas and a variety of other solid tumours. Previous analyses of sarcomas have indicated the presence of at least two separate amplicons within this region, one located in 1q21 and one located near the apolipoprotein A-II (APOA2) gene in 1q23. In this study we have mapped and characterized the amplicon in 1q23 in more detail.
Results
We have used fluorescence in situ hybridisation (FISH) and microarray-based comparative genomic hybridisation (array CGH) to map and define the borders of the amplicon in 10 sarcomas. A subregion of approximately 800 kb was identified as the core of the amplicon. The amplification patterns of nine possible candidate target genes located to this subregion were determined by Southern blot analysis. The genes activating transcription factor 6 (ATF6) and dual specificity phosphatase 12 (DUSP12) showed the highest level of amplification, and they were also shown to be over-expressed by quantitative real-time reverse transcription PCR (RT-PCR). In general, the level of expression reflected the level of amplification in the different tumours. DUSP12 was expressed significantly higher than ATF6 in a subset of the tumours. In addition, two genes known to be transcriptionally activated by ATF6, glucose-regulated protein 78 kDa and -94 kDa (GRP78 and GRP94), were shown to be over-expressed in the tumours that showed over-expression of ATF6.
Conclusion
ATF6 and DUSP12 seem to be the most likely candidate target genes for the 1q23 amplification in sarcomas. Both genes have possible roles in promoting cell growth, which makes them interesting candidate targets.
doi:10.1186/1476-4598-4-39
PMCID: PMC1308856  PMID: 16274472
21.  A second major histocompatibility complex susceptibility locus for multiple sclerosis 
Annals of Neurology  2007;61(3):228-236.
Objective
Variation in the major histocompatibility complex (MHC) on chromosome 6p21 is known to influence susceptibility to multiple sclerosis with the strongest effect originating from the HLA-DRB1 gene in the class II region. The possibility that other genes in the MHC independently influence susceptibility to multiple sclerosis has been suggested but remains unconfirmed.
Methods
Using a combination of microsatellite, single nucleotide polymorphism, and human leukocyte antigen (HLA) typing, we screened the MHC in trio families looking for evidence of residual association above and beyond that attributable to the established DRB1*1501 risk haplotype. We then refined this analysis by extending the genotyping of classical HLA loci into independent cases and control subjects.
Results
Screening confirmed the presence of residual association and suggested that this was maximal in the region of the HLA-C gene. Extending analysis of the classical loci confirmed that this residual association is partly due to allelic heterogeneity at the HLA-DRB1 locus, but also reflects an independent effect from the HLA-C gene. Specifically, the HLA-C*05 allele, or a variant in tight linkage disequilibrium with it, appears to exert a protective effect (p = 3.3 × 10−5).
Interpretation
Variation in the HLA-C gene influences susceptibility to multiple sclerosis independently of any effect attributable to the nearby HLA-DRB1 gene. Ann Neurol 2007
doi:10.1002/ana.21063
PMCID: PMC2737610  PMID: 17252545
22.  Interactions between Social/ behavioral factors and ADRB2 genotypes may be associated with health at advanced ages in China 
BMC Geriatrics  2013;13:91.
Background
Existing literature indicates that ADRB2 gene is associated with health and longevity, but none of previous studies investigated associations of carrying the ADRB2 minor alleles and interactions between ADRB2 genotypes and social/behavioral factors(GxE) with health outcomes at advanced ages. This study intends to fill in this research gap.
Method
We conducted an exploratory analysis, using longitudinal survey phenotype/genotype data from 877 oldest-old aged 90+. To estimate association of GxE interactions with health outcome, adjusted for the potential correlation between genotypes and social/behavioral factors and various other potentially confounding factors, we develop and test an innovative three-step procedure which combines logistic regression and structural equation methods.
Results
Interaction between regular exercise and carrying rs1042718 minor allele is significantly and positively associated with good cognitive function; interaction between regular exercise and carrying rs1042718 or rs1042719 minor allele is significantly and positively associated with self-reported good health; and interaction between social-leisure activities and carrying rs1042719 minor allele is significantly and positively associated with self-reported good health. Carrying rs1042718 or rs1042719 minor alleles is significantly and negatively associated with negative emotion, but the ADRB2 SNPs are not significantly associated with cognitive function and self-reported health. Our structural equation analysis found that, adjusted for the confounding effects of correlation of the ADRB2 SNPs with negative emotion, interaction between negative emotion and carrying rs1042718 or rs1042719 minor allele is significantly and negatively associated with cognitive function. The positive association of regular exercise and social-leisure activities with cognitive function and self-reported health, and negative association of negative emotion with cognitive function, were much stronger among carriers of rs1042718 or rs1042719 alleles, compared to the non-carriers.
Conclusions
The results indicate significant positive associations of interactions between social/behavioral factors and the ADRB2 genotypes with health outcomes of cognitive function and self-reported health, and negative associations of carrying rs1042718 or rs1042719 minor alleles with negative emotion, at advanced ages in China. Our findings are exploratory rather than causal conclusions. This study implies that near-future health promotion programs considering individuals’ genetic profiles, with appropriate protection of privacy/confidentiality, would yield increased benefits and reduced costs to the programs and their participants.
doi:10.1186/1471-2318-13-91
PMCID: PMC3846634  PMID: 24016068
Health aging; Oldest-old; Social/behavioral factors; ADRB2 genotypes; GxE Interactions; Cognitive function; Self-reported health; Regular exercise; Social-leisure activities; Negative emotion
23.  Stratified Whole Genome Linkage Analysis of Chiari Type I Malformation Implicates Known Klippel-Feil Syndrome Genes as Putative Disease Candidates 
PLoS ONE  2013;8(4):e61521.
Chiari Type I Malformation (CMI) is characterized by displacement of the cerebellar tonsils below the base of the skull, resulting in significant neurologic morbidity. Although multiple lines of evidence support a genetic contribution to disease, no genes have been identified. We therefore conducted the largest whole genome linkage screen to date using 367 individuals from 66 families with at least two individuals presenting with nonsyndromic CMI with or without syringomyelia. Initial findings across all 66 families showed minimal evidence for linkage due to suspected genetic heterogeneity. In order to improve power to localize susceptibility genes, stratified linkage analyses were performed using clinical criteria to differentiate families based on etiologic factors. Families were stratified on the presence or absence of clinical features associated with connective tissue disorders (CTDs) since CMI and CTDs frequently co-occur and it has been proposed that CMI patients with CTDs represent a distinct class of patients with a different underlying disease mechanism. Stratified linkage analyses resulted in a marked increase in evidence of linkage to multiple genomic regions consistent with reduced genetic heterogeneity. Of particular interest were two regions (Chr8, Max LOD = 3.04; Chr12, Max LOD = 2.09) identified within the subset of “CTD-negative” families, both of which harbor growth differentiation factors (GDF6, GDF3) implicated in the development of Klippel-Feil syndrome (KFS). Interestingly, roughly 3–5% of CMI patients are diagnosed with KFS. In order to investigate the possibility that CMI and KFS are allelic, GDF3 and GDF6 were sequenced leading to the identification of a previously known KFS missense mutation and potential regulatory variants in GDF6. This study has demonstrated the value of reducing genetic heterogeneity by clinical stratification implicating several convincing biological candidates and further supporting the hypothesis that multiple, distinct mechanisms are responsible for CMI.
doi:10.1371/journal.pone.0061521
PMCID: PMC3631233  PMID: 23620759
24.  Polymorphic variants in tenascin-C (TNC) are associated with atherosclerosis and coronary artery disease 
Human genetics  2011;129(6):641-654.
Tenascin-C (TNC) is an extracellular matrix protein implicated in biological processes important for atherosclerotic plaque development and progression, including smooth muscle cell migration and proliferation. Previously, we observed differential expression of TNC in atherosclerotic aortas compared with healthy aortas. The goal of this study was to investigate whether common genetic variation within TNC is associated with risk of atherosclerosis and coronary artery disease (CAD) in three independent datasets. We genotyped 35 single nucleotide polymorphisms (SNPs), including 21 haplotype tagging SNPs, in two of these datasets: human aorta tissue samples (n = 205) and the CATHGEN cardiovascular study (n = 1,325). Eleven of these 35 SNPs were then genotyped in a third dataset, the GENECARD family study of early-onset CAD (n = 879 families). Three SNPs representing a block of linkage disequilibrium, rs3789875, rs12347433, and rs4552883, were significantly associated with athero sclerosis in multiple datasets and demonstrated consistent, but suggestive, genetic effects in all analyses. In combined analysis rs3789875 and rs12347433 were statistically significant after Bonferroni correction for 35 comparisons, p = 2 × 10−6 and 5 × 10−6, respectively. The SNP rs12347433 is a synonymous coding SNP and may be biologically relevant to the mechanism by which tenascin-C influences the pathophysiology of CAD and atherosclerosis. This is the first report of genetic association between polymorphisms in TNC and atherosclerosis or CAD.
doi:10.1007/s00439-011-0959-z
PMCID: PMC3576662  PMID: 21298289
25.  Fine mapping of a linkage peak with integration of lipid traits identifies novel coronary artery disease genes on chromosome 5 
BMC Genetics  2012;13:12.
Background
Coronary artery disease (CAD), and one of its intermediate risk factors, dyslipidemia, possess a demonstrable genetic component, although the genetic architecture is incompletely defined. We previously reported a linkage peak on chromosome 5q31-33 for early-onset CAD where the strength of evidence for linkage was increased in families with higher mean low density lipoprotein-cholesterol (LDL-C). Therefore, we sought to fine-map the peak using association mapping of LDL-C as an intermediate disease-related trait to further define the etiology of this linkage peak. The study populations consisted of 1908 individuals from the CATHGEN biorepository of patients undergoing cardiac catheterization; 254 families (N = 827 individuals) from the GENECARD familial study of early-onset CAD; and 162 aorta samples harvested from deceased donors. Linkage disequilibrium-tagged SNPs were selected with an average of one SNP per 20 kb for 126.6-160.2 MB (region of highest linkage) and less dense spacing (one SNP per 50 kb) for the flanking regions (117.7-126.6 and 160.2-167.5 MB) and genotyped on all samples using a custom Illumina array. Association analysis of each SNP with LDL-C was performed using multivariable linear regression (CATHGEN) and the quantitative trait transmission disequilibrium test (QTDT; GENECARD). SNPs associated with the intermediate quantitative trait, LDL-C, were then assessed for association with CAD (i.e., a qualitative phenotype) using linkage and association in the presence of linkage (APL; GENECARD) and logistic regression (CATHGEN and aortas).
Results
We identified four genes with SNPs that showed the strongest and most consistent associations with LDL-C and CAD: EBF1, PPP2R2B, SPOCK1, and PRELID2. The most significant results for association of SNPs with LDL-C were: EBF1, rs6865969, p = 0.01; PPP2R2B, rs2125443, p = 0.005; SPOCK1, rs17600115, p = 0.003; and PRELID2, rs10074645, p = 0.0002). The most significant results for CAD were EBF1, rs6865969, p = 0.007; PPP2R2B, rs7736604, p = 0.0003; SPOCK1, rs17170899, p = 0.004; and PRELID2, rs7713855, p = 0.003.
Conclusion
Using an intermediate disease-related quantitative trait of LDL-C we have identified four novel CAD genes, EBF1, PRELID2, SPOCK1, and PPP2R2B. These four genes should be further examined in future functional studies as candidate susceptibility loci for cardiovascular disease mediated through LDL-cholesterol pathways.
doi:10.1186/1471-2156-13-12
PMCID: PMC3309961  PMID: 22369142
Cardiovascular Disease; Positional Cloning; Intermediate Phenotype; Linkage; Fine Mapping

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