Koustubh Ranade

Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations.

BACKGROUND A gene on chromosome 9p, p16INK4, has been implicated in the pathogenesis of cutaneous malignant melanoma in 19 melanoma-prone families. In 10 of these kindreds mutations that impaired the function of the p16INK4 protein (p16M alleles) cosegregated with the disease. By contrast, in the other nine kindreds the mutation did not alter the function of p16INK4 (p16W alleles). We looked for differences in clinical and genetic epidemiologic features in these two groups of families. METHODS We compared the median ages at diagnosis of melanoma, number of melanomas, thickness of the tumors, and number of nevi in the kindreds. We estimated prospectively the risks of melanoma or other cancers in families followed for 6 to 18 years and the risks of other cancers since 1925 (the entire period) by comparing the number of cancer cases observed with the number expected. RESULTS The risk of invasive melanoma was increased by a factor of 75 in kindreds with p16M alleles and a factor of 38 in kindreds with p16W alleles. Although this difference was not significant (P = 0.14), there was a striking difference in the risk of other tumors. In kindreds with p16M alleles, the risk of pancreatic cancer was increased by a factor of 13 in the prospective period (2 cases observed, 0.15 expected; standardized incidence ratio, 13.1; 95 percent confidence interval, 1.5 to 47.4) and by a factor of 22 in the entire period (7 cases observed, 0.32 expected; standardized incidence ratio, 21.8; 95 percent confidence interval, 8.7 to 44.8). In contrast, we found no cases of pancreatic cancer in kindred with p16W alleles. CONCLUSIONS The development of pancreatic cancer in kindreds prone to melanoma may require a p16M mutation. Genetic factors, such as the kind of mutation found in p16INK4, may explain the inconsistent occurrence of other cancers in these kindreds.

Polymorphism in KIF6 gene and benefit from statins after acute coronary syndromes: results from the PROVE IT-TIMI 22 study

OBJECTIVES We explored whether the benefit of intensive versus moderate statin therapy would be greater in carriers of KIF6 719Arg than in noncarriers. BACKGROUND The 719Arg variant of Trp719Arg (rs20455), a polymorphism in kinesin-like protein 6, is associated with greater risk of coronary events and greater benefit from pravastatin versus placebo. METHODS We genotyped 1,778 acute coronary syndrome patients within the PROVE IT-TIMI 22 (Pravastatin or Atorvastatin Evaluation and Infection Therapy: Thrombolysis in Myocardial Infarction 22) trial and investigated different intensities of statin therapy in carriers of 719Arg and in noncarriers using Cox proportional hazards models that adjusted for traditional risk factors. RESULTS Benefit from intensive, compared with moderate, statin therapy was significantly greater in the 59% of the cohort who were carriers (hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.45 to 0.77) than in those who were noncarriers (HR 0.94, 95% CI 0.70 to 1.27; p = 0.018 for interaction between 719Arg carrier status and treatment). Absolute risk reduction was 10.0% in carriers versus 0.8% in noncarriers. The benefit of intensive therapy in carriers was significant as early as day 30 of therapy. Carriers and noncarriers did not differ in on-treatment low-density lipoprotein cholesterol, triglyceride, or C-reactive protein (CRP) levels. CONCLUSIONS Carriers of 719Arg receive significantly greater benefit from intensive statin therapy than do noncarriers, a superior benefit that appears to be due to a mechanism distinct from lipid or CRP lowering. Functional studies of the KIF6 kinesin are warranted, given the consistent association of Trp719Arg with risk of coronary events and statin benefit.

Evaluation of the Paraoxonases as Candidate Genes for Stroke. Gln192Arg Polymorphism in the Paraoxonase 1 Gene Is Associated With Increased Risk of Stroke

Background and Purpose— The paraoxonases are involved in protecting low-density lipoprotein (LDL) from lipid oxidation. Paraoxonase 1 (PON1) was implicated in susceptibility to coronary artery disease and stroke in previous studies. We evaluated, in a comprehensive way, all 3 paraoxonase genes for association with stroke observed in the Cholesterol and Recurrent Events (CARE) trial. Methods— Over 2500 subjects enrolled in the CARE trial were genotyped for 14 single nucleotide polymorphisms, including 7 newly identified in this study, in the 3 paraoxonase genes. Results— A glutamine (Gln)/arginine (Arg) polymorphism at amino acid residue 192 in PON1 was significantly associated with stroke (P=0.003 in multivariate analysis, including age, sex, LDL, hypertension, diabetes, smoking, and pravastatin treatment as covariates). The odds ratios were 2.28 (95% CI, 1.38 to 3.79) for Gln/Arg heterozygotes and 2.47 (95% CI, 1.18 to 5.19) for Arg/Arg homozygotes compared with Gln/Gln homozygotes. These results are consistent with 2 of 3 other published studies. In combined analysis of all 4 studies, the association between Gln192Arg SNP and stroke was highly significant (&khgr;28df=45.58, P<0.000001). Sequence analysis of the PON1 gene from seventy stroke cases revealed a novel nonsense mutation at codon 32 in one stroke case, which was not detected in over 2500 unaffected individuals. Polymorphisms in the PON2 and PON3 genes were not associated with stroke. Conclusions— These results suggest that Gln192Arg genotype is an important risk factor for stroke.

Genotyping Using the TaqMan Assay

The 5′‐nuclease allelic discrimination assay, or TaqMan assay, is a PCR‐based assay for genotyping single nucleotide polymorphisms (SNPs). The region flanking the SNP is amplified in the presence of two allele‐specific fluorescent probes. The probes do not fluoresce in solution because of a quencher at the 3′ end. The presence of two probes allows the detection of both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post‐PCR processing, a feature that is unavailable with most other genotyping methods. This unit describes probe and primer design and PCR conditions. Curr. Protoc. Hum. Genet. 56:2.10.1‐2.10.8.

Association Between ADAMTS1 Matrix Metalloproteinase Gene Variation, Coronary Heart Disease, and Benefit of Statin Therapy

Objective—The purpose of this study was to investigate the association between the Ala227Pro polymorphism in the ADAMTS1 metalloproteinase gene and coronary heart disease and benefit from statin therapy in 2 independent cohorts. Methods and Results—The frequency of the ADAMTS1 227Pro minor allele was 0.24 in 2421 male subjects from CARE, a randomized trial of pravastatin versus placebo. In the placebo arm, homozygotes (6.3% of study population) had a significantly increased risk of fatal coronary disease or nonfatal myocardial infarction (D/MI) compared with noncarriers (OR 2.12, 95% CI 1.07 to 4.19, P=0.03), and in the entire study the benefit of pravastatin in reducing the risk of D/MI was greater in these subjects (OR 0.21, 95% CI 0.06 to 0.69) than in heterozygotes (OR 0.74, 95% CI 0.48 to 1.14) or noncarriers (OR 0.99, 95% CI 0.68 to 1.42; Pinteraction=0.044). Results were tested in 1565 male subjects from WOSCOPS, also a randomized trial of pravastatin versus placebo. Similar to the results in CARE, in the placebo arm subjects homozygous for the minor allele were at increased risk of D/MI (OR 1.72, P=0.052) and in the entire study the benefit of pravastatin in reducing D/MI was greater in these subjects (OR 0.24, 95% CI 0.09 to 0.68) than in heterozygotes (OR 0.73, 95% CI 0.48 to 1.11) or noncarriers (OR 0.65, 95% CI 0.20 to 2.09) (Pinteraction=0.029). Conclusions—In men not on pravastatin, those homozygous for the 227Pro allele of ADAMTS1 have a nearly 2-fold increased risk of coronary heart disease events compared with noncarriers. In this high-risk group, treatment with pravastatin is highly efficacious, reducing the odds of fatal coronary disease or nonfatal MI by approximately 75%, as compared with 25% in noncarriers or heterozygotes.

Genetic analysis implicates resistin in HIV lipodystrophy.

Objectives:To investigate the role of genetic variation in influencing the risk of metabolic complications associated with highly active antiretroviral therapy (HAART). Methods:Cluster analysis of metabolic traits of 189 patients enrolled in ACTG5005s, the metabolic substudy of ACTG384, a clinical trial of HAART, was performed to identify a subgroup of individuals with increased risk of developing a cluster of metabolic abnormalities after exposure to HAART. Almost 300 single nucleotide polymorphisms in 135 candidate genes were evaluated for their association with this subgroup. Results:A subgroup of patients was identified that had a normal metabolic profile at baseline but developed significantly elevated lipids and insulin resistance on HAART. This high-risk subgroup of patients also experienced significant body composition changes, particularly limb fat loss. Candidate gene analysis revealed that a single nucleotide polymorphism in resistin, a gene previously implicated in obesity and insulin resistance, was associated with this high-risk group (P = 0.0003). Conclusion:Genetic variation in resistin is associated with metabolic complications caused by HAART.

Genetic and gene expression studies implicate renin and endothelin-1 in edema caused by peroxisome proliferator-activated receptor γ agonists

Objective Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) agonists can cause peripheral edema in susceptible individuals. To investigate the mechanistic basis underlying this adverse event, we performed a candidate gene analysis of patients enrolled in clinical trials of muraglitazar, an investigational PPAR&agr;/&ggr; dual agonist, and developed a cell culture-based gene expression assay and nonhuman primate model of edema to study the edemagenic properties of PPAR&ggr; agonists. Methods A total of 213 single nucleotide polymorphisms (SNPs) in 63 genes were genotyped in 730 participants. Chi-square and logistic regression analyses were used to test for association with edema. Transcriptional responses to PPAR&ggr; agonists were evaluated in Calu-6 cells using quantitative real-time PCR. Male Cynomolgus monkeys were treated with PPAR agonists and were evaluated for edema using MRI. Results SNPs in renin (rs2368564) and endothelin-1 (rs5370) were associated with reduced risk of edema (P=0.003 and P=0.028, respectively) and an SNP in &bgr;1 adrenergic receptor (rs1801253) was associated with increased susceptibility to edema (P=0.034). Gene expression studies revealed that renin and endothelin-1 were regulated by PPAR&ggr; in Calu-6 cells. A survey of 10 PPAR&ggr; agonists further revealed that a compounds in vitro potency was correlated with its edemagenic potential leading to the prediction that one of three previously uncharacterized PPAR&ggr; agonists would cause less edema. This prediction was validated in a nonhuman primate model of PPAR&ggr; agonist-induced edema. Conclusion Our results implicate a key role for renin and endothelin-1 in the edema caused by PPAR&ggr; agonists and demonstrate how knowledge gained from pharmacogenetic studies can be applied in drug discovery.

High‐Throughput Genotyping Using the TaqMan Assay

This unit describes the use of the 5‐nuclease allelic discrimination assay, or TaqMan, for genotyping with single nucleotide polymorphisms (SNPs).