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1.  Genetic evaluation and application of posterior cranial fossa traits as endophenotypes for Chiari Type I Malformation 
Annals of human genetics  2013;78(1):1-12.
Chiari Type I Malformation (CMI) is characterized by herniation of the cerebellar tonsils through the base of the skull. Although cerebellar tonsillar herniation (CTH) is hypothesized to result from an underdeveloped posterior cranial fossa (PF), patients are frequently diagnosed by the extent of CTH without cranial morphometric assessment. We recently completed the largest CMI whole genome qualitative linkage screen to date. Despite an initial lack of statistical evidence, stratified analyses using clinical criteria to reduce heterogeneity resulted in a striking increase in evidence for linkage. The present study focused on the use of cranial base morphometrics to further dissect this heterogeneity and increase power to identify disease genes. We characterized the genetic contribution for a series of PF traits and evaluated the use of heritable, disease-relevant PF traits in ordered subset analysis (OSA). Consistent with a genetic hypothesis for CMI, much of the PF morphology was found to be heritable and multiple genomic regions were strongly implicated from OSA, including regions on chromosomes 1 (LOD=3.07, p=3×10−3) and 22 (LOD=3.45, p=6×10−5) containing several candidates warranting further investigation. This study underscores the genetic heterogeneity of CMI and the utility of PF traits in CMI genetic studies.
PMCID: PMC4041368  PMID: 24359474
Posterior cranial fossa; Chiari Type I Malformation; Endophenotypes; Heritability; Ordered subset analysis
2.  Mitochondrial Polymorphism A10398G and Haplogroup I Are Associated With Fuchs' Endothelial Corneal Dystrophy 
We investigated whether mitochondrial DNA (mtDNA) variants affect the susceptibility of Fuchs endothelial corneal dystrophy (FECD).
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).
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).
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.
The nine European mitochondrial haplogroups were investigated for the susceptibility of Fuchs endothelial corneal dystrophy (FECD). A10398G and Haplogroup I were found significantly decreasing the risk of FECD.
PMCID: PMC4109404  PMID: 24917144
mitochondrial haplogroup; genetic association; oxidative stress; TCF4; smoking
3.  Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis 
Human Molecular Genetics  2013;22(25):5107-5120.
Smooth muscle cell (SMC) proliferation is a hallmark of vascular injury and disease. Global hypomethylation occurs during SMC proliferation in culture and in vivo during neointimal formation. Regardless of the programmed or stochastic nature of hypomethylation, identifying these changes is important in understanding vascular disease, as maintenance of a cells' epigenetic profile is essential for maintaining cellular phenotype. Global hypomethylation of proliferating aortic SMCs and concomitant decrease of DNMT1 expression were identified in culture during passage. An epigenome screen identified regions of the genome that were hypomethylated during proliferation and a region containing Collagen, type XV, alpha 1 (COL15A1) was selected by ‘genomic convergence’ for characterization. COL15A1 transcript and protein levels increased with passage-dependent decreases in DNA methylation and the transcript was sensitive to treatment with 5-Aza-2′-deoxycytidine, suggesting DNA methylation-mediated gene expression. Phenotypically, knockdown of COL15A1 increased SMC migration and decreased proliferation and Col15a1 expression was induced in an atherosclerotic lesion and localized to the atherosclerotic cap. A sequence variant in COL15A1 that is significantly associated with atherosclerosis (rs4142986, P = 0.017, OR = 1.434) was methylated and methylation of the risk allele correlated with decreased gene expression and increased atherosclerosis in human aorta. In summary, hypomethylation of COL15A1 occurs during SMC proliferation and the consequent increased gene expression may impact SMC phenotype and atherosclerosis formation. Hypomethylated genes, such as COL15A1, provide evidence for concomitant epigenetic regulation and genetic susceptibility, and define a class of causal targets that sit at the intersection of genetic and epigenetic predisposition in the etiology of complex disease.
PMCID: PMC3842173  PMID: 23912340
4.  Gene–smoking interactions in multiple Rho-GTPase pathway genes in an early-onset coronary artery disease cohort 
Human genetics  2013;132(12):10.1007/s00439-013-1339-7.
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.
PMCID: PMC3835376  PMID: 23907653
5.  Missing Genetic Risk in Neural Tube Defects: Can Exome Sequencing Yield an Insight? 
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.
Next generation sequencing platforms are facilitating the production of massive amounts of sequencing data, primarily from the protein coding regions of the genome, at a faster rate and cheaper cost than has previously been possible. These platforms are permitting the identification of variants (de novo, rare, and common) that are drivers of NYTD etiology, and the cost of the approach allows for the screening of increased numbers of affected and unaffected individuals from NTD families and in simplex cases.
The next generation sequencing platforms represent a powerful tool in the armory of the genetics researcher to identify the causal genetic basis of NTDs.
PMCID: PMC4169137  PMID: 25044326
next generation exome sequencing; de novo; rare and common variation; compound heterozygosity
6.  Genetic Association Analyses of Nitric Oxide Synthase Genes and Neural Tube Defects Vary by Phenotype 
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.
PMCID: PMC4169175  PMID: 24323870
NOS1; NOS2; NOS3; MTHFR; neural tube defect; NTD; anencephaly; spina bifida
7.  Outcome and Life Satisfaction of Adults with Myelomeningocele 
Disability and health journal  2013;6(3):236-243.
Myelomeningocele (MMC) commonly causes impairments in body structure and functions as well as cognitive disabilities that can have an adverse effect on adult life. Improved medical care has resulted in increased numbers of individuals with MMC surviving to adulthood, however little is known about the impact of MMC on the lives of adults age 25 years or older.
To gain a better understanding of outcomes in education, employment, relationships, reproduction and life satisfaction of adults with MMC.
A primarily quantitative multiple-choice questionnaire designed to capture outcomes in education, employment, relationships and reproduction, along with a previously validated life satisfaction checklist (LiSat-11), was completed by adults with MMC. Relationships between demographic variables, outcomes and life satisfaction were determined using cross tabulation analysis, logistic regression and linear regression.
Ninety adults with MMC, age 25 to 85 years (median age 32), reported a diverse range of outcomes in education, employment, relationships and reproduction. The most consistent variable associated with difficulty attaining adult milestones was hydrocephalus, the presence of which reduced the likelihood of living independently (p=<0.001), having a partner (p=0.003) and reproducing (p=<0.001), but did not contribute to reduced life satisfaction.
Adults with MMC, especially those without hydrocephalus, can obtain gainful employment, live independently, form partner relationships and have children, and these achievements contribute to life satisfaction. While MMC does not affect overall reported life satisfaction for adults, attention should be paid to specific domains with less reported satisfaction.
PMCID: PMC3687213  PMID: 23769483
myelomeningocele; spina bifida; adults; life satisfaction
8.  Identification of Chiari Type I Malformation subtypes using whole genome expression profiles and cranial base morphometrics 
BMC Medical Genomics  2014;7:39.
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.
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.
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.
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.
PMCID: PMC4082616  PMID: 24962150
Chiari Type I Malformation; Posterior fossa; Disease subtypes; Whole genome expression; Cranial base morphometrics; Clustering
9.  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.
PMCID: PMC3820424  PMID: 22815244
Fumonisin; Fusarium verticillioides; Urinary fumonisin B1
10.  Interactions between Social/ behavioral factors and ADRB2 genotypes may be associated with health at advanced ages in China 
BMC Geriatrics  2013;13:91.
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.
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.
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.
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.
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
11.  ALOX5AP Variants are Associated with In-Stent Restenosis After Percutaneous Coronary Intervention 
Atherosclerosis  2008;201(1):148-154.
Use of drug-eluting stents (DES) has reduced in-stent restenosis after percutaneous coronary intervention (PCI); however, DES are associated with late stent thrombosis. There is no accurate way to predict in-stent restenosis, although risk factors for atherosclerosis overlap those for in-stent restenosis. Therefore, we evaluated atherosclerosis candidate genes for association with in-stent restenosis.
We identified 46 consecutive cases that had undergone PCI with bare-metal stents who subsequently developed symptomatic in-stent restenosis of the target lesion (≥75% luminal narrowing) within six months. Forty-six age-, race-, vessel-diameter- and sex-matched controls without in-stent restenosis after PCI with bare-metal stent were also identified. Single-nucleotide polymorphisms (SNPs, N=82) from 39 candidate atherosclerosis genes were genotyped. Multivariable logistic regression models were used to test for association.
Five SNPs were associated with in-stent restenosis. Three ALOX5AP SNPs were most strongly associated, two with increased risk (OR 3.74, p=0.01; OR 3.46, p=0.02), and the third with decreased risk of in-stent restenosis (OR 0.09, p=0.004). Two ALOX5AP haplotypes were associated with in-stent restenosis (HapB: OR 3.13, p=0.03); and a haplotype similar to HapA: OR 0.14, p=0.0009).
ALOX5AP, a gene within the inflammatory leukotriene pathway linked to and associated with coronary atherosclerosis, is also associated with in-stent restenosis. Genotyping these variants may help identify those at risk for in-stent restenosis who would benefit most from use of DES.
PMCID: PMC3733458  PMID: 18374923
Genetics; Stent Restenosis; Coronary Artery Disease; Inflammation
12.  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.
PMCID: PMC3732259  PMID: 23936524
13.  Genome-Wide Linkage Analysis of Quantitative Biomarker Traits of Osteoarthritis in a Large Multigenerational Extended Family 
Arthritis and rheumatism  2010;62(3):781-790.
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.
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.
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).
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.
PMCID: PMC3684272  PMID: 20187133
whole-genome scan; osteoarthritis; biomarkers; quantitative trait; linkage; heritability
14.  Clinical, radiological, and genetic similarities between patients with Chiari Type I and Type 0 malformations 
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.
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.
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.
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.
PMCID: PMC3678957  PMID: 22462700
Chiari malformation; idiopathic syringomyelia; genetics
15.  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.
PMCID: PMC3631233  PMID: 23620759
16.  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.
PMCID: PMC3628753  PMID: 21857681
17.  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.
PMCID: PMC3576662  PMID: 21298289
18.  Deletion or epigenetic silencing of AJAP1 on 1p36 in glioblastoma 
Molecular Cancer Research  2012;10(2):208-217.
Glioblastoma is universally fatal because of its propensity for rapid recurrence due to highly migratory tumor cells. Unraveling the genomic complexity that underlies this migratory characteristic could provide therapeutic targets that would greatly complement current surgical therapy. Using multiple high-resolution genomic screening methods, we identified a single locus, Adherens Junctional Associated Protein 1 (AJAP1) on chromosome 1p36, that is lost or epigenetically silenced in many glioblastomas. We found AJAP1 expression absent or reduced in 86% and 100% of primary glioblastoma tumors and cell lines, respectively, and the loss of expression correlates with AJAP1 methylation. Restoration of AJAP1 gene expression by transfection or demethylation agents results in decreased tumor cell migration in glioblastoma cell lines. This work demonstrates the significant loss of expression of AJAP1 in glioblastoma and provides evidence of its role in the highly migratory characteristic of these tumors.
PMCID: PMC3288240  PMID: 22241217
AJAP1 (Adherens junctional associated protein); glioblastoma; malignant glioma; methylation; gene deletion; epigenetics; migration
19.  Genetic screen of African Americans with Fuchs endothelial corneal dystrophy 
Molecular Vision  2013;19:2508-2516.
Fuchs endothelial corneal dystrophy (FECD) is a genetically heterogeneous disorder that has been primarily studied in patients of European or Asian ancestry. Given the sparse literature on African Americans with FECD, we sought to characterize the genetic variation in three known FECD candidate genes in African American patients with FECD.
Over an 8-year period, we enrolled 47 African American probands with FECD. All participants were clinically examined with slit-lamp biomicroscopy, and when corneal tissue specimens were available, histopathologic confirmation of the clinical diagnosis was obtained. The coding regions of known FECD susceptibility genes collagen, type VIII, alpha 2 (COL8A2); solute carrier family 4, sodium borate transporter, member 11 (SLC4A11); and zinc finger E-box binding homeobox 1 (ZEB1 [also known as TCF8]) were Sanger sequenced in the 47 probands using DNA isolated from blood samples.
Twenty-two coding variants were detected across the COL8A2, SLC4A11, and ZEB1 genes; six were nonsynonymous variants. Three novel coding variants were detected: a synonymous variant each in COL8A2 and SLC4A11 and one nonsynonymous variant in ZEB1 (p.P559S), which is predicted to be benign and tolerated, thus making its physiologic consequence uncertain.
Variation in the COL8A2, SLC4A11, and ZEB1 genes is present in only a small fraction of our African American cases and as such does not appear to significantly contribute to the genetic risk of FECD in African Americans. This observation is on par with findings from previous sequencing studies involving European or Asian ancestry patients with FECD.
PMCID: PMC3859630  PMID: 24348007
20.  The S1103Y Cardiac Sodium Channel Variant Is Associated with ICD Events in African Americans with Heart Failure and Reduced Ejection Fraction 
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.
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.
PMCID: PMC3086077  PMID: 21498565
heart failure; death; sudden; genetics; ion channels; race/ethnicity
21.  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.
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).
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.
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.
PMCID: PMC3309961  PMID: 22369142
Cardiovascular Disease; Positional Cloning; Intermediate Phenotype; Linkage; Fine Mapping
22.  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.
PMCID: PMC2940701  PMID: 20472715
chemoresistance; GBM; HDMX; oncogene; p53
23.  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.
PMCID: PMC2893804  PMID: 20421368
24.  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.
PMCID: PMC2940668  PMID: 20164239
array-CGH; astrocytoma; centromere; deletion; microarray; oligodendroglioma; translocation
25.  Replication of TCF4 through Association and Linkage Studies in Late-Onset Fuchs Endothelial Corneal Dystrophy 
PLoS ONE  2011;6(4):e18044.
Fuchs endothelial corneal dystrophy (FECD) is a common, late-onset disorder of the corneal endothelium. Although progress has been made in understanding the genetic basis of FECD by studying large families in which the phenotype is transmitted in an autosomal dominant fashion, a recently reported genome-wide association study identified common alleles at a locus on chromosome 18 near TCF4 which confer susceptibility to FECD. Here, we report the findings of our independent validation study for TCF4 using the largest FECD dataset to date (450 FECD cases and 340 normal controls). Logistic regression with sex as a covariate was performed for three genetic models: dominant (DOM), additive (ADD), and recessive (REC). We found significant association with rs613872, the target marker reported by Baratz et al.(2010), for all three genetic models (DOM: P = 9.33×10−35; ADD: P = 7.48×10−30; REC: P = 5.27×10−6). To strengthen the association study, we also conducted a genome-wide linkage scan on 64 multiplex families, composed primarily of affected sibling pairs (ASPs), using both parametric and non-parametric two-point and multipoint analyses. The most significant linkage region localizes to chromosome 18 from 69.94cM to 85.29cM, with a peak multipoint HLOD = 2.5 at rs1145315 (75.58cM) under the DOM model, mapping 1.5 Mb proximal to rs613872. In summary, our study presents evidence to support the role of the intronic TCF4 single nucleotide polymorphism rs613872 in late-onset FECD through both association and linkage studies.
PMCID: PMC3080358  PMID: 21533127

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