Utilization of Positive and Negative Controls to Examine Comorbid Associations in Observational Database Studies.

BACKGROUND: Opportunities to leverage observational data for precision medicine research are hampered by underlying sources of bias and paucity of methods to handle resulting uncertainty. We outline an approach to account for bias in identifying comorbid associations between 2 rare genetic disorders and type 2 diabetes (T2D) by applying a positive and negative control disease paradigm. RESEARCH DESIGN: Association between 10 common and 2 rare genetic disorders [Hereditary Fructose Intolerance (HFI) and α-1 antitrypsin deficiency] and T2D was compared with the association between T2D and 7 negative control diseases with no established relationship with T2D in 4 observational databases. Negative controls were used to estimate how much bias and variance existed in datasets when no effect should be observed. RESULTS: Unadjusted association for common and rare genetic disorders and T2D was positive and variable in magnitude and distribution in all 4 databases. However, association between negative controls and T2D was 200% greater than expected indicating the magnitude and confidence intervals for comorbid associations are sensitive to systematic bias. A meta-analysis using this method demonstrated a significant association between HFI and T2D but not for α-1 antitrypsin deficiency. CONCLUSIONS: For observational studies, when covariate data are limited or ambiguous, positive and negative controls provide a method to account for the broadest level of systematic bias, heterogeneity, and uncertainty. This provides greater confidence in assessing associations between diseases and comorbidities. Using this approach we were able to demonstrate an association between HFI and T2D. Leveraging real-world databases is a promising approach to identify and corroborate potential targets for precision medicine therapies.

Genes contributing to pain sensitivity in the normal population: an exome sequencing study.

Sensitivity to pain varies considerably between individuals and is known to be heritable. Increased sensitivity to experimental pain is a risk factor for developing chronic pain, a common and debilitating but poorly understood symptom. To understand mechanisms underlying pain sensitivity and to search for rare gene variants (MAF<5%) influencing pain sensitivity, we explored the genetic variation in individuals' responses to experimental pain. Quantitative sensory testing to heat pain was performed in 2,500 volunteers from TwinsUK (TUK): exome sequencing to a depth of 70× was carried out on DNA from singletons at the high and low ends of the heat pain sensitivity distribution in two separate subsamples. Thus in TUK1, 101 pain-sensitive and 102 pain-insensitive were examined, while in TUK2 there were 114 and 96 individuals respectively. A combination of methods was used to test the association between rare variants and pain sensitivity, and the function of the genes identified was explored using network analysis. Using causal reasoning analysis on the genes with different patterns of SNVs by pain sensitivity status, we observed a significant enrichment of variants in genes of the angiotensin pathway (Bonferroni corrected p = 3.8×10(-4)). This pathway is already implicated in animal models and human studies of pain, supporting the notion that it may provide fruitful new targets in pain management. The approach of sequencing extreme exome variation in normal individuals has provided important insights into gene networks mediating pain sensitivity in humans and will be applicable to other common complex traits.

Baseline Serum and Stool Microbiome Biomarkers Predict Clinical Efficacy and Tissue Molecular Response After Ritlecitinib Induction Therapy in Ulcerative Colitis.

BACKGROUND AND AIMS: Ritlecitinib, an oral JAK3/TEC family kinase inhibitor, was well-tolerated and efficacious in the phase 2b VIBRATO study in participants with moderate-to-severe ulcerative colitis [UC]. The aim of this study was to identify baseline serum and microbiome markers that predict subsequent clinical efficacy and to develop noninvasive serum signatures as potential real-time noninvasive surrogates of clinical efficacy after ritlecitinib. METHODS: Tissue and peripheral blood proteomics, transcriptomics, and faecal metagenomics were performed on samples before and after 8 weeks of oral ritlecitinib induction therapy [20 mg, 70 mg, 200 mg, or placebo once daily, N = 39, 41, 33, and 18, respectively]. Linear mixed models were used to identify baseline and longitudinal protein markers associated with efficacy. The combined predictivity of these proteins was evaluated using a logistic model with permuted efficacy data. Differential expression of faecal metagenomics was used to differentiate responders and nonresponders. RESULTS: Peripheral blood serum proteomics identified four baseline serum markers [LTA, CCL21, HLA-E, MEGF10] predictive of modified clinical remission [MR], endoscopic improvement [EI], histological remission [HR], and integrative score of tissue molecular improvement. In responders, 37 serum proteins significantly changed at Week 8 compared with baseline [false discovery rate of <0.05]; of these, changes in four [IL4R, TNFRSF4, SPINK4, and LAIR-1] predicted concurrent EI and HR responses. Faecal metagenomics analysis revealed baseline and treatment response signatures that correlated with EI, MR, and tissue molecular improvement. CONCLUSIONS: Blood and microbiome biomarkers stratify endoscopic, histological, and tissue molecular responses to ritlecitinib, which may help guide future precision medicine approaches to UC treatment. ClinicalTrials.gov NCT02958865.

Genome-wide pharmacogenetics of anti-drug antibody response to bococizumab highlights key residues in HLA DRB1 and DQB1.

In this largest to-date genetic analysis of anti-drug antibody (ADA) response to a therapeutic monoclonal antibody (MAb), genome-wide association was performed for five measures of ADA status among 8844 individuals randomized to bococizumab, which targets PCSK9 for LDL-C lowering and cardiovascular protection. Index associations prioritized specific amino acid substitutions at the DRB1 and DQB1 MHC class II genes rather than canonical haplotypes. Two clusters of missense variants at DRB1 were associated with general ADA measures (residues 9, 11, 13; and 96, 112, 120, 180) and a third cluster of missense variants in DQB1 was associated with ADA measures including neutralizing antibody (NAb) titers (residues 66, 67, 71, 74, 75). The structural disposition of the missense substitutions implicates peptide antigen binding and CD4 effector function, mechanisms that are potentially generalizable to other therapeutic mAbs.Clinicaltrials.gov: NCT01968954, NCT01968967, NCT01968980, NCT01975376, NCT01975389, NCT02100514.

Antitumor Necrosis Factor-like Ligand 1A Therapy Targets Tissue Inflammation and Fibrosis Pathways and Reduces Gut Pathobionts in Ulcerative Colitis.

BACKGROUND: The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known. METHODS: Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis. RESULTS: Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy. CONCLUSIONS: The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.

Self-report data as a tool for subtype identification in genetically-defined Parkinson's Disease.

Through a targeted recruitment 23andMe has collected DNA and patient-reported symptoms from more than 10,000 subjects reporting a physician-verified diagnosis of PD. This study evaluated the potential of self-report, web-based questionnaires to rapidly assess disease natural history and symptomology in genetically-defined PD populations. While average age-at-diagnosis was significantly lower in GBA mutation carriers compared to idiopathic PD, or iPD (idiopathic PD, defined as no GBA mutations and no LRRK2 G2019S mutation), there were no significant differences in symptoms. Conversely, LRRK2 G2019S carrier status significantly associated with reporting of milder daily symptoms of lightheadedness and several differences were observed at a false discovery rate < 0.1, including increased reporting of changes in walking as an initial symptom of disease, decreased reporting of lightheadedness upon standing, and milder symptoms related to daily functioning. The subclinical differences in symptoms reported by LRRK2 G2019S carriers suggest differences in underlying pathophysiology and/or disease progression in LRRK2 carriers compared to iPD. Importantly, we confirm previous findings in PD genetic subsets where disease characteristics were ascertained through clinical exam. Overall, these data support the effective use of self-report and genetic data to rapidly analyze information from a large disease population or difficult to identify genetic subgroups.

Association of White Matter With Core Cognitive Deficits in Patients With Schizophrenia.

Importance: Efforts to remediate the multiple cognitive function impairments in schizophrenia should consider white matter as one of the underlying neural mechanisms. Objective: To determine whether altered structural brain connectivity is responsible for 2 of the core cognitive deficits in schizophrenia- reduced information processing speed and impaired working memory. Design, Setting, and Participants: This cross-sectional study design took place in outpatient clinics from August 1, 2004, to August 31, 2015. Participants included 166 patients with schizophrenia and 213 healthy control individuals. These participants were from 3 independent cohorts, each of which had its own healthy control group. No participant had current or past neurological conditions or major medical conditions. Patients were diagnosed with either schizophrenia or schizoaffective disorder as defined by the DSM-IV. Controls had no Axis I psychiatric disorder. Main Outcomes and Measures: Mediation analyses and structural equation modeling were used to analyze the associations among processing speed, working memory, and white matter microstructures. Whole-brain and regional diffusion tensor imaging fractional anisotropy were used to measure white matter microstructures. Results: Of the study participants, the 166 patients with schizophrenia had a mean (SD) age of 38.2 (13.3) years and the 213 healthy controls had a mean (SD) age of 39.2 (14.0) years. There were significantly more male patients than controls in each of the 3 cohorts (117 [70%] vs 91 [43%]), but there were no significant differences in sex composition among the 3 cohorts. Patients had significantly reduced processing speed (Cohen d = 1.24; P = 6.91 × 10-30) and working memory deficits (Cohen d = 0.83; P = 1.10 × 10-14) as well as a significant whole-brain fractional anisotropy deficit (Cohen d = 0.63; P = 2.20 × 10-9). In schizophrenia, working memory deficit was mostly accounted for by processing speed deficit, but this deficit remained when accounting for working memory (Cohen d = 0.89; P = 2.21 × 10-17). Mediation analyses showed a significant association pathway from fractional anisotropy to processing speed to working memory (P = 5.01 × 10-7). The strength of this brain-to-cognition pathway in different white matter tracts was strongly associated with the severity of schizophrenia-associated fractional anisotropy deficits in the corresponding white matter tracts as determined by a meta-analysis (r = 0.85-0.94; all P < .001). The same pattern was observed in patients and controls either jointly or independently. Conclusions and Relevance: Study findings suggest that (1) processing speed contributes to the association between white matter microstructure and working memory in schizophrenia and (2) white matter impairment in schizophrenia is regional tract-specific, particularly in tracts normally supporting processing speed performance.

Identification of 15 genetic loci associated with risk of major depression in individuals of European descent.

Despite strong evidence supporting the heritability of major depressive disorder (MDD), previous genome-wide studies were unable to identify risk loci among individuals of European descent. We used self-report data from 75,607 individuals reporting clinical diagnosis of depression and 231,747 individuals reporting no history of depression through 23andMe and carried out meta-analysis of these results with published MDD genome-wide association study results. We identified five independent variants from four regions associated with self-report of clinical diagnosis or treatment for depression. Loci with a P value <1.0 × 10(-5) in the meta-analysis were further analyzed in a replication data set (45,773 cases and 106,354 controls) from 23andMe. A total of 17 independent SNPs from 15 regions reached genome-wide significance after joint analysis over all three data sets. Some of these loci were also implicated in genome-wide association studies of related psychiatric traits. These studies provide evidence for large-scale consumer genomic data as a powerful and efficient complement to data collected from traditional means of ascertainment for neuropsychiatric disease genomics.

An integrated epigenomic analysis for type 2 diabetes susceptibility loci in monozygotic twins.

DNA methylation has a great potential for understanding the aetiology of common complex traits such as Type 2 diabetes (T2D). Here we perform genome-wide methylated DNA immunoprecipitation sequencing (MeDIP-seq) in whole-blood-derived DNA from 27 monozygotic twin pairs and follow up results with replication and integrated omics analyses. We identify predominately hypermethylated T2D-related differentially methylated regions (DMRs) and replicate the top signals in 42 unrelated T2D cases and 221 controls. The strongest signal is in the promoter of the MALT1 gene, involved in insulin and glycaemic pathways, and related to taurocholate levels in blood. Integrating the DNA methylome findings with T2D GWAS meta-analysis results reveals a strong enrichment for DMRs in T2D-susceptibility loci. We also detect signals specific to T2D-discordant twins in the GPR61 and PRKCB genes. These replicated T2D associations reflect both likely causal and consequential pathways of the disease. The analysis indicates how an integrated genomics and epigenomics approach, utilizing an MZ twin design, can provide pathogenic insights as well as potential drug targets and biomarkers for T2D and other complex traits.

An atlas of genetic influences on human blood metabolites.

Genome-wide association scans with high-throughput metabolic profiling provide unprecedented insights into how genetic variation influences metabolism and complex disease. Here we report the most comprehensive exploration of genetic loci influencing human metabolism thus far, comprising 7,824 adult individuals from 2 European population studies. We report genome-wide significant associations at 145 metabolic loci and their biochemical connectivity with more than 400 metabolites in human blood. We extensively characterize the resulting in vivo blueprint of metabolism in human blood by integrating it with information on gene expression, heritability and overlap with known loci for complex disorders, inborn errors of metabolism and pharmacological targets. We further developed a database and web-based resources for data mining and results visualization. Our findings provide new insights into the role of inherited variation in blood metabolic diversity and identify potential new opportunities for drug development and for understanding disease.

Sequence analysis of the IL28A/IL28B inverted gene duplication that contains polymorphisms associated with treatment response in hepatitis C patients.

Several SNPs located in or around the IL28B gene are associated with response of patients infected with Hepatitis C virus to treatment with pegylated interferon-α ⁺/⁻ ribavirin or with spontaneous clearance of the virus. The results of such studies are so compelling that future treatment approaches are likely to involve clinical decisions being made on the basis of a patient's genotype. Since IL28B is a paralogue of IL28A with greater than 95% sequence identity, it is possible that without genotyping assay specificity, sequences in IL28A may contribute to genotype identification, and potentially confound treatment decisions. This study aimed to 1) examine DNA sequences in IL28B surrounding each of the reported associated SNPs and the corresponding regions in IL28A; and 2) develop a robust assay for rs12979860, the most 'cosmopolitan' SNP most strongly associated with treatment response across all global populations studied to date. Bioinformatic analysis of genomic regions surrounding IL28A and IL28B demonstrated that 3 SNPs were unique to IL28B, whereas the remaining 6 SNP regions shared >93% identity between IL28A and IL28B. Using a panel of DNA samples, PCR amplification followed by Sanger sequencing was used to examine IL28B SNPs and the corresponding regions in IL28A. For the overlapping SNPs, all 6 in IL28B were confirmed to be polymorphic whereas the corresponding positions in IL28A were monomorphic. Based upon IL28A and IL28B sequence data, a specific TaqMan® assay was developed for SNP rs12979860 that was 100% concordant to the sequence-derived genotypes. Analysis using a commercial assay identified one discordant result which led to a change in their genotype-calling algorithm. Where future treatment decisions are made upon the results of genotyping assays, it is very important that results are concordant with data from a sequence-based format. This is especially so in situations where designing specific PCR primers is a challenge.

The 719Arg variant of KIF6 and cardiovascular outcomes in statin-treated, stable coronary patients of the treating to new targets and incremental decrease in end points through aggressive lipid-lowering prospective studies.

BACKGROUND: Carriers of the KIF6 719Arg variant may be at increased risk for CVD and may benefit more from statin therapy, in terms of CVD risk reduction, than noncarriers. Our objective was to investigate whether carriers of the KIF6 719Arg genetic variant (rs20455) are at increased cardiovascular risk and obtain more benefit from high-dose statin therapy than do noncarriers. METHODS AND RESULTS: We used an adjusted Cox proportional hazard model to assess the hazard ratio (HR) for the reduction of major cardiovascular events by 80 mg/d atorvastatin over 10 mg/d atorvastatin in 4599 patients of the Treating to New Targets (TNT) study and by 80 mg/d atorvastatin over 20-40 mg/d simvastatin in 6541 patients of the Incremental Decrease in End Points Through Aggressive Lipid-Lowering (IDEAL) study. A total of 381 and 648 patients had a cardiovascular event during follow-up in TNT and IDEAL, respectively. Heterozygotes and homozygotes for the minor allele were not at increased risk compared with noncarriers. In TNT, for noncarriers of the 719Arg allele, the HR for high- versus low-dose atorvastatin was 0.81 (95% confidence interval, 0.59-1.11). In carriers of 1 or 2 minor alleles, the HR was 0.85 (0.66-1.11) and carriers of 2 copies of the minor allele obtained a significant risk reduction (HR: 0.44, 95% confidence interval, 0.23-0.84). In IDEAL, the respective HRs were 0.85 (0.67-1.10), 0.88 (0.62-1.07) and 0.91 (0.58-1.43). The interaction term for carrier status by treatment was also nonsignificant (P=0.810 in TNT and P=0.909 in IDEAL). CONCLUSIONS: In these 2 large, randomized clinical trials, carriers of the KIF6 719Arg allele were not at increased cardiovascular risk and did not obtain consistent cardiovascular benefit from high-dose statin therapy compared with noncarriers.

Genetic association of the CCR5 region with lipid levels in at-risk cardiovascular patients.

BACKGROUND: There is mounting evidence to suggest that chemokine receptor 5 (CCR5) plays an important role in the development and progression of atherosclerosis. A naturally occurring variant of the CCR5 gene CCR532, exists at allele frequencies of typically 10% in European populations and results in a nonfunctional CCR5 receptor. METHODS AND RESULTS: The CCR5Delta32 deletion and 26 other variants within the chemokine receptor 2-CCR5-chemokine receptor-like protein 2 (CCRL2) gene cluster spanning 59 kilobases of chromosome 3 were genotyped in 5748 subjects from the Treating to New Targets atorvastatin trial to determine whether genetic associations could be identified with circulating lipid values and cardiovascular disease. Our results demonstrate an association between the CCR5Delta32 deletion and increased plasma high-density lipoprotein cholesterol and decreased plasma triglycerides, both of which are beneficial from a cardiovascular perspective. Three single-nucleotide polymorphisms (rs1154428, rs6808835, and rs6791599) in CCRL2 in linkage disequilibrium (r(2)> or =0.65) with CCR5Delta32 and located up to 45 kilobases distal to it were associated with high-density lipoprotein cholesterol. The high-density lipoprotein cholesterol and triglycerides findings were replicated in an additional set of >6000 individuals from the Incremental Decrease in Endpoints through Aggressive Lipid Lowering atorvastatin trial. CONCLUSIONS: Our study provides evidence that a locus within the region of the genome encompassing the CCR5-CCRL2 region is associated with lipid levels and suggests that chemokine activity influences lipid levels in populations with preexisting cardiovascular disease. CLINICAL TRIAL REGISTRATION- clinicaltrials.gov. Identifier: TNT, NCT00327691; IDEAL, NCT00159835.

Genome-wide association of lipid-lowering response to statins in combined study populations.

BACKGROUND: Statins effectively lower total and plasma LDL-cholesterol, but the magnitude of decrease varies among individuals. To identify single nucleotide polymorphisms (SNPs) contributing to this variation, we performed a combined analysis of genome-wide association (GWA) results from three trials of statin efficacy. METHODS AND PRINCIPAL FINDINGS: Bayesian and standard frequentist association analyses were performed on untreated and statin-mediated changes in LDL-cholesterol, total cholesterol, HDL-cholesterol, and triglyceride on a total of 3932 subjects using data from three studies: Cholesterol and Pharmacogenetics (40 mg/day simvastatin, 6 weeks), Pravastatin/Inflammation CRP Evaluation (40 mg/day pravastatin, 24 weeks), and Treating to New Targets (10 mg/day atorvastatin, 8 weeks). Genotype imputation was used to maximize genomic coverage and to combine information across studies. Phenotypes were normalized within each study to account for systematic differences among studies, and fixed-effects combined analysis of the combined sample were performed to detect consistent effects across studies. Two SNP associations were assessed as having posterior probability greater than 50%, indicating that they were more likely than not to be genuinely associated with statin-mediated lipid response. SNP rs8014194, located within the CLMN gene on chromosome 14, was strongly associated with statin-mediated change in total cholesterol with an 84% probability by Bayesian analysis, and a p-value exceeding conventional levels of genome-wide significance by frequentist analysis (P = 1.8 x 10(-8)). This SNP was less significantly associated with change in LDL-cholesterol (posterior probability = 0.16, P = 4.0 x 10(-6)). Bayesian analysis also assigned a 51% probability that rs4420638, located in APOC1 and near APOE, was associated with change in LDL-cholesterol. CONCLUSIONS AND SIGNIFICANCE: Using combined GWA analysis from three clinical trials involving nearly 4,000 individuals treated with simvastatin, pravastatin, or atorvastatin, we have identified SNPs that may be associated with variation in the magnitude of statin-mediated reduction in total and LDL-cholesterol, including one in the CLMN gene for which statistical evidence for association exceeds conventional levels of genome-wide significance. TRIAL REGISTRATION: PRINCE and TNT are not registered. CAP is registered at Clinicaltrials.gov NCT00451828.

Comprehensive whole-genome and candidate gene analysis for response to statin therapy in the Treating to New Targets (TNT) cohort.

BACKGROUND: Statins are effective at lowering low-density lipoprotein cholesterol and reducing risk of cardiovascular disease, but variability in response is not well understood. To address this, 5745 individuals from the Treating to New Targets (TNT) trial were genotyped in a combination of a whole-genome and candidate gene approach to identify associations with response to atorvastatin treatment. METHODS AND RESULTS: A total of 291 988 single-nucleotide polymorphisms (SNPs) from 1984 individuals were analyzed for association with statin response, followed by genotyping top hits in 3761 additional individuals. None was significant at the whole-genome level in either the initial or follow-up test sets for association with low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, or triglyceride response. In addition to the whole-genome platform, 23 candidate genes previously associated with statin response were analyzed in these 5745 individuals. Three SNPs in apoE were most highly associated with low-density lipoprotein cholesterol response, followed by 1 in PCSK9 with a similar effect size. At the candidate gene level, SNPs in HMGCR were also significant though the effect was less than with those in apoE and PCSK9. rs7412/apoE had the most significant association (P=6x10(-30)), and its high significance in the whole-genome study (P=4x10(-9)) confirmed the suitability of this population for detecting effects. Age and gender were found to influence low-density lipoprotein cholesterol response to a similar extent as the most pronounced genetic effects. CONCLUSIONS: Among SNPs tested with an allele frequency of at least 5%, only SNPs in apoE are found to influence statin response significantly. Less frequent variants in PCSK9 and smaller effect sizes in SNPs in HMGCR were also revealed.

Genome-wide scan identifies variation in MLXIPL associated with plasma triglycerides.

We tested over 267,000 SNPs in 1,005 Northern Europeans and 248,000 in 1,006 Indian Asians for association with triglycerides and HDL cholesterol, with replication in 10,536 subjects. We found association of a nonsynonymous SNP (rs3812316, G771C, Gln241His) in MLXIPL with plasma triglyceride levels (combined P = 1.4 x 10(-10)). MLXIPL coordinates transcriptional regulation of enzymes that channel glycolytic end-products into lipogenesis and energy storage, making MLXIPL a plausible 'thrifty gene'.

High-density genotyping and functional SNP localization in the CETP gene.

The cholesteryl ester transfer protein gene (CETP) has been the subject of hundreds of genetic analyses that typically focus on a small number of polymorphisms within a single ethnic group. Furthermore, the extent of DNA beyond the transcribed sequence from which single nucleotide polymorphisms (SNPs) may influence CETP expression has not been well defined. To better understand the role of natural variation in modulating CETP and high density lipoprotein-cholesterol (HDL-C) levels, dense genotyping of CETP and regions up to 15 kb on either side of the gene was carried out on >2,000 individuals. A complex, nonlinear set of linkage disequilibrium bins was found, with many bins interspersed along the DNA sequence and spread over large regions of the gene. Bins assigned based on large numbers of individuals matched the small subset of SNPs that had been assigned to bins previously with a small number of individuals. Associations of known functional SNPs with HDL-C were found, but there were suggestions that there are additional functional SNPs not characterized previously. Narrowing of the set of likely functional SNPs was accomplished by comparing associations observed in different ethnic groups. The promoter SNP most highly associated with HDL-C that is likely to be functional, position -4,502, alters a consensus transcription factor binding site.