AIM:To optimally address the interindividual variability observed in pharmacokinetic drug response, we have created a custom genotyping panel that interrogates most of the key genetic variations present in a set of 181 prioritized genes responsible for the absorption, distribution, metabolism and excretion (ADME) of many therapeutic agents. This consensus list of genes and variants was based on the ADME core and extended gene lists compiled by a group of pharmaceutical companies as having relevance. Although these pharmacokinetic genes and pathways are well known, tools that can interrogate a large number of these genes simultaneously within a single experiment are not currently available.METHODS:Using novel design strategies, we have developed an optimized and validated ADME genotyping panel, encompassing approximately 3000 variants, that has broad applicability to any study or clinical trial that would benefit from the evaluation of an extensive list of ADME genes.RESULTS & CONCLUSION:Over the course of three design iterations, overall assay conversion rates were improved from 83 to 97% resulting in a panel that fills in many of the gaps in coverage present on currently available commercial genotyping assays. The utility of the assay has been demonstrated by the screening of more than 1000 samples resulting in the discovery of novel pharmacogenomic associations. The assay, and the underlying methods, will continue to be a valuable tool for use in future pharmacogenomic studies.

Background: Many mutations among genes encoding sarcomeric proteins have been identified in hypertrophic cardiomyopathy (HCM). Data on specific genotype-phenotype correlations are limited and highly variable. Methods: Identified mutations in genes TNNI3 (troponin I), TNNT2 (troponin T), MYBPC3 (myosin-binding protein C) and MYH7 (myosin heavy chain) were screened in 284 patients at the Cardiovascular Genetic Clinic of the Montreal Heart Institute. Mutations were identified in 113 patients (40%) and 84 patients (74%) were included for analysis. Five cardiac transplant recipients without previous echocardiographic images available were analysed according to clinical characteristics only. We excluded 16 patients without HCM phenotype and 13 patients with follow-up outside of our center. Results: MYH7 (40%) and MYBPC3 (40%) mutations were the most common. Only one patient was identified with a TNNI3 (1%) mutation and was excluded. Patients with MYH7 mutations were younger at diagnosis and presented more often with a familial history of sudden cardiac death (76%) and defibrillator implantation (59%). Patients with MYH7 mutations were also more likely to progress to “burn-out” HCM (27%) and cardiac transplantation (18%). Significant mitral insufficiency was more prevalent in patients with MYBPC3 mutations (33%). Patients with TNNT2 (18%) mutations showed an increased probability of having a significant resting and provocable left outflow tract obstruction (47%) with systolic anterior movement of the mitral valve (53%) leading to septal myomectomy (27%). Genotype did not affect septal morphology with an overall predominance of the midseptal form (73% overall). Conclusion: This retrospective study suggests that TNNT2 mutations are associated with obstructive HCM while MYH7 mutations are related with arrhythmic complications. Moreover, septal morphologies do not appear to be influenced by any specific gene mutation.

BACKGROUND AND AIMS:The familial Mediterranean fever (FMF) gene (MEFV) encodes pyrin, a major regulator of the inflammasome platform controlling caspase-1 activation and IL-1beta processing. Pyrin has been shown to interact with the gene product of NLRP3, NALP3/cryopyrin, also an important active member of the inflammasome. The NLRP3 region was recently reported to be associated with Crohn's disease (CD) susceptibility. We therefore sought to evaluate MEFV as an inflammatory bowel disease (IBD) susceptibility gene. METHODOLOGY AND RESULTS:MEFV colonic mucosal gene expression was significantly increased in experimental colitis mice models (TNBS p<0.0003; DSS p<0.006), in biopsies from CD (p<0.02) and severe ulcerative colitis (UC) patients (p<0.008). Comprehensive genetic screening of the MEFV region in the Belgian exploratory sample set (440 CD trios, 137 UC trios, 239 CD cases, 96 UC cases, and 107 healthy controls) identified SNPs located in the MEFV 5' haplotype block that were significantly associated with UC (rs224217; p = 0.003; A allele frequency: 56% cases, 45% controls), while no CD associations were observed. Sequencing and subsequent genotyping of variants located in this associated haplotype block identified three synonymous variants (D102D/rs224225, G138G/rs224224, A165A/rs224223) and one non-synonymous variant (R202Q/rs224222) located in MEFV exon 2 that were significantly associated with UC (rs224222: p = 0.0005; A allele frequency: 32% in cases, 23% in controls). No consistent associations were observed in additional Canadian (256 CD trios, 91 UC trios) and Scottish (495 UC, 370 controls) sample sets. We note that rs224222 showed marginal association (p = 0.012; G allele frequency: 82% in cases, 70% in controls) in the Canadian sample, but with a different risk allele. None of the NLRP3 common variants were associated with UC in the Belgian-Canadian UC samples and no significant interactions were observed between NLRP3 and MEFV that could explain the observed flip-flop of the rs224222 risk allele. CONCLUSION:The differences in association levels observed between the sample sets may be a consequence of distinct founder effects or of the relative small sample size of the cohorts evaluated in this study. However, the results suggest that common variants in the MEFV region do not contribute to CD and UC susceptibility.

Initiation of warfarin therapy is associated with bleeding owing to its narrow therapeutic window and unpredictable therapeutic dose. Pharmacogenetic-based dosing algorithms can improve accuracy of initial warfarin dosing but require rapid genotyping for cytochrome P-450 2C9 (CYP2C9) *2 and *3 single nucleotide polymorphisms (SNPs) and a vitamin K epoxide reductase (VKORC1) SNP. We evaluated 4 commercial systems: INFINITI analyzer (AutoGenomics, Carlsbad, CA), Invader assay (Third Wave Technologies, Madison, WI), Tag-It Mutation Detection assay (Luminex Molecular Diagnostics, formerly Tm Bioscience, Toronto, Canada), and Pyrosequencing (Biotage, Uppsala, Sweden). We genotyped 112 DNA samples and resolved any discrepancies with bidirectional sequencing. The INFINITI analyzer was 100% accurate for all SNPs and required 8 hours. Invader and Tag-It were 100% accurate for CYP2C9 SNPs, 99% accurate for VKORC1 -1639/3673 SNP, and required 3 hours and 8 hours, respectively. Pyrosequencing was 99% accurate for CYP2C9 *2, 100% accurate for CYP2C9 *3, and 100% accurate for VKORC1 and required 4 hours. Current commercial platforms provide accurate and rapid genotypes for pharmacogenetic dosing during initiation of warfarin therapy.

Susceptibility to coronary heart disease (CHD) has long been known to exhibit familial aggregation, with heritability estimated to be greater than 50%. The French Canadian population of the Saguenay-Lac Saint-Jean region of Quebec, Canada is descended from a founder population that settled this region 300-400 years ago and this may provide increased power to detect genes contributing to complex traits such as CHD. Probands with early-onset CHD, defined by angiographically determined coronary stenosis, and their relatives were recruited from this population (average sibship size of 6.4). Linkage analysis was performed following a genome-wide microsatellite marker scan on 42 families with 284 individuals. Nonparametric linkage (NPL) analysis provided suggestive evidence for a CHD susceptibility locus on chromosome 8 with an NPL score of 3.14 (P=0.001) at D8S1106. Linkage to this locus was verified by fine mapping in an enlarged sample of 50 families with 320 individuals. This analysis provided evidence of linkage at D8S552 (NPL score=3.53, P=0.0003), a marker that maps to the same location as D8S1106. Candidate genes in this region, including macrophage scavenger receptor 1, farnesyl-diphosphate farnesyltransferase 1, fibrinogen-like 1, and GATA-binding protein 4, were resequenced in all coding exons in both affected and unaffected individuals. Association studies with variants in these and five other genes did not identify a disease-associated mutation. In conclusion, a genome-wide scan and additional fine mapping provide evidence for a locus on chromosome 8 that contributes to CHD in a French Canadian population.

The relative contribution of phenotypic measures and CYP2C9-vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms to warfarin dose requirements at day 14 was determined in 132 hospitalized, heavily medicated patients. Phenotypic measures were (1) the urinary losartan metabolic ratio before the first dose of warfarin, (2) the S:R-warfarin ratio at day 1, and (3) a dose-adjusted international normalized ratio (INR) at day 4. CYP2C9 and VKORC1 genotypes were determined by gene chip analysis. In multivariate analyses, the dose-adjusted INR at day 4 explained 31% of variability observed in warfarin doses at day 14, whereas genotypic measures (CYP2C9-VKORC1) contributed 6.5%. When S:R-warfarin ratio was used, genotypes contributed more significantly (23.5%). Finally, urinary losartan metabolic ratio was of low predictive value. The best models obtained explained 51% of intersubject variability in warfarin dose requirements. Thus, combination of a phenotypic measure to CYP2C9-VKORC1 genotypes represents a useful strategy to predict warfarin doses in patients receiving multiple drugs (11 +/- 4 drugs/day).

CX3CR1, a fractalkine receptor, mediates cell-adhesive and migratory functions in inflammation. Based on CX3CR1 expression observed in bronchial tissues of asthmatic subjects, we hypothesized that genetic variation at this locus may affect susceptibility to asthma. We carried out an association study and a haplotypic analysis with selected polymorphisms of the CX3CR1 in a familial asthmatic sample from a founder population. Genetic analyses performed by FBAT software showed five CX3CR1 single nucleotide polymorphisms (rs938203, rs2669849, rs1050592, T280M and V249I) with significant associations between their common alleles and asthma (P<0.004) in a dominant model. A haplotype formed with common alleles of rs1050592, T280M and V249I is also overtransmitted in asthmatic subjects (P=0.005) under a dominant model. The associations of V249I and rs2669849 have been validated in an independent case–control sample. For V249I, odds ratios (OR) are 2.16 (common homozygous) and 2.11 (heterozygous) in dominant model (P=0.031). For rs2669849, OR are 2.75 (common homozygous) and 1.86 (heterozygous) in additive model (P=0.007) and dominant model (P=0.059). These results suggest an asthma protective effect of the minor alleles in healthy control carriers. Further functional studies of CX3CR1 are needed to document its role in the pathophysiology of asthma.

The original article to which this Erratum refers was published in Human Mutation 22:67–73 Human Mutation(2003) 22(1) 67–73

Toll-like receptor 4 (TLR4) is the principal receptor for bacterial endotoxin recognition, and functional variants in the gene confer endotoxin-hyporesponsiveness in humans. Furthermore, there is evidence that endotoxin exposure during early life is protective against the development of atopy and asthma, although this relationship remains poorly understood. It is therefore possible that genetic variation in the TLR4 locus contributes to asthma susceptibility. In this study we characterize the genetic diversity in the TLR4 locus and test for association between the common genetic variants and asthma-related phenotypes. In a cohort of 90 ethnically diverse subjects, we resequenced the TLR4 locus and identified a total of 29 single nucleotide polymorphisms. We assessed five common polymorphisms for evidence of association with asthma in two large family-based cohorts: a heterogeneous North American cohort (589 families), and a more homogenous population from northeastern Quebec, Canada (167 families). Using the transmission-disequilibrium test, we found no evidence of association for any of the polymorphisms tested, including two functional variants. Furthermore, we found no evidence for association between the TLR4 variants and four quantitative intermediate asthma- and atopy-related phenotypes. Based on these results, we found no evidence that genetic variation in TLR4 contributes to asthma susceptibility.

Diabetes and obesity have long been known to be related. The recently characterized adipocyte hormone resistin (also called FIZZ3/ADSF) has been implicated as a molecular link between impaired glucose tolerance (IGT) and obesity in mice. A search for sequence variants at the human resistin locus identified nine single-nucleotide polymorphisms (SNPs) but no coding variants. An investigation into the association of these SNPs with diabetes and obesity revealed two 5' flanking variants (g.-537 and g.-420), in strong linkage disequilibrium, that are associated with BMI. In nondiabetic individuals from the Quebec City area and the Saguenay-Lac-St-Jean region of Quebec, the g.-537 mutation (allelic frequency = 0.04) was significantly associated with an increase in BMI (P = 0.03 and P = 0.01, respectively). When the data from these two populations were combined and adjusted for age and sex, both the g.-537 (odds ratio [OR] 2.72, 95% CI 1.28-5.81) and the g.-420 variants (1.58, 1.06-2.35) were associated with an increased risk for a BMI > or =30 kg/m(2). In contrast, in case/control and family-based study populations from Scandinavia, we saw no effect on BMI with either of these promoter variants. No association was seen with diabetes in any of the population samples.