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1.  SNP genotyping on a genome-wide amplified DOP-PCR template 
Nucleic Acids Research  2002;30(22):e125.
With the increasing demand for higher throughput single nucleotide polymorphism (SNP) genotyping, the quantity of genomic DNA often falls short of the number of assays required. We investigated the use of degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) to generate a template for our SNP genotyping methodology of fluorescence polarization template-directed dye-terminator incorporation detection. DOP-PCR employs a degenerate primer (5′-CCGACTCGAGNNNNNNATGTGG-3′) to produce non-specific uniform amplification of DNA. This approach has been successfully applied to microsatellite genotyping. We compared genotyping of DOP-PCR-amplified genomic DNA to genomic DNA as a template. Results were analyzed with respect to feasibility, allele loss of alleles, genotyping accuracy and storage conditions in a high-throughput genotyping environment. DOP-PCR yielded overall satisfactory results, with a certain loss in accuracy and quality of the genotype assignments. Accuracy and quality of genotypes generated from the DOP-PCR template also depended on storage conditions. Adding carrier DNA to a final concentration of 10 ng/µl improved results. In conclusion, we have successfully used DOP-PCR to amplify our genomic DNA collection for subsequent SNP genotyping as a standard process.
PMCID: PMC137182  PMID: 12434007
2.  GWAS of blood cell traits identifies novel associated loci and epistatic interactions in Caucasian and African-American children 
Human Molecular Genetics  2012;22(7):1457-1464.
Hematological traits are important clinical indicators, the genetic determinants of which have not been fully investigated. Common measures of hematological traits include red blood cell (RBC) count, hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), mean corpuscular volume (MCV), platelet count (PLT) and white blood cell (WBC) count. We carried out a genome-wide association study of the eight common hematological traits among 7943 African-American children and 6234 Caucasian children. In African Americans, we report five novel associations of HBE1 variants with HCT and MCHC, the alpha-globin gene cluster variants with RBC and MCHC, and a variant at the ARHGEF3 locus with PLT, as well as replication of four previously reported loci at genome-wide significance. In Caucasians, we report a novel association of variants at the COPZ1 locus with PLT as well as replication of four previously reported loci at genome-wide significance. Extended analysis of an association observed between MCH and the alpha-globin gene cluster variants demonstrated independent effects and epistatic interaction at the locus, impacting the risk of iron deficiency anemia in African Americans with specific genotype states. In summary, we extend the understanding of genetic variants underlying hematological traits based on analyses in African-American children.
doi:10.1093/hmg/dds534
PMCID: PMC3657475  PMID: 23263863
3.  Genetic Variation in Genes Encoding Airway Epithelial Potassium Channels Is Associated with Chronic Rhinosinusitis in a Pediatric Population 
PLoS ONE  2014;9(3):e89329.
Background
Apical potassium channels regulate ion transport in airway epithelial cells and influence air surface liquid (ASL) hydration and mucociliary clearance (MCC). We sought to identify whether genetic variation within genes encoding airway potassium channels is associated with chronic rhinosinusitis (CRS).
Methods
Single nucleotide polymorphism (SNP) genotypes for selected potassium channels were derived from data generated on the Illumnia HumanHap550 BeadChip or Illumina Human610-Quad BeadChip for 828 unrelated individuals diagnosed with CRS and 5,083 unrelated healthy controls from the Children's Hospital of Philadelphia (CHOP). Statistical analysis was performed with set-based tests using PLINK, and corrected for multiple testing.
Results
Set-based case control analysis revealed the gene KCNMA1 was associated with CRS in our Caucasian subset of the cohort (598 CRS cases and 3,489 controls; p = 0.022, based on 10,000 permutations). In addition there was borderline evidence that the gene KCNQ5 (p = 0.0704) was associated with the trait in our African American subset of the cohort (230 CRS cases and 1,594 controls). In addition to the top significant SNPs rs2917454 and rs6907229, imputation analysis uncovered additional genetic variants in KCNMA1 and in KCNQ5 that were associated with CRS.
Conclusions
We have implicated two airway epithelial potassium channels as novel susceptibility loci in contributing to the pathogenesis of CRS.
doi:10.1371/journal.pone.0089329
PMCID: PMC3940609  PMID: 24595210
4.  A genome wide association study of plasma uric acid levels in obese cases and never-overweight controls 
Obesity (Silver Spring, Md.)  2013;21(9):E490-E494.
Objective
To identify plasma uric acid related genes in extremely obese and normal weight individuals using genome wide association studies (GWAS).
Design and Methods
Using genotypes from a GWAS focusing on obesity and thinness, we performed quantitative trait association analyses (PLINK) for plasma uric acid levels in 1,060 extremely obese individuals [body mass index (BMI) >35 kg/m2] and normal-weight controls (BMI<25kg/m2). In 961 samples with uric acid data, 924 were females.
Results
Significant associations were found in SLC2A9 gene SNPs and plasma uric acid levels (rs6449213, P=3.15×10−12). DIP2C gene SNP rs877282 also reached genome wide significance(P=4,56×10−8). Weaker associations (P<1×10−5) were found in F5, PXDNL, FRAS1, LCORL, and MICAL2genes. Besides SLC2A9, 3 previously identified uric acid related genes ABCG2 (rs2622605, P=0.0026), SLC17A1 (rs3799344, P=0.0017), and RREB1 (rs1615495, P =0.00055) received marginal support in our study.
Conclusions
Two genes/chromosome regions reached genome wide association significance (P< 1× 10−7, 550K SNPs) in our GWAS : SLC2A9, the chromosome 2 60.1 Mb region (rs6723995), and the DIP2C gene region. Five other genes (F5, PXDNL, FRAS1, LCORL, and MICAL2) yielded P<1× 10−5. Four previous reported associations were replicated in our study, including SLC2A9, ABCG2, RREB, and SLC17A1.
doi:10.1002/oby.20303
PMCID: PMC3762924  PMID: 23703922
uric acid; genome wide association study; obesity
5.  Obesity-susceptibility loci and the tails of the pediatric BMI distribution 
Obesity (Silver Spring, Md.)  2013;21(6):1256-1260.
Objective
We aimed to determine if previously identified adult obesity susceptibility loci were associated uniformly with childhood BMI across the BMI distribution.
Design and Methods
Children were recruited through the Children's Hospital of Philadelphia (n=7225). Associations between the following loci and BMI were assessed using quantile regression: FTO (rs3751812), MC4R (rs12970134), TMEM18 (rs2867125), BDNF (rs6265), TNNI3K (rs1514175), NRXN3 (rs10146997), SEC16B (rs10913469), and GNPDA2 (rs13130484). BMI z-score (age and gender adjusted) was modeled as the dependent variable, and genotype risk score (sum of risk alleles carried at the 8 loci) was modeled as the independent variable.
Results
Each additional increase in genotype risk score was associated with an increase in BMI z-score at the 5th, 15th, 25th, 50th, 75th, 85th and 95th BMI z-score percentiles by 0.04 (±0.02, p=0.08), 0.07 (±0.01, p=9.58 × 10-7), 0.07 (±0.01, p=1.10 × 10-8), 0.09 (±0.01, p=3.13 × 10-22), 0.11 (±0.01, p=1.35 × 10-25), 0.11 (±0.01, p=1.98 × 10-20), and 0.06 (±0.01, p=2.44 × 10-6), respectively. Each additional increase in genotype risk score was associated with an increase in mean BMI z-score by 0.08 (±0.01, p=4.27 × 10-20).
Conclusion
Obesity risk alleles were more strongly associated with increases in BMI z-score at the upper tail compared to the lower tail of the distribution.
doi:10.1002/oby.20319
PMCID: PMC3661695  PMID: 23408508
6.  The missense variation landscape of FTO, MC4R and TMEM18 in obese children of African ancestry 
Obesity (Silver Spring, Md.)  2013;21(1):159-163.
Common variation at the loci harboring FTO, MC4R and TMEM18 is consistently reported as being statistically the most strongly associated with obesity. We investigated if these loci also harbor rarer missense variants that confer substantially higher risk of common childhood obesity in African American (AA) children. We sequenced the exons of FTO, MC4R and TMEM18 in an initial subset of our cohort i.e. 200 obese (BMI≥95th percentile) and 200 lean AA children (BMI≤5th percentile). Any missense exonic variants that were uncovered went on to be further genotyped in a further 768 obese and 768 lean (BMI≤50th percentile) children of the same ethnicity. A number of exonic variants were observed from our sequencing effort: seven in FTO, of which four were non-synonymous (A163T, G182A, M400V and A405V), thirteen in MC4R, of which six were non-synonymous (V103I, N123S, S136A, F202L, N240S and I251L) and four in TMEM18, of which two were non-synonymous (P2S and V113L). Follow-up genotyping of these missense variants revealed only one significant difference in allele frequency between cases and controls, namely with N240S in MC4R(Fisher's Exact P = 0.0001). In summary, moderately rare missense variants within the FTO, MC4R and TMEM18 genes observed in our study did not confer risk of common childhood obesity in African Americans except for a degree of evidence for one known loss-of-function variant in MC4R.
doi:10.1002/oby.20147
PMCID: PMC3605748  PMID: 23505181
Obesity; Pediatrics; Genomics
7.  Physical Activity And Physical Fitness 
The focus of the PhenX (Phenotypes and eXposures) Toolkit is to provide researchers whose expertise lies outside a particular area with key measures identified by experts for uniform use in large-scale genetic studies and other extensive epidemiologic efforts going forward. The current paper specifically addresses the PhenX Toolkit research domain of physical activity and physical fitness (PA/PF), which are often associated with health outcomes. A Working Group (WG) of content experts completed a 6-month consensus process in which they identified a set of 14 high-priority, low-burden, and scientifically supported measures. During this process the WG considered self-reported and objective measures which included the latest technology (e.g., accelerometers, pedometers, heart-rate monitors). They also sought the input of measurement experts and other members of the research community during their deliberations. A majority of the measures include protocols for children (or adolescents), adults, and older adults or are applicable to all ages.
Measures from the PA/PF domain and 20 other domains are publicly available and found at the PhenX Toolkit website, www.phenxtoolkit.org. The use of common measures and protocols across large studies enhances the capacity to combine or compare data across studies, benefitting both PA/PF experts and non-experts. Use of these common measures by the research community should increase statistical power and enhance the ability to answer scientific questions that might have previously gone unanswered.
doi:10.1016/j.amepre.2011.11.017
PMCID: PMC3331998  PMID: 22516489
8.  Integrative genomics identifies LMO1 as a neuroblastoma oncogene 
Nature  2010;469(7329):216-220.
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths1,2. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10−16, odds ratio of risk allele = 1.34 (95% confidence interval 1.25–1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
doi:10.1038/nature09609
PMCID: PMC3320515  PMID: 21124317
9.  Genome-wide association studies of adolescent idiopathic scoliosis suggest candidate susceptibility genes 
Human Molecular Genetics  2011;20(7):1456-1466.
Adolescent idiopathic scoliosis (AIS) is an unexplained and common spinal deformity seen in otherwise healthy children. Its pathophysiology is poorly understood despite intensive investigation. Although genetic underpinnings are clear, replicated susceptibility loci that could provide insight into etiology have not been forthcoming. To address these issues, we performed genome-wide association studies (GWAS) of ∼327 000 single nucleotide polymorphisms (SNPs) in 419 AIS families. We found strongest evidence of association with chromosome 3p26.3 SNPs in the proximity of the CHL1 gene (P < 8 × 10−8 for rs1400180). We genotyped additional chromosome 3p26.3 SNPs and tested replication in two follow-up case–control cohorts, obtaining strongest results when all three cohorts were combined (rs10510181 odds ratio = 1.49, 95% confidence interval = 1.29–1.73, P = 2.58 × 10−8), but these were not confirmed in a separate GWAS. CHL1 is of interest, as it encodes an axon guidance protein related to Robo3. Mutations in the Robo3 protein cause horizontal gaze palsy with progressive scoliosis (HGPPS), a rare disease marked by severe scoliosis. Other top associations in our GWAS were with SNPs in the DSCAM gene encoding an axon guidance protein in the same structural class with Chl1 and Robo3. We additionally found AIS associations with loci in CNTNAP2, supporting a previous study linking this gene with AIS. Cntnap2 is also of functional interest, as it interacts directly with L1 and Robo class proteins and participates in axon pathfinding. Our results suggest the relevance of axon guidance pathways in AIS susceptibility, although these findings require further study, particularly given the apparent genetic heterogeneity in this disease.
doi:10.1093/hmg/ddq571
PMCID: PMC3049353  PMID: 21216876
10.  Association of RASGRP1 with type 1 diabetes is revealed by combined follow-up of two genome-wide studies 
Journal of Medical Genetics  2009;46(8):553-554.
Background
The two genome-wide association studies published by us and by the Wellcome Trust Case-Control Consortium (WTCCC) revealed a number of novel loci but neither had the statistical power to elucidate all of the genetic components of type 1 diabetes risk, a task for which larger effective sample sizes are needed.
Methods
We analyzed data from two sources: 1) The previously published second stage of our study, with a total sample size of the two stages consisting of 1,046 Canadian case-parent trios and 538 multiplex families with 929 affected offspring from the Type 1 Diabetes Genetics Consortium (T1DGC); 2) The RR2 project of the T1DGC, which genotyped 4,417 individuals from 1,062 non-overlapping families, including 2,059 affected individuals (mostly sibling pairs) for the 1,536 markers with the highest statistical significance for type 1 diabetes in the WTCCC results.
Results
One locus, mapping to an LD block at chr15q14, reached statistical significance by combining results from two markers (rs17574546 and rs7171171) in perfect linkage disequilibrium (LD) with each other (r2=1). We obtained a joint p value of 1.3 ×10−6, which exceeds by an order of magnitude the conservative threshold of 3.26×10−5 obtained by correcting for the 1,536 SNPs tested in our study. Meta-analysis with the original WTCCC genome-wide data produced a p value of 5.83×10−9.
Conclusions
A novel type 1 diabetes locus was discovered. It involves RASGRP1, a gene known to play a crucial role in thymocyte differentiation and TCR signaling by activating the Ras signaling pathway.
doi:10.1136/jmg.2009.067140
PMCID: PMC3272492  PMID: 19465406
Etiology; Genetic susceptibility; Type 1 diabetes; RASGRP1
11.  Large Copy-Number Variations Are Enriched in Cases With Moderate to Extreme Obesity 
Diabetes  2010;59(10):2690-2694.
OBJECTIVE
Obesity is an increasingly common disorder that predisposes to several medical conditions, including type 2 diabetes. We investigated whether large and rare copy-number variations (CNVs) differentiate moderate to extreme obesity from never-overweight control subjects.
RESEARCH DESIGN AND METHODS
Using single nucleotide polymorphism (SNP) arrays, we performed a genome-wide CNV survey on 430 obese case subjects (BMI >35 kg/m2) and 379 never-overweight control subjects (BMI <25 kg/m2). All subjects were of European ancestry and were genotyped on the Illumina HumanHap550 arrays with ∼550,000 SNP markers. The CNV calls were generated by PennCNV software.
RESULTS
CNVs >1 Mb were found to be overrepresented in case versus control subjects (odds ratio [OR] = 1.5 [95% CI 0.5–5]), and CNVs >2 Mb were present in 1.3% of the case subjects but were absent in control subjects (OR = infinity [95% CI 1.2–infinity]). When focusing on rare deletions that disrupt genes, even more pronounced effect sizes are observed (OR = 2.7 [95% CI 0.5–27.1] for CNVs >1 Mb). Interestingly, obese case subjects who carry these large CNVs have moderately high BMI and do not appear to be extreme cases. Several CNVs disrupt known candidate genes for obesity, such as a 3.3-Mb deletion disrupting NAP1L5 and a 2.1-Mb deletion disrupting UCP1 and IL15.
CONCLUSIONS
Our results suggest that large CNVs, especially rare deletions, confer risk of obesity in patients with moderate obesity and that genes impacted by large CNVs represent intriguing candidates for obesity that warrant further study.
doi:10.2337/db10-0192
PMCID: PMC3279563  PMID: 20622171
12.  Pathway-Wide Association Study Implicates Multiple Sterol Transport and Metabolism Genes in HDL Cholesterol Regulation 
Pathway-based association methods have been proposed to be an effective approach in identifying disease genes, when single-marker association tests do not have sufficient power. The analysis of quantitative traits may be benefited from these approaches, by sampling from two extreme tails of the distribution. Here we tested a pathway association approach on a small genome-wide association study (GWAS) on 653 subjects with extremely high high-density lipoprotein cholesterol (HDL-C) levels and 784 subjects with low HDL-C levels. We identified 102 genes in the sterol transport and metabolism pathways that collectively associate with HDL-C levels, and replicated these association signals in an independent GWAS. Interestingly, the pathways include 18 genes implicated in previous GWAS on lipid traits, suggesting that genuine HDL-C genes are highly enriched in these pathways. Additionally, multiple biologically relevant loci in the pathways were not detected by previous GWAS, including genes implicated in previous candidate gene association studies (such as LEPR, APOA2, HDLBP, SOAT2), genes that cause Mendelian forms of lipid disorders (such as DHCR24), and genes expressing dyslipidemia phenotypes in knockout mice (such as SOAT1, PON1). Our study suggests that sampling from two extreme tails of a quantitative trait and examining genetic pathways may yield biological insights from smaller samples than are generally required using single-marker analysis in large-scale GWAS. Our results also implicate that functionally related genes work together to regulate complex quantitative traits, and that future large-scale studies may benefit from pathway-association approaches to identify novel pathways regulating HDL-C levels.
doi:10.3389/fgene.2011.00041
PMCID: PMC3268595  PMID: 22303337
GWAS; lipid; HDL-C; pathway analysis; cholesterol; sterol transport; sterol metabolism; genetic association
13.  A Genome-Wide Association Study on Obesity and Obesity-Related Traits 
PLoS ONE  2011;6(4):e18939.
Large-scale genome-wide association studies (GWAS) have identified many loci associated with body mass index (BMI), but few studies focused on obesity as a binary trait. Here we report the results of a GWAS and candidate SNP genotyping study of obesity, including extremely obese cases and never overweight controls as well as families segregating extreme obesity and thinness. We first performed a GWAS on 520 cases (BMI>35 kg/m2) and 540 control subjects (BMI<25 kg/m2), on measures of obesity and obesity-related traits. We subsequently followed up obesity-associated signals by genotyping the top ∼500 SNPs from GWAS in the combined sample of cases, controls and family members totaling 2,256 individuals. For the binary trait of obesity, we found 16 genome-wide significant signals within the FTO gene (strongest signal at rs17817449, P = 2.5×10−12). We next examined obesity-related quantitative traits (such as total body weight, waist circumference and waist to hip ratio), and detected genome-wide significant signals between waist to hip ratio and NRXN3 (rs11624704, P = 2.67×10−9), previously associated with body weight and fat distribution. Our study demonstrated how a relatively small sample ascertained through extreme phenotypes can detect genuine associations in a GWAS.
doi:10.1371/journal.pone.0018939
PMCID: PMC3084240  PMID: 21552555
14.  Examination of All Type 2 Diabetes GWAS Loci Reveals HHEX-IDE as a Locus Influencing Pediatric BMI 
Diabetes  2009;59(3):751-755.
OBJECTIVE
A number of studies have found that BMI in early life influences the risk of developing type 2 diabetes later in life. Our goal was to investigate if any type 2 diabetes variants uncovered through genome-wide association studies (GWAS) impact BMI in childhood.
RESEARCH DESIGN AND METHODS
Using data from an ongoing GWAS of pediatric BMI in our cohort, we investigated the association of pediatric BMI with 20 single nucleotide polymorphisms at 18 type 2 diabetes loci uncovered through GWAS, consisting of ADAMTS9, CDC123-CAMK1D, CDKAL1, CDKN2A/B, EXT2, FTO, HHEX-IDE, IGF2BP2, the intragenic region on 11p12, JAZF1, KCNQ1, LOC387761, MTNR1B, NOTCH2, SLC30A8, TCF7L2, THADA, and TSPAN8-LGR5. We randomly partitioned our cohort exactly in half in order to have a discovery cohort (n = 3,592) and a replication cohort (n = 3,592).
RESULTS
Our data show that the major type 2 diabetes risk–conferring G allele of rs7923837 at the HHEX-IDE locus was associated with higher pediatric BMI in both the discovery (P = 0.0013 and survived correction for 20 tests) and replication (P = 0.023) sets (combined P = 1.01 × 10−4). Association was not detected with any other known type 2 diabetes loci uncovered to date through GWAS except for the well-established FTO.
CONCLUSIONS
Our data show that the same genetic HHEX-IDE variant, which is associated with type 2 diabetes from previous studies, also influences pediatric BMI.
doi:10.2337/db09-0972
PMCID: PMC2828649  PMID: 19933996
15.  Association Between a High-Risk Autism Locus on 5p14 and Social Communication Spectrum Phenotypes in the General Population 
The American journal of psychiatry  2010;167(11):1364-1372.
Objective
Recent genome-wide analysis identified a genetic variant on 5p14.1 (rs4307059), which is associated with risk for autism spectrum disorder. This study investigated whether rs4307059 also operates as a quantitative trait locus underlying a broader autism phenotype in the general population, focusing specifically on the social communication aspect of the spectrum.
Method
Study participants were 7,313 children from the Avon Longitudinal Study of Parents and Children. Single-trait and joint-trait genotype associations were investigated for 29 measures related to language and communication, verbal intelligence, social interaction, and behavioral adjustment, assessed between ages 3 and 12 years. Analyses were performed in one-sided or directed mode and adjusted for multiple testing, trait interrelatedness, and random genotype dropout.
Results
Single phenotype analyses showed that an increased load of rs4307059 risk allele is associated with stereotyped conversation and lower pragmatic communication skills, as measured by the Children's Communication Checklist (at a mean age of 9.7 years). In addition a trend toward a higher frequency of identification of special educational needs (at a mean age of 11.8 years) was observed. Variation at rs4307059 was also associated with the phenotypic profile of studied traits. This joint signal was fully explained neither by single-trait associations nor by overall behavioral adjustment problems but suggested a combined effect, which manifested through multiple subthreshold social, communicative, and cognitive impairments.
Conclusions
Our results suggest that common variation at 5p14.1 is associated with social communication spectrum phenotypes in the general population and support the role of rs4307059 as a quantitative trait locus for autism spectrum disorder.
doi:10.1176/appi.ajp.2010.09121789
PMCID: PMC3008767  PMID: 20634369
16.  Examination of Type 2 Diabetes Loci Implicates CDKAL1 as a Birth Weight Gene 
Diabetes  2009;58(10):2414-2418.
OBJECTIVE
A number of studies have found that reduced birth weight is associated with type 2 diabetes later in life; however, the underlying mechanism for this correlation remains unresolved. Recently, association has been demonstrated between low birth weight and single nucleotide polymorphisms (SNPs) at the CDKAL1 and HHEX-IDE loci, regions that were previously implicated in the pathogenesis of type 2 diabetes. In order to investigate whether type 2 diabetes risk–conferring alleles associate with low birth weight in our Caucasian childhood cohort, we examined the effects of 20 such loci on this trait.
RESEARCH DESIGN AND METHODS
Using data from an ongoing genome-wide association study in our cohort of 5,465 Caucasian children with recorded birth weights, we investigated the association of the previously reported type 2 diabetes–associated variation at 20 loci including TCF7L2, HHEX-IDE, PPARG, KCNJ11, SLC30A8, IGF2BP2, CDKAL1, CDKN2A/2B, and JAZF1 with birth weight.
RESULTS
Our data show that the minor allele of rs7756992 (P = 8 × 10−5) at the CDKAL1 locus is strongly associated with lower birth weight, whereas a perfect surrogate for variation previously implicated for the trait at the same locus only yielded nominally significant association (P = 0.01; r2 rs7756992 = 0.677). However, association was not detected with any of the other type 2 diabetes loci studied.
CONCLUSIONS
We observe association between lower birth weight and type 2 diabetes risk–conferring alleles at the CDKAL1 locus. Our data show that the same genetic locus that has been identified as a marker for type 2 diabetes in previous studies also influences birth weight.
doi:10.2337/db09-0506
PMCID: PMC2750235  PMID: 19592620
17.  The role of obesity-associated loci identified in genome wide association studies in the determination of pediatric BMI 
Obesity (Silver Spring, Md.)  2009;17(12):2254-2257.
The prevalence of obesity in children and adults in the United States has increased dramatically over the past decade. Besides environmental factors, genetic factors are known to play an important role in the pathogenesis of obesity. A number of genetic determinants of adult BMI have already been established through genome wide association studies. In this study, we examined 25 single nucleotide polymorphisms (SNPs) corresponding to thirteen previously reported genomic loci in 6,078 children with measures of BMI. Fifteen of these SNPs yielded at least nominally significant association to BMI, representing nine different loci including INSIG2, FTO, MC4R, TMEM18, GNPDA2, NEGR1, BDNF, KCTD15 and 1q25. Other loci revealed no evidence for association, namely at MTCH2, SH2B1, 12q13 and 3q27. For the 15 associated variants, the genotype score explained 1.12% of the total variation for BMI z-score. We conclude that among thirteen loci that have been reported to associate with adult BMI, at least nine also contribute to the determination of BMI in childhood as demonstrated by their associations in our pediatric cohort.
doi:10.1038/oby.2009.159
PMCID: PMC2860782  PMID: 19478790
18.  Investigation of the locus near MC4R with childhood obesity in Americans of European and African ancestry 
Obesity (Silver Spring, Md.)  2009;17(7):1461-1465.
Recently a modest, but consistently, replicated association was demonstrated between obesity and the single nucleotide polymorphism (SNP), rs17782313, 3’ of the MC4R locus as a consequence of a meta-analysis of genome wide association (GWA) studies of the disease in Caucasian populations. We investigated the association in the context of the childhood form of the disease utilizing data from our ongoing GWA study in a cohort of 728 European American (EA) obese children (BMI ≥ 95th percentile) and 3,960 EA controls (BMI < 95th percentile), as well as 1,008 African American (AA) obese children and 2,715 AA controls. rs571312, rs10871777 and rs476828 (perfect surrogates for rs17782313) yielded odds ratios in the EA cohort of 1.142 (P = 0.045), 1.137 (P = 0.054) and 1.145 (P = 0.042); however, there was no significant association with these SNPs in the AA cohort. When investigating all thirty SNPs present on the Illumina BeadChip at this locus, again there was no evidence for association in AA cases when correcting for the number of tests employed. As such, variants 3’ to the MC4R locus present on the genotyping platform utilized confer a similar magnitude of risk of obesity in Caucasian children as to their adult Caucasian counterparts but this observation did not extend to African Americans.
doi:10.1038/oby.2009.53
PMCID: PMC2860794  PMID: 19265794
19.  SNP array mapping of 20p deletions: Genotypes, Phenotypes and Copy Number Variation 
Human mutation  2009;30(3):371-378.
The use of array technology to define chromosome deletions and duplications is bringing us closer to establishing a genotype/phenotype map of genomic copy number alterations. We studied 21 patients and 5 relatives with deletions of the short arm of chromosome 20 using the Illumina HumanHap550 SNP array to 1) more accurately determine the deletion sizes, 2) identify and compare breakpoints, 3) establish genotype/phenotype correlations and 4) investigate the use of the HumanHap550 platform for analysis of chromosome deletions. Deletions ranged from 95kb to 14.62Mb, and all of the breakpoints were unique. Eleven patients had deletions between 95kb and 4Mb and these individuals had normal development, with no anomalies outside of those associated with Alagille syndrome. The proximal and distal boundaries of these eleven deletions constitute a 5.4MB region, and we propose that haploinsufficiency for only 1 of the 12 genes in this region causes phenotypic abnormalities. This defines the JAG1 associated critical region, in which deletions do not confer findings other than those associated with Alagille syndrome. The other 10 patients had deletions between 3.28Mb and 14.62Mb, which extended outside the critical region, and notably, all of these patients, had developmental delay. This group had other findings such as autism, scoliosis and bifid uvula. We identified 47 additional polymorphic genome-wide copy number variants (>20 SNPs), with 0–5 variants called per patient. Deletions of the short arm of chromosome 20 are associated with relatively mild and limited clinical anomalies. The use of SNP arrays provides accurate high-resolution definition of genomic abnormalities.
doi:10.1002/humu.20863
PMCID: PMC2650004  PMID: 19058200
SNP array analysis; 20p deletion; copy number variants; Alagille syndrome; haploinsufficiency; JAG1
20.  ATOM: a powerful gene-based association test by combining optimally weighted markers 
Bioinformatics  2008;25(4):497-503.
Background: Large-scale candidate-gene and genome-wide association studies genotype multiple SNPs within or surrounding a gene, including both tag and functional SNPs. The immense amount of data generated in these studies poses new challenges to analysis. One particularly challenging yet important question is how to best use all genetic information to test whether a gene or a region is associated with the trait of interest.
Methods: Here we propose a powerful gene-based Association Test by combining Optimally Weighted Markers (ATOM) within a genomic region. Due to variation in linkage disequilibrium, different markers often associate with the trait of interest at different levels. To appropriately apportion their contributions, we assign a weight to each marker that is proportional to the amount of information it captures about the trait locus. We analytically derive the optimal weights for both quantitative and binary traits, and describe a procedure for estimating the weights from a reference database such as the HapMap. Compared with existing approaches, our method has several distinct advantages, including (i) the ability to borrow information from an external database to increase power, (ii) the theoretical derivation of optimal marker weights and (iii) the scalability to simultaneous analysis of all SNPs in candidate genes and pathways.
Results: Through extensive simulations and analysis of the FTO gene in our ongoing genome-wide association study on childhood obesity, we demonstrate that ATOM increases the power to detect genetic association as compared with several commonly used multi-marker association tests.
Contact: mingyao@mail.med.upenn.edu; chun.li@vanderbilt.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btn641
PMCID: PMC2642636  PMID: 19074959
21.  Follow-Up Analysis of Genome-Wide Association Data Identifies Novel Loci for Type 1 Diabetes 
Diabetes  2009;58(1):290-295.
OBJECTIVE—Two recent genome-wide association (GWA) studies have revealed novel loci for type 1 diabetes, a common multifactorial disease with a strong genetic component. To fully utilize the GWA data that we had obtained by genotyping 563 type 1 diabetes probands and 1,146 control subjects, as well as 483 case subject–parent trios, using the Illumina HumanHap550 BeadChip, we designed a full stage 2 study to capture other possible association signals.
RESEARCH DESIGN AND METHODS—From our existing datasets, we selected 982 markers with P < 0.05 in both GWA cohorts. Genotyping these in an independent set of 636 nuclear families with 974 affected offspring revealed 75 markers that also had P < 0.05 in this third cohort. Among these, six single nucleotide polymorphisms in five novel loci also had P < 0.05 in the Wellcome Trust Case-Control Consortium dataset and were further tested in 1,303 type 1 diabetes probands from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) plus 1,673 control subjects.
RESULTS—Two markers (rs9976767 and rs3757247) remained significant after adjusting for the number of tests in this last cohort; they reside in UBASH3A (OR 1.16; combined P = 2.33 × 10−8) and BACH2 (1.13; combined P = 1.25 × 10−6).
CONCLUSIONS—Evaluation of a large number of statistical GWA candidates in several independent cohorts has revealed additional loci that are associated with type 1 diabetes. The two genes at these respective loci, UBASH3A and BACH2, are both biologically relevant to autoimmunity.
doi:10.2337/db08-1022
PMCID: PMC2606889  PMID: 18840781
22.  Association Analysis of Type 2 Diabetes Loci in Type 1 Diabetes 
Diabetes  2008;57(7):1983-1986.
OBJECTIVE—To search for a possible association of type 1 diabetes with 10 validated type 2 diabetes loci, i.e., PPARG, KCNJ11, WFS1, HNF1B, IDE/HHEX, SLC30A8, CDKAL1, CDKN2A/B, IGF2BP2, and FTO/RPGRIP1L.
RESEARCH DESIGN AND METHODS—Two European population samples were studied: 1) one case-control cohort of 514 type 1 diabetic subjects and 2,027 control subjects and 2) one family cohort of 483 complete type 1 diabetic case-parent trios (total 997 affected). A total of 13 tag single nucleotide polymorphisms (SNPs) from the 10 type 2 diabetes loci were analyzed for type 1 diabetes association.
RESULTS—No association of type 1 diabetes was found with any of the 10 type 2 diabetes loci, and no age-at-onset effect was detected. By combined analysis using the Wellcome Trust Case-Control Consortium type 1 diabetes data, SNP rs1412829 in the CDKN2A/B locus bordered on significance (P = 0.039) (odds ratio 0.929 [95% CI 0.867–0.995]), which did not reach the statistical significance threshold adjusted for 13 tests (α = 0.00385).
CONCLUSIONS—This study suggests that the type 2 diabetes loci do not play any obvious role in type 1 diabetes genetic susceptibility. The distinct molecular mechanisms of the two diseases highlighted the importance of differentiation diagnosis and different treatment principles.
doi:10.2337/db08-0270
PMCID: PMC2453613  PMID: 18426861
23.  Concept, Design and Implementation of a Cardiovascular Gene-Centric 50 K SNP Array for Large-Scale Genomic Association Studies 
PLoS ONE  2008;3(10):e3583.
A wealth of genetic associations for cardiovascular and metabolic phenotypes in humans has been accumulating over the last decade, in particular a large number of loci derived from recent genome wide association studies (GWAS). True complex disease-associated loci often exert modest effects, so their delineation currently requires integration of diverse phenotypic data from large studies to ensure robust meta-analyses. We have designed a gene-centric 50 K single nucleotide polymorphism (SNP) array to assess potentially relevant loci across a range of cardiovascular, metabolic and inflammatory syndromes. The array utilizes a “cosmopolitan” tagging approach to capture the genetic diversity across ∼2,000 loci in populations represented in the HapMap and SeattleSNPs projects. The array content is informed by GWAS of vascular and inflammatory disease, expression quantitative trait loci implicated in atherosclerosis, pathway based approaches and comprehensive literature searching. The custom flexibility of the array platform facilitated interrogation of loci at differing stringencies, according to a gene prioritization strategy that allows saturation of high priority loci with a greater density of markers than the existing GWAS tools, particularly in African HapMap samples. We also demonstrate that the IBC array can be used to complement GWAS, increasing coverage in high priority CVD-related loci across all major HapMap populations. DNA from over 200,000 extensively phenotyped individuals will be genotyped with this array with a significant portion of the generated data being released into the academic domain facilitating in silico replication attempts, analyses of rare variants and cross-cohort meta-analyses in diverse populations. These datasets will also facilitate more robust secondary analyses, such as explorations with alternative genetic models, epistasis and gene-environment interactions.
doi:10.1371/journal.pone.0003583
PMCID: PMC2571995  PMID: 18974833
24.  Modeling genetic inheritance of copy number variations 
Nucleic Acids Research  2008;36(21):e138.
Copy number variations (CNVs) are being used as genetic markers or functional candidates in gene-mapping studies. However, unlike single nucleotide polymorphism or microsatellite genotyping techniques, most CNV detection methods are limited to detecting total copy numbers, rather than copy number in each of the two homologous chromosomes. To address this issue, we developed a statistical framework for intensity-based CNV detection platforms using family data. Our algorithm identifies CNVs for a family simultaneously, thus avoiding the generation of calls with Mendelian inconsistency while maintaining the ability to detect de novo CNVs. Applications to simulated data and real data indicate that our method significantly improves both call rates and accuracy of boundary inference, compared to existing approaches. We further illustrate the use of Mendelian inheritance to infer SNP allele compositions in each of the two homologous chromosomes in CNV regions using real data. Finally, we applied our method to a set of families genotyped using both the Illumina HumanHap550 and Affymetrix genome-wide 5.0 arrays to demonstrate its performance on both inherited and de novo CNVs. In conclusion, our method produces accurate CNV calls, gives probabilistic estimates of CNV transmission and builds a solid foundation for the development of linkage and association tests utilizing CNVs.
doi:10.1093/nar/gkn641
PMCID: PMC2588508  PMID: 18832372
25.  Association Analysis of the FTO Gene with Obesity in Children of Caucasian and African Ancestry Reveals a Common Tagging SNP 
PLoS ONE  2008;3(3):e1746.
Recently an association was demonstrated between the single nucleotide polymorphism (SNP), rs9939609, within the FTO locus and obesity as a consequence of a genome wide association (GWA) study of type 2 diabetes in adults. We examined the effects of two perfect surrogates for this SNP plus 11 other SNPs at this locus with respect to our childhood obesity cohort, consisting of both Caucasians and African Americans (AA). Utilizing data from our ongoing GWA study in our cohort of 418 Caucasian obese children (BMI≥95th percentile), 2,270 Caucasian controls (BMI<95th percentile), 578 AA obese children and 1,424 AA controls, we investigated the association of the previously reported variation at the FTO locus with the childhood form of this disease in both ethnicities. The minor allele frequencies (MAF) of rs8050136 and rs3751812 (perfect surrogates for rs9939609 i.e. both r2 = 1) in the Caucasian cases were 0.448 and 0.443 respectively while they were 0.391 and 0.386 in Caucasian controls respectively, yielding for both an odds ratio (OR) of 1.27 (95% CI 1.08–1.47; P = 0.0022). Furthermore, the MAFs of rs8050136 and rs3751812 in the AA cases were 0.449 and 0.115 respectively while they were 0.436 and 0.090 in AA controls respectively, yielding an OR of 1.05 (95% CI 0.91–1.21; P = 0.49) and of 1.31 (95% CI 1.050–1.643; P = 0.017) respectively. Investigating all 13 SNPs present on the Illumina HumanHap550 BeadChip in this region of linkage disequilibrium, rs3751812 was the only SNP conferring significant risk in AA. We have therefore replicated and refined the association in an AA cohort and distilled a tag-SNP, rs3751812, which captures the ancestral origin of the actual mutation. As such, variants in the FTO gene confer a similar magnitude of risk of obesity to children as to their adult counterparts and appear to have a global impact.
doi:10.1371/journal.pone.0001746
PMCID: PMC2262153  PMID: 18335027

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