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1.  Whole Exome Sequence Analysis Implicates Rare Il17REL Variants In Familial And Sporadic Inflammatory Bowel Disease 
Inflammatory bowel diseases  2016;22(1):20-27.
Rare variants (<1%) likely contribute significantly to risk for common diseases such as inflammatory bowel disease (IBD) in specific patient subsets, such as those with high familiality. They are, however, extraordinarily challenging to identify.
To discover candidate rare variants associated with IBD, we performed whole exome sequencing (WES) on six members of a pediatric-onset IBD family with multiple affected individuals. To determine whether the variants discovered in this family are also associated with non-familial IBD, we investigated their influence on disease in two large case-control (CC) series.
We identified two rare variants, rs142430606 and rs200958270, both in the established IBD-susceptibility gene IL17REL, carried by all four affected family members and their obligate-carrier parents. We then demonstrated that both variants are associated with sporadic ulcerative colitis (UC) in two independent datasets. For UC in CC 1: rs142430606 (OR=2.99, Padj=0.028; MAFcases=0.0063, MAFcontrols=0.0021); rs200958270 (OR=2.61, Padj=0.082; MAFcases=0.0045, MAFcontrols=0.0017). For UC in CC 2: rs142430606 (OR=1.94, P=0.0056; MAFcases=0.0071, MAFcontrols=0.0045); rs200958270 (OR=2.08, P=0.0028; MAFcases=0.0071, MAFcontrols=0.0042).
We discover in a family and replicate in two CC datasets two rare susceptibility variants for IBD, both in IL17REL. Our results illustrate that WES performed on disease-enriched families to guide association testing can be an efficient strategy for the discovery of rare disease-associated variants. We speculate that rare variants identified in families and confirmed in the general population may be important modifiers of disease risk for patients with a family history, and that genetic testing of these variants may be warranted in this patient subset.
PMCID: PMC4679526  PMID: 26480299
Ulcerative colitis; familial inflammatory bowel disease; inflammatory bowel disease risk; complex disease genetics; whole exome sequencing
2.  Genetic Predisposition to Weight Loss and Regain With Lifestyle Intervention: Analyses From the Diabetes Prevention Program and the Look AHEAD Randomized Controlled Trials 
Diabetes  2015;64(12):4312-4321.
Clinically relevant weight loss is achievable through lifestyle modification, but unintentional weight regain is common. We investigated whether recently discovered genetic variants affect weight loss and/or weight regain during behavioral intervention. Participants at high-risk of type 2 diabetes (Diabetes Prevention Program [DPP]; N = 917/907 intervention/comparison) or with type 2 diabetes (Look AHEAD [Action for Health in Diabetes]; N = 2,014/1,892 intervention/comparison) were from two parallel arm (lifestyle vs. comparison) randomized controlled trials. The associations of 91 established obesity-predisposing loci with weight loss across 4 years and with weight regain across years 2–4 after a minimum of 3% weight loss were tested. Each copy of the minor G allele of MTIF3 rs1885988 was consistently associated with greater weight loss following lifestyle intervention over 4 years across the DPP and Look AHEAD. No such effect was observed across comparison arms, leading to a nominally significant single nucleotide polymorphism×treatment interaction (P = 4.3 × 10−3). However, this effect was not significant at a study-wise significance level (Bonferroni threshold P < 5.8 × 10−4). Most obesity-predisposing gene variants were not associated with weight loss or regain within the DPP and Look AHEAD trials, directly or via interactions with lifestyle.
PMCID: PMC4657576  PMID: 26253612
3.  Genetic predictors of depressive symptoms in the Look AHEAD Trial 
Psychosomatic medicine  2015;77(9):982-992.
Numerous studies find elevated depressive symptoms among individuals with type 2 diabetes, yet the mechanisms remain unclear. We examined whether genetic loci previously associated with depressive symptoms predict depressive symptoms among overweight/obese individuals with type 2 diabetes or change in depressive symptoms during behavioral weight loss.
The Illumina CARe iSelect (IBC) chip and Cardiometabochip were characterized in 2,118 overweight or obese participants with type 2 diabetes from Look AHEAD (Action for Health in Diabetes), a randomized trial to determine the effects of intensive lifestyle intervention (ILI) and Diabetes Support and Education (DSE) on cardiovascular morbidity and mortality. Primary analyses focused on baseline Beck Depression Inventory (BDI) scores and depressive symptom change at one year.
Of eight single nucleotide polymorphisms (SNPs) in six loci, three a priori SNPs in two loci (Chr5: rs60271; LBR: rs2230419, rs1011319) were associated with baseline BDI scores, but in the opposite direction of prior research. In joint analysis of 90,003 IBC and Cardiometabochip SNPs, rs1543654 in the region of KCNE1 predicted change in BDI scores at year 1 in DSE (beta= −1.05, SE=0.21, p=6.9 × 10−7) at the level of chip-wide significance, while also showing a nominal association with baseline BDI (beta=0.35, SE=0.16, p=0.026). Adjustment for antidepressant medication and/or limiting analyses to Non-Hispanic White individuals did not meaningfully alter results.
Previously reported genetic associations with depressive symptoms did not replicate in this cohort of overweight/obese individuals with type 2 diabetes. We identified KCNE1 as a potential novel locus associated with depressive symptoms.
PMCID: PMC4643359  PMID: 26489030
genetics; depression; diabetes; weight loss; obesity
4.  Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease 
Background & Aims
Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestinal NADPH oxidases might be present in children with IBD.
After targeted exome sequencing of epithelial NADPH oxidases NOX1 and DUOX2 on 209 children with very early onset inflammatory bowel disease (VEOIBD), the identified mutations were validated using Sanger Sequencing. A structural analysis of NOX1 and DUOX2 variants was performed by homology in silico modeling. The functional characterization included ROS generation in model cell lines and in in vivo transduced murine crypts, protein expression, intracellular localization, and cell-based infection studies with the enteric pathogens Campylobacter jejuni and enteropathogenic Escherichia coli.
We identified missense mutations in NOX1 (c.988G>A, p.Pro330Ser; c.967G>A, p.Asp360Asn) and DUOX2 (c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys) in 5 of 209 VEOIBD patients. The NOX1 p.Asp360Asn variant was replicated in a male Ashkenazi Jewish ulcerative colitis cohort. All NOX1 and DUOX2 variants showed reduced ROS production compared with wild-type enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated translocation of altered oxidases, cells harboring NOX1 or DUOX2 variants had defective host resistance to infection with C. jejuni.
This study identifies the first inactivating missense variants in NOX1 and DUOX2 associated with VEOIBD. Defective ROS production from intestinal epithelial cells constitutes a risk factor for developing VEOIBD.
PMCID: PMC4539615  PMID: 26301257
Inflammatory Bowel Disease; NADPH Oxidase; NOX1; DUOX2; Reactive Oxygen Species; VEOIBD
5.  C-Reactive protein reactions to glucose-insulin-potassium infusion and relations to infarct size in patients with acute coronary syndromes 
Some benefits of glucose-insulin-potassium (GIK) in patients with acute coronary syndromes (ACS) may be from an anti-inflammatory effect. The primary aim of this study was to assess the impact of GIK administration early in the course of ACS on inflammatory marker C-reactive protein (CRP) levels. A secondary aim was to investigate the association between CRP and 30-day infarct size.
Methods and Results
Retrospective analysis of participants with ACS randomly assigned to GIK or placebo for at least 8 h in the IMMEDIATE Trial biological mechanism cohort (n = 143). High sensitivity CRP (hs-CRP) was measured at emergency department presentation, and 6 and 12 h into infusion. Logarithmically transformed hs-CRP values at 12-hours were lower with GIK vs. placebo (mean =0.65 mg/L in GIK, 0.84 mg/L in placebo), with a marginal trend toward significance (P = 0.053). Furthermore, using mixed models of hs-CRP, time, and study group, there was a significant increase in hs-CRP levels over time, but the rate of change did not differ between treatment arms (P = 0.3). Multivariable analysis showed that an elevation in hs-CRP, measured at 12 h, was an independent predictor of 30-day infarct size (β coefficient, 6.80; P = 0.04) using sestamibi SPECT imaging.
The results of this study show no significant effect of GIK on hs-CRP. In addition our results show that in patients with ACS, hs-CRP measured as early as 12 h can predict 30-day infarct size.
Electronic supplementary material
The online version of this article (doi:10.1186/s12872-015-0153-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4668670  PMID: 26631004
Acute coronary syndromes; Glucose-insulin-potassium (GIK); Inflammation; C-reactive protein; Metabolic therapy
6.  Acute Porphyrias in the USA: Features of 108 Subjects from Porphyria Consortium 
The American journal of medicine  2014;127(12):1233-1241.
Recent descriptions of the clinical and laboratory features of subjects with acute porphyrias in the US are lacking. Our aim was to describe clinical, biochemical and genetic features of 108 subjects.
Between Sep 2010—Dec 2012, 108 subjects with acute porphyrias [90 acute intermittent porphyria, 9 hereditary coproporphyria, 9 variegate porphyria] were enrolled into an observational study. Genetic testing was performed at a central genetic testing laboratory and clinical information entered into a central data base. Selected features were compared to data for adults in the USA.
Most subjects [88/108, 81%] were female with self-reported onset of symptoms in 2nd–4th decades of life. The most common symptom was abdominal pain. Appendectomies and cholecystectomies were common prior to a diagnosis of porphyria. The diagnosis was delayed by a mean of 15 years. Anxiety and depression were common, and 18% complained of chronic symptoms, especially neuropathic and other pains. The incidences of and systemic arterial hypertension, chronic kidney disease, seizure disorders, and psychiatric conditions were markedly increased. Mutations of the known causative genes were found in 102/105 of those tested, with novel mutations being found in 37 including in 7/8 subjects with hereditary coproporphyria. Therapy with intravenous hematin was the most effective therapy both for treatment of acute attacks and for prevention of recurrent attacks.
Acute porphyrias often remain undiagnosed for more than a decade after first symptoms develop. Intravenous hematin is the treatment of choice, both for treatment of acute attacks and for prevention of recurrent attacks.
Trial Registration identifier: NCT01561157
PMCID: PMC4563803  PMID: 25016127
acute intermittent porphyria; clinical features; delta-aminolevulinic acid; hematin; heme; hereditary coproporphyria; hydroxymethylbilane synthase; porphobilinogen; porphobilinogen deaminase; porphyrins; variegate porphyria
7.  Pharmacogenetics in Jewish populations 
Spanning over 2000 years, the Jewish population has a long history of migration, population bottlenecks, expansions, and geographical isolation, which has resulted in a unique genetic architecture among the Jewish people. As such, many Mendelian disease genes and founder mutations for autosomal recessive diseases have been discovered in several Jewish groups, which have prompted recent genomic studies in the Jewish population on common disease susceptibility and other complex traits. Although few studies on the genetic determinants of drug response variability have been reported in the Jewish population, a number of unique pharmacogenetic variants have been discovered that are more common in Jewish populations than in other major racial groups. Notable examples identified in the Ashkenazi Jewish (AJ) population include the vitamin K epoxide reductase complex subunit 1 (VKORC1) c.106G>T (p.D36Y) variant associated with high warfarin dosing requirements and the recently reported cytochrome P450 2C19 (CYP2C19) allele, CYP2C19*4B, that harbors both loss-of-function [*4 (c.1A>G)] and increased-function [*17 (c.−806C>T)] variants on the same haplotype. These data are encouraging in that like other ethnicities and subpopulations, the Jewish population likely harbors numerous pharmacogenetic variants that are uncommon or absent in other larger racial groups and ethnicities. In addition to unique variants, common multi-ethnic variants in key drug metabolism genes (e.g., ABCB1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, NAT2) have also been detected in the AJ and other Jewish groups. This review aims to summarize the currently available pharmacogenetics literature and discuss future directions for related research with this unique population.
PMCID: PMC4221483  PMID: 24867283
Ashkenazi Jewish; CYP2C19*4B; Jewish genetics; pharmacogenetics; pharmacogenomics; VKORC1 p.D36Y
8.  Abnormal Newborn Screening Data and Acylcarnitines in HIV/ARV-Exposed Infants 
Antiretroviral drugs (ARV), specifically nucleoside analogs, are toxic to mitochondrial oxidative phosphorylation (OXPHOS). Other metabolic pathways, such as fatty acid oxidation, organic acid metabolism and amino acid metabolism, are dependent on normal OXPHOS but remain unexamined as potential points of ARV toxicity.
We analyzed newborn screening data from New York and compared proportions of abnormal newborn metabolic screens in HIV antibody screen-positive and HIV screen-negative neonates. Subsequently, we compared acylcarnitine levels in ARV-exposed (n=16) and ARV-unexposed (n=14) HIV-exposed infants to assess for dysfunctional fatty and organic acid metabolism.
The rate of abnormal newborn metabolic screens in HIV screen-positive infants was higher than that in the general population (2.2% versus 1.2%, p=0.00025), most of which were for disorders of mitochondria-related metabolism. Abnormal acylcarnitine levels occurred more frequently in ARV-exposed compared to ARV-unexposed infants (43% versus 0%, p=0.02).
A higher proportion of positive metabolic screens in HIV screen-positive neonates suggests that HIV or ARV exposure is associated with dysfunctional intermediary metabolism in newborns. Abnormal acylcarnitine levels were more frequent in ARV-exposed infants suggesting that ARV may perturb normal fatty acid oxidation in some infants. Studies designed to validate and determine the clinical significance of these findings are warranted.
PMCID: PMC4648253  PMID: 22935866
HIV/AIDS; mitochondrial toxicity; newborn screening; metabolism
10.  Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering 
Microbiome  2015;3:43.
High-throughput bacterial 16S rRNA gene sequencing followed by clustering of short sequences into operational taxonomic units (OTUs) is widely used for microbiome profiling. However, clustering of short 16S rRNA gene reads into biologically meaningful OTUs is challenging, in part because nucleotide variation along the 16S rRNA gene is only partially captured by short reads. The recent emergence of long-read platforms, such as single-molecule real-time (SMRT) sequencing from Pacific Biosciences, offers the potential for improved taxonomic and phylogenetic profiling. Here, we evaluate the performance of long- and short-read 16S rRNA gene sequencing using simulated and experimental data, followed by OTU inference using computational pipelines based on heuristic and complete-linkage hierarchical clustering.
In simulated data, long-read sequencing was shown to improve OTU quality and decrease variance. We then profiled 40 human gut microbiome samples using a combination of Illumina MiSeq and Blautia-specific SMRT sequencing, further supporting the notion that long reads can identify additional OTUs. We implemented a complete-linkage hierarchical clustering strategy using a flexible computational pipeline, tailored specifically for PacBio circular consensus sequencing (CCS) data that outperforms heuristic methods in most settings:
Our data demonstrate that long reads can improve OTU inference; however, the choice of clustering algorithm and associated clustering thresholds has significant impact on performance.
Electronic supplementary material
The online version of this article (doi:10.1186/s40168-015-0105-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4593230  PMID: 26434730
Microbiome; 16S rRNA gene sequencing; OTU picking
11.  Genetic Variation at the LDL Receptor and HMG CoA Reductase Gene Loci, Lipid Levels, Statin Response, and Cardiovascular Disease Incidence in PROSPER 
Atherosclerosis  2008;200(1):109-114.
Our purpose was to evaluate associations of single nucleotide polymorphisms (SNPs) at the low density lipoprotein (LDL) receptor (LDLR C44857T, minor allele frequency (MAF) 0.26, and A44964G, MAF 0.25, both in the untranslated region) and HMG-CoA reductase (HMGCR i18T>G, MAF 0.019) gene loci with baseline lipid values, statin induced LDL- cholesterol (C) lowering response, and incident coronary heart disease (CHD) and cardiovascular disease on trial (CVD). Our population consisted of 5804 elderly men and women with vascular disease or one or more vascular disease risk factors, who were randomly allocated to pravastatin or placebo. Other risk factors and apolipoprotein (apo) E phenotype were controlled for in the analysis. Despite a prior report, no relationships with the HMGCR SNP were noted. For the LDLR SNPs C44857T and A44964G we noted significant associations of the rare alleles with baseline LDL-C and triglyceride levels, a modest association of the C44857T with LDL-C lowering to pravastatin in men, and significant associations with incident CHD and CVD of both SNPs, especially in men on pravastatin. Our data indicate that genetic variation at the LDLR locus can affect baseline lipids, response to pravastatin, and CVD risk in subjects placed on statin treatment.
PMCID: PMC4565152  PMID: 18261733
Genetics; Statins; Low density lipoproteins (LDL); Coronary Heart Disease (CHD); HMGCR gene; LDLR gene
12.  Genome-wide mapping of IBD segments in an Ashkenazi PD cohort identifies associated haplotypes 
Human Molecular Genetics  2014;23(17):4693-4702.
The recent series of large genome-wide association studies in European and Japanese cohorts established that Parkinson disease (PD) has a substantial genetic component. To further investigate the genetic landscape of PD, we performed a genome-wide scan in the largest to date Ashkenazi Jewish cohort of 1130 Parkinson patients and 2611 pooled controls. Motivated by the reduced disease allele heterogeneity and a high degree of identical-by-descent (IBD) haplotype sharing in this founder population, we conducted a haplotype association study based on mapping of shared IBD segments. We observed significant haplotype association signals at three previously implicated Parkinson loci: LRRK2 (OR = 12.05, P = 1.23 × 10−56), MAPT (OR = 0.62, P = 1.78 × 10−11) and GBA (multiple distinct haplotypes, OR > 8.28, P = 1.13 × 10−11 and OR = 2.50, P = 1.22 × 10−9). In addition, we identified a novel association signal on chr2q14.3 coming from a rare haplotype (OR = 22.58, P = 1.21 × 10−10) and replicated it in a secondary cohort of 306 Ashkenazi PD cases and 2583 controls. Our results highlight the power of our haplotype association method, particularly useful in studies of founder populations, and reaffirm the benefits of studying complex diseases in Ashkenazi Jewish cohorts.
PMCID: PMC4119402  PMID: 24842889
13.  Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease 
Cell  2014;158(5):1000-1010.
Specific members of the intestinal microbiota dramatically affect inflammatory bowel disease (IBD) in mice. In humans, however, identifying bacteria that preferentially affect disease susceptibility and severity remains a major challenge. Here, we used flow cytometry-based bacterial cell sorting and 16S sequencing to characterize taxa-specific coating of the intestinal microbiota with immunoglobulin A (IgA−SEQ) and show that high IgA−coating uniquely identifies colitogenic intestinal bacteria in a mouse model of microbiota-driven colitis. We then used IgA−SEQ and extensive anaerobic culturing of fecal bacteria from IBD patients to create personalized disease-associated gut microbiota culture collections with pre-defined levels of IgA coating. Using these collections, we found that intestinal bacteria selected on the basis of high coating with IgA conferred dramatic susceptibility to colitis in germ-free mice. Thus, our studies suggest that IgA−coating identifies inflammatory commensals that preferentially drive intestinal disease. Targeted elimination of such bacteria may reduce, reverse, or even prevent disease development.
PMCID: PMC4174347  PMID: 25171403
14.  Closing the Gap: Genetic and Genomic Continuum from Syndromic to Nonsyndromic Craniosynostoses 
Current genetic medicine reports  2014;2(3):135-145.
Craniosynostosis, a condition that includes the premature fusion of one or multiple cranial sutures, is a relatively common birth defect in humans and the second most common craniofacial anomaly after orofacial clefts. There is a significant clinical variation among different sutural synostoses as well as significant variation within any given single-suture synostosis. Craniosynostosis can be isolated (i.e., nonsyndromic) or occurs as part of a genetic syndrome (e.g., Crouzon, Pfeiffer, Apert, Muenke, and Saethre-Chotzen syndromes). Approximately 85 % of all cases of craniosynostosis are nonsyndromic. Several recent genomic discoveries are elucidating the genetic basis for nonsyndromic cases and implicate the newly identified genes in signaling pathways previously found in syndromic craniosynostosis. Published epidemiologic and phenotypic studies clearly demonstrate that nonsyndromic craniosynostosis is a complex and heterogeneous condition supporting a strong genetic component accompanied by environmental factors that contribute to the pathogenetic network of this birth defect. Large population, rather than single-clinic or hospital-based studies is required with phenotypically homogeneous subsets of patients to further understand the complex genetic, maternal, environmental, and stochastic factors contributing to nonsyndromic craniosynostosis. Learning about these variables is a key in formulating the basis of multidisciplinary and lifelong care for patients with these conditions.
PMCID: PMC4489147  PMID: 26146596
Craniosynostosis; Suture; Sagittal synostosis; Coronal synostosis; Genome wide association study; Whole exome sequencing
15.  Cell-free DNA Fragmentation Patterns in Amniotic Fluid Identify Genetic Abnormalities and Changes due to Storage 
Circulating cell-free DNA (cfDNA) has become a promising biomarker in prenatal diagnosis. However, despite extensive studies in different body fluids, cfDNA predictive value is uncertain owing to the confounding factors that can affect its levels, such as gestational age, maternal weight, smoking status, and medications. Residual fresh and archived amniotic fluid (AF) supernatants were obtained from gravid women (mean gestational age 17 wk) carrying euploid (N = 36) and aneuploid (N = 29) fetuses, to characterize cfDNA-fragmentation patterns with regard to aneuploidy and storage time (−80°C). AF cfDNA was characterized by the real-time quantitative polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and pattern recognition of the DNA fragmentation. The distributions of cfDNA fragment lengths were compared using 6 measures that defined the locations and slopes for the first and last peaks, after elimination of the confounding variables. This method allowed for the unique classification of euploid and aneuploid cfDNA samples in AF, which had been matched for storage time. In addition, we showed that archived euploid AF samples gradually lose long cfDNA fragments: this loss accurately distinguishes them from the fresh samples. We present preliminary data using cfDNA-fragmentation patterns, to uniquely distinguish between AF samples of pregnant women with regard to aneuploidy and storage time, independent of gestational age and initial DNA amount. In addition to potential applications in prenatal diagnosis, these data suggest that archived AF samples consist of large amounts of short cfDNA fragments, which are undetectable using standard real-time polymerase chain reaction amplification.
PMCID: PMC4459511  PMID: 18382362
cell-free DNA; DNA fragmentation; storage; karyotype
16.  Evaluation of New and Established Age-Related Macular Degeneration Susceptibility Genes in the Women’s Health Initiative Sight Exam (WHI-SE) Study 
American journal of ophthalmology  2011;152(6):1005-1013.e1.
To assess whether established and newly reported genetic variants, independent of known lifestyle factors, are associated with the risk of age-related macular degeneration (AMD) among women participating in the Women’s Health Initiative Sight Exam (WHI-SE) Genetic Ancillary Study.
Multicenter case-control study.
One hundred and forty-six women with intermediate and late stages of AMD and 1269 subjects without AMD underwent ocular examinations and fundus photography to determine stage of AMD. Fourteen polymorphisms at or near 11 genes, including previously confirmed genes CFH, ARMS2/HTRA1, C2, C3, and CFI; recently reported AMD genes in the high-density lipoprotein cholesterol (HDL) pathway LIPC, ABCA1, CETP, and LPL; TIMP3/SYN3, a known ocular gene recently linked with AMD; and APOE, were assessed using logistic regression analysis.
After adjustment for demographic, behavioral, and other genetic factors, a protective effect was detected among TT carriers compared with non-carriers for the HDL pathway gene, LIPC rs493258, for intermediate and late AMD (OR [95% confidence interval]: 0.3 [0.2–-0.7], P = .003). Variants in CFH rs1410996, ARMS2/HTRA1 A69S, and C3 R102G were significantly associated with an increased risk of AMD. Individuals with the homozygous CFI rs10033900 TT genotype had a 2.9 [1.2–7.2]-fold increased risk, and those with the CFH Y402H GG genotype had a 2.2 [1.0–4.8]-fold higher risk of developing AMD compared with non-carriers. APOE4 carriers may have a reduced risk of intermediate/late AMD (OR = 0.5 [0.3–0.9], P = .015. Suggestive associations were seen between AMD and the HDL pathway genes CETP and LPL.
In this unique national cohort of women, we found associations with established AMD-related genetic factors and the recently reported LIPC gene in the HDL pathway. These findings may help develop novel therapeutic targets to treat or delay the onset of the disease.
PMCID: PMC4446967  PMID: 21906714
17.  Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS) 
BMC Genomics  2015;16(1):350.
DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS).
Optimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625–1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r = 0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r = 0.906; 42 CpG sites) and second generation sequencing (r = 0.933; 174 CpG sites); however, longer SMRT-BS amplicons (>1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r = 0.836-0.897 and r = 0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r = 0.940-0.951 and r = 0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702–866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines.
SMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (>1.0 kb) and the previously reported bisulfite sequencing PCR bias towards unmethylated DNA should be considered when measuring intermediately methylated regions. Coupled with an optimized bisulfite PCR protocol, SMRT-BS is capable of interrogating ~1.5 kb amplicons, which theoretically can cover ~91% of CpG islands in the human genome.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1572-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4422326  PMID: 25943404
DNA methylation; CpG islands; Bisulfite sequencing; Long-read sequencing; Third generation sequencing; Single-molecule real-time (SMRT) sequencing; Pacific Bioscience
18.  Gene-centric meta-analyses for central adiposity traits in up to 57 412 individuals of European descent confirm known loci and reveal several novel associations 
Yoneyama, Sachiko | Guo, Yiran | Lanktree, Matthew B. | Barnes, Michael R. | Elbers, Clara C. | Karczewski, Konrad J | Padmanabhan, Sandosh | Bauer, Florianne | Baumert, Jens | Beitelshees, Amber | Berenson, Gerald S. | Boer, Jolanda M.A. | Burke, Gregory | Cade, Brian | Chen, Wei | Cooper-Dehoff, Rhonda M. | Gaunt, Tom R. | Gieger, Christian | Gong, Yan | Gorski, Mathias | Heard-Costa, Nancy | Johnson, Toby | Lamonte, Michael J. | Mcdonough, Caitrin | Monda, Keri L. | Onland-Moret, N. Charlotte | Nelson, Christopher P. | O'Connell, Jeffrey R. | Ordovas, Jose | Peter, Inga | Peters, Annette | Shaffer, Jonathan | Shen, Haiqinq | Smith, Erin | Speilotes, Liz | Thomas, Fridtjof | Thorand, Barbara | Monique Verschuren, W. M. | Anand, Sonia S. | Dominiczak, Anna | Davidson, Karina W. | Hegele, Robert A. | Heid, Iris | Hofker, Marten H. | Huggins, Gordon S. | Illig, Thomas | Johnson, Julie A. | Kirkland, Susan | König, Wolfgang | Langaee, Taimour Y. | Mccaffery, Jeanne | Melander, Olle | Mitchell, Braxton D. | Munroe, Patricia | Murray, Sarah S. | Papanicolaou, George | Redline, Susan | Reilly, Muredach | Samani, Nilesh J. | Schork, Nicholas J. | Van Der Schouw, Yvonne T. | Shimbo, Daichi | Shuldiner, Alan R. | Tobin, Martin D. | Wijmenga, Cisca | Yusuf, Salim | Hakonarson, Hakon | Lange, Leslie A. | Demerath, Ellen W | Fox, Caroline S. | North, Kari E | Reiner, Alex P. | Keating, Brendan | Taylor, Kira C.
Human Molecular Genetics  2013;23(9):2498-2510.
Waist circumference (WC) and waist-to-hip ratio (WHR) are surrogate measures of central adiposity that are associated with adverse cardiovascular events, type 2 diabetes and cancer independent of body mass index (BMI). WC and WHR are highly heritable with multiple susceptibility loci identified to date. We assessed the association between SNPs and BMI-adjusted WC and WHR and unadjusted WC in up to 57 412 individuals of European descent from 22 cohorts collaborating with the NHLBI's Candidate Gene Association Resource (CARe) project. The study population consisted of women and men aged 20–80 years. Study participants were genotyped using the ITMAT/Broad/CARE array, which includes ∼50 000 cosmopolitan tagged SNPs across ∼2100 cardiovascular-related genes. Each trait was modeled as a function of age, study site and principal components to control for population stratification, and we conducted a fixed-effects meta-analysis. No new loci for WC were observed. For WHR analyses, three novel loci were significantly associated (P < 2.4 × 10−6). Previously unreported rs2811337-G near TMCC1 was associated with increased WHR (β ± SE, 0.048 ± 0.008, P = 7.7 × 10−9) as was rs7302703-G in HOXC10 (β = 0.044 ± 0.008, P = 2.9 × 10−7) and rs936108-C in PEMT (β = 0.035 ± 0.007, P = 1.9 × 10−6). Sex-stratified analyses revealed two additional novel signals among females only, rs12076073-A in SHC1 (β = 0.10 ± 0.02, P = 1.9 × 10−6) and rs1037575-A in ATBDB4 (β = 0.046 ± 0.01, P = 2.2 × 10−6), supporting an already established sexual dimorphism of central adiposity-related genetic variants. Functional analysis using ENCODE and eQTL databases revealed that several of these loci are in regulatory regions or regions with differential expression in adipose tissue.
PMCID: PMC3988452  PMID: 24345515
19.  Reciprocal duplication of the Williams-Beuren syndrome deletion on chromosome 7q11.23 is associated with schizophrenia 
Biological psychiatry  2013;75(5):371-377.
Several copy number variants (CNVs) have been implicated as susceptibility factors for schizophrenia (SZ). Some of these same CNV also increase risk for autism spectrum disorders (ASD), suggesting an etiologic overlap between these conditions. Recently, de novo duplications of a region on chromosome 7q11.23 were associated with ASD. The reciprocal deletion of this region causes Williams-Beuren syndrome (WBS).
We assayed an Ashkenazi Jewish cohort of 554 SZ cases and 1014 controls for copy number variation (CNV), using a high-density genome-wide array. An excess of large rare and de novo CNV were observed, including a 1.4 Mb duplication on chromosome 7q11.23 identified in two unrelated patients. To test whether this 7q11.23 duplication is also associated with SZ, we obtained data for 14,387 SZ cases and 28,139 controls from seven additional studies with high-resolution genome-wide CNV detection. We performed a meta-analysis, correcting for study population of origin, to assess whether the 7q11.23 duplication is associated with SZ.
We find duplications at 7q11.23 in 11 of 14,387 SZ cases with only 1 in 28,139 controls (unadjusted odds ratio, 21.52, 95% CI: 3.13-922.6, p-value 5.5×10-5; adjusted odds ratio 10.8, 95% CI: 1.46-79.62, p-value 0.007). Of three SZ duplication carriers with available detailed retrospective data, all show social anxiety and language delay premorbid to SZ onset, consistent with both human studies and animal models of the 7q11.23 duplication.
We have identified a new CNV associated with SZ. Reciprocal duplication of the Williams syndrome deletion at chromosome 7q11.23 confers an approximately 10-fold increase in risk for SZ.
PMCID: PMC3838485  PMID: 23871472
Schizophrenia; 7q11.23 duplication syndrome; Psychiatric genetics; Schizophrenia genetics; Autism; Williams-Beuren syndrome
20.  Human Cardiovascular Disease IBC Chip-Wide Association with Weight Loss and Weight Regain in the Look AHEAD Trial 
Human heredity  2013;75(0):160-174.
The present study identified genetic predictors of weight change during behavioral weight loss treatment.
Participants were 3,899 overweight/obese individuals with type 2 diabetes from Look AHEAD, a randomized controlled trial to determine the effects of intensive lifestyle intervention (ILI), including weight loss and physical activity, relative to diabetes support and education, on cardiovascular outcomes. Analyses focused on associations of single nucleotide polymorphisms (SNPs) on the Illumina CARe iSelect (IBC) chip (minor allele frequency >5%; n = 31,959) with weight change at year 1 and year 4, and weight regain at year 4, among individuals who lost ≥ 3% at year 1.
Two novel regions of significant chip-wide association with year-1 weight loss in ILI were identified (p < 2.96E-06). ABCB11 rs484066 was associated with 1.16 kg higher weight per minor allele at year 1, whereas TNFRSF11A, or RANK, rs17069904 was associated with 1.70 kg lower weight per allele at year 1.
This study, the largest to date on genetic predictors of weight loss and regain, indicates that SNPs within ABCB11, related to bile salt transfer, and TNFRSF11A, implicated in adipose tissue physiology, predict the magnitude of weight loss during behavioral intervention. These results provide new insights into potential biological mechanisms and may ultimately inform weight loss treatment.
PMCID: PMC4257841  PMID: 24081232
Type 2 diabetes; Obesity; Weight loss; Diet; Genetics
21.  Sequencing an Ashkenazi reference panel supports population-targeted personal genomics and illuminates Jewish and European origins 
Nature Communications  2014;5:4835.
The Ashkenazi Jewish (AJ) population is a genetic isolate close to European and Middle Eastern groups, with genetic diversity patterns conducive to disease mapping. Here we report high-depth sequencing of 128 complete genomes of AJ controls. Compared with European samples, our AJ panel has 47% more novel variants per genome and is eightfold more effective at filtering benign variants out of AJ clinical genomes. Our panel improves imputation accuracy for AJ SNP arrays by 28%, and covers at least one haplotype in ≈67% of any AJ genome with long, identical-by-descent segments. Reconstruction of recent AJ history from such segments confirms a recent bottleneck of merely ≈350 individuals. Modelling of ancient histories for AJ and European populations using their joint allele frequency spectrum determines AJ to be an even admixture of European and likely Middle Eastern origins. We date the split between the two ancestral populations to ≈12–25 Kyr, suggesting a predominantly Near Eastern source for the repopulation of Europe after the Last Glacial Maximum.
Ashkenazi Jews are a genetically isolated population with distinct patterns of genetic diversity. Here, the authors sequence the genomes of 128 Ashkenazi Jewish individuals and use the sequence information to provide insight into the population's European and Middle Eastern origins.
PMCID: PMC4164776  PMID: 25203624
22.  Frequency of the Cholesteryl Ester Storage Disease Common LIPA E8SJM Mutation (c.894G>A) in Various Racial and Ethnic Groups 
Hepatology (Baltimore, Md.)  2013;58(3):958-965.
Cholesteryl Ester Storage Disease (CESD) and Wolman disease are autosomal recessive later-onset and severe infantile disorders, respectively, which result from the deficient activity of lysosomal acid lipase (LAL). LAL is encoded by LIPA (10q23.31) and the most common mutation associated with CESD is an exon 8 splice junction mutation (c.894G>A; E8SJM), which expresses only ~3–5% of normally spliced LAL. However, the frequency of c.894G>A is unknown in most populations. To estimate the prevalence of CESD in different populations, the frequencies of the c.894G>A mutation were determined in 10,000 LIPA alleles from healthy African-American, Asian, Caucasian, Hispanic and Ashkenazi Jewish individuals from the greater New York metropolitan area and 6,578 LIPA alleles from African-American, Caucasian, and Hispanic subjects enrolled in the Dallas Heart Study. The combined c.894G>A allele frequencies from the two cohorts ranged from 0.0005 (Asian) to 0.0017 (Caucasian and Hispanic), which translated to carrier frequencies of 1 in 1,000 to ~1 in 300, respectively. No African-American heterozygotes were detected. Additionally, by surveying the available literature, c.894G>A was estimated to account for 60% (95% CI: 51%–69%) of reported mutations among multi-ethnic CESD patients. Using this estimate, the predicted prevalence of CESD in the Caucasian and Hispanic populations is ~0.8 per 100,000 (~1 in 130,000; 95% CI: ~1 in 90,000 to 1 in 170,000).
These data indicate that CESD may be under-diagnosed in the general Caucasian and Hispanic populations, which is important since clinical trials of enzyme replacement therapy for LAL deficiency are currently being developed. Moreover, future studies on CESD prevalence in African and Asian populations may require full-gene LIPA sequencing to determine heterozygote frequencies since c.894G>A is not common in these racial groups.
PMCID: PMC3690149  PMID: 23424026
lysosomal acid lipase deficiency; allele frequency; carrier frequency; disease prevalence; splice junction mutation; genotyping
23.  Evidence of expression variation and allelic imbalance in Crohn’s disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells 
Gene  2013;527(2):496-502.
Human dendritic cells (DCs) play an important role in induction and progression of Crohn’s disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~40% and ~70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.
PMCID: PMC4135475  PMID: 23850724
Allelic imbalance; NOD2; ATG16L1; Human dendritic cells; NDV; Crohn’s disease
24.  Do Genetic Modifiers of HDL-C and Triglyceride Levels also Modify Their Response to a Lifestyle Intervention in the Setting of Obesity and Type-2 Diabetes Mellitus? The Look AHEAD Study 
High-density lipoprotein cholesterol (HDL-C) and triglycerides are cardiovascular risk factors susceptible to lifestyle behavior modification and genetics. We hypothesized that genetic variants identified by genome-wide association studies (GWASs) as associated with HDL-C or triglyceride levels will modify 1-year treatment response to an intensive lifestyle intervention (ILI), relative to a usual care of diabetes support and education (DSE).
Methods and Results
We evaluated 82 SNPs, representing 31 loci demonstrated by GWAS to be associated with HDL-C and/or triglycerides, in 3,561 participants who consented for genetic studies and met eligibility criteria. Variants associated with higher baseline HDL-C levels, cholesterol ester transfer protein (CETP) rs3764261 and hepatic lipase (LIPC) rs8034802, were found to be associated with HDL-C increases with ILI (p=0.0038 and 0.013, respectively) and had nominally significant treatment interactions (p=0.047 and 0.046, respectively). The fatty acid desaturase-2 (FADS-2) rs1535 variant, associated with low baseline HDL-C (p=0.017), was associated with HDL-C increases with ILI (0.0037) and had a nominal treatment interaction (p= 0.035). ApoB (rs693) and LIPC (rs8034802) SNPs showed nominally significant associations with HDL-C and triglyceride changes with ILI and a treatment interaction (p<0.05). A PGS1 SNP (rs4082919) showed the most significant triglyceride treatment interaction in the full cohort (p=0.0009).
This is the first study to identify genetic variants modifying lipid responses to a randomized lifestyle behavior intervention in overweight/obese diabetic individuals. The effect of genetic factors on lipid changes may differ from the effects on baseline lipids and are modifiable by behavioral intervention.
PMCID: PMC4077278  PMID: 23861364
genomics; physiological; cholesterylester transfer protein genetics; triglycerides; behavior modification; lipoprotein
25.  FTO predicts weight regain in the Look AHEAD Clinical Trial 
International journal of obesity (2005)  2013;37(12):10.1038/ijo.2013.54.
Genome-wide association studies have provided new insights into the genetic factors that contribute to the development of obesity. We hypothesized that these genetic markers would also predict magnitude of weight loss and weight regain after initial weight loss.
Established obesity risk alleles available on the Illumina CARe iSelect (IBC) chip were characterized in 3,899 overweight or obese participants with type 2 diabetes from the Look AHEAD (Action for Health in Diabetes), a randomized trial to determine the effects of intensive lifestyle intervention (ILI) and Diabetes Support and Education (DSE) on cardiovascular morbidity and mortality. Primary analyses examined the interaction between 13 obesity-risk polymorphisms in 8 genes and randomized treatment arm in predicting weight change at year 1, and weight regain at year 4 among individuals who lost 3% or more of their baseline weight by year 1.
No SNPs were significantly associated with magnitude of weight loss or interacted with treatment arm at year 1. However, FTO rs3751812 predicted weight regain within DSE (1.56 kg per risk allele, p = 0.005), but not ILI (p = 0.761), resulting in SNP×treatment arm interaction (p = 0.009). In a partial replication of prior research, the obesity risk (G) allele at BDNF rs6265 was associated with greater weight regain across treatment arms (0.773 kg per risk allele), although results were of borderline statistical significance (p=0.051).
Variations in the FTO and BDNF loci may contribute risk of weight regain after weight loss.
PMCID: PMC3750057  PMID: 23628854
type 2 diabetes; obesity; weight loss, diet, genetics

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