Clinical-pharmacogenetic predictive models for MTX discontinuation due to adverse events in rheumatoid arthritis.

We describe a novel approach to investigate and evaluate combined effect of a large number of clinical and pharmacogenetic factors on treatment outcome. We have used this approach to investigate predictors of methotrexate (MTX)-induced adverse events (AEs) leading to treatment discontinuation in rheumatoid arthritis (RA) patients. In total, 333 RA patients were genotyped for 34 polymorphisms in MTX transporters, folate and adenosine pathways. The effect of clinical and pharmacogenetic factors was assessed with penalized regression in the cause-specific Cox proportional hazards model. The predictive capacity was evaluated with the area under time-dependent receiver operating characteristic curve where cross-validation was applied. SLC19A1, ABCG2, ADORA3 and TYMS were associated with discontinuation because of AEs in clinical-pharmacogenetic model. Cross-validation showed that both clinical-pharmacogenetic model and nongenetic model had worthless predictive ability for MTX discontinuation because of AEs. These models could be further improved, either with additional polymorphisms or with epigenetic predictors.

The influence of platinum pathway polymorphisms on the outcome in patients with malignant mesothelioma.

BACKGROUND: Platinum-based therapy is widely used in the treatment of malignant mesothelioma (MM); however, the efficacy and toxicity of platinum agents vary greatly between patients. The aim of our study was to evaluate the influence of platinum pathway polymorphisms on treatment outcome in patients with MM. PATIENTS AND METHODS: In total, 133 patients with MM treated with (n = 97) or without (n = 36) platinum-based therapy were genotyped for common XPD, ERCC1, and GSTP1 polymorphisms, as well as for GSTM1 and GSTT1 gene deletion. Haplotype analysis was carried out to assess the combined effect of nucleotide excision repair (NER) polymorphisms. RESULTS: GST polymorphisms were not associated with treatment outcome in patients with MM. In the group of platinum-treated patients with MM, ERCC1 8092C/C wild-type genotype significantly influenced progression-free survival (PFS) in multivariable analysis accounting for clinical variables (P = 0.034). XPD 312Asp/Asp and ERCC1 8092C/C wild-type genotypes also increased the odds of treatment-related toxic effects in univariable as well as multivariable analysis. The association of wild-type NER genotypes with better PFS and higher susceptibility to treatment-related toxic effects was confirmed in haplotype analysis. CONCLUSIONS: Our results suggest that polymorphisms in NER pathway influence platinum-treatment efficacy and toxicity; therefore, these should be further evaluated as potential markers for the prediction of clinical outcome in patients with MM.

Genetic polymorphisms of glutathione S-transferases and disease activity of rheumatoid arthritis.

OBJECTIVES: Glutathione S-transferases (GST); GST-mu1 (GSTM1), GST-pi1 (GSTP1) and GST-theta1 (GSTT1) have peroxidase activity towards cytotoxic metabolites produced in inflammatory reactions, the main feature of rheumatoid arthritis (RA). Genetic polymorphisms in GSTM1, GSTP1 and GSTT1 modify the enzyme conjugation capacity and may be associated with the activity of RA. METHODS: A genotyping approach was used to analyze GSTM1-0, GSTT1-0 and GSTP1 Ile105Val and Ala114Val polymorphisms in 213 RA patients. Disease activity was assessed by the disease activity score of 28 joint counts (DAS28) twice for each patient and mean DAS28 values were calculated. RESULTS: The patients with GSTT1-0 genotype had a higher risk for developing high activity RA than the patients with GSTT1 genes present (p=0.028, OR=2.761, 95% CI=1.114-6.843). An interaction between the GSTT1 polymorphism and smoking was observed. In the group of smokers, the carriers of a homozygous deletion GSTT1 had an 8.5-fold higher risk for developing high disease activity than the patients with the GSTT1-1 genotype (p=0.004, OR=8.640, 95% CI=1.995-37.426). GSTM1 and GSTP1 polymorphisms were not associated with the disease activity. CONCLUSION: Our results suggest that the presence of the GSTT1-0 genotype contributed to higher disease activity in RA patients. The risk for developing highly active RA was the highest in smokers with the GSTT1-0 genotype.

Polymorphisms in genes encoding the serotonin and dopamine pathways in two sisters with metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a metabolic disease that has recently been investigated as a model for the study of psychosis. We report on two sisters with adult-type MLD who developed psychiatric symptomatology, but differed in their expression of psychotic and depressive symptoms. Association studies have indicated that polymorphisms in genes encoding the serotonin and dopamine transporters and receptors are related to the symptomatology of schizophrenia and/or depression; hence both sisters were genotyped for some of these candidate genes. The sisters shared dopamine receptor D(2) (DRD(2)) c.1047GG (p.311Ser/Ser) and c.-141Cins/ins polymorphisms, which are significantly associated with schizophrenia, but differed in the serotonin transporter gene-linked polymorphic region and serotonin receptor 1A (5-HT(1A)) c.-1019C to G polymorphisms, which may have increased the elder sister's susceptibility to depressive symptoms. Much bigger samples would be needed to gain enough statistical power to develop any hypotheses. This is the first report on genotyping MLD patients for candidate genes for psychiatric disorders, although MLD has been proposed as a model for schizophrenia.

Implementing Pharmacogenomics in Europe: Design and Implementation Strategy of the Ubiquitous Pharmacogenomics Consortium.

Despite scientific and clinical advances in the field of pharmacogenomics (PGx), application into routine care remains limited. Opportunely, several implementation studies and programs have been initiated over recent years. This article presents an overview of these studies and identifies current research gaps. Importantly, one such gap is the undetermined collective clinical utility of implementing a panel of PGx-markers into routine care, because the evidence base is currently limited to specific, individual drug-gene pairs. The Ubiquitous Pharmacogenomics (U-PGx) Consortium, which has been funded by the European Commission's Horizon-2020 program, aims to address this unmet need. In a prospective, block-randomized, controlled clinical study (PREemptive Pharmacogenomic testing for prevention of Adverse drug REactions [PREPARE]), pre-emptive genotyping of a panel of clinically relevant PGx-markers, for which guidelines are available, will be implemented across healthcare institutions in seven European countries. The impact on patient outcomes and cost-effectiveness will be investigated. The program is unique in its multicenter, multigene, multidrug, multi-ethnic, and multihealthcare system approach.

Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia.

OBJECTIVE: To analyze the mutational spectrum of steroid 21-hydroxylase (CYP21) and the genotype- phenotype correlation in patients with congenital adrenal hyperplasia (CAH) registered in the Middle European Society for Pediatric Endocrinology CAH database, and to design a reliable and rational approach for CYP21 mutation detection in Middle European populations. DESIGN AND METHODS: Molecular analysis of the CYP21 gene was performed in 432 CAH patients and 298 family members. Low-resolution genotyping was performed to detect the eight most common point mutations. High-resolution genotyping, including Southern blotting and sequencing was performed to detect CYP21 gene deletions, conversions, point mutations or other sequence changes. RESULTS: CYP21 gene deletion and In2 and Ile172Asn mutation accounted for 72.7% of the affected alleles in the whole study group. A good genotype-phenotype correlation was observed, with the exception of Ile172Asn and Pro30Leu mutations. In 37% of patients low resolution genotyping could not identify the causative mutation or distinguish homozygosity from hemizygosity. Using high-resolution genotyping, the causative mutations could be identified in 341 out of 348 analyzed patients. A novel mutation Gln315Stop was found in one simple virilising CAH (SV-CAH) patient from Austria. In the remaining seven patients polymorphisms were identified as the leading sequence alteration. The presence of elevated basal and ACTH-stimulated 17-hydroxyprogesterone, premature pubarche, advanced bone age and clitoral hypertrophy directly implicated Asn493Ser polymorphism in the manifestation of nonclassical- (NC) and even SV-CAH. CONCLUSIONS: By genotyping for the most common point mutations, CYP21 gene deletion/conversion and the 8 bp deletion in exon 3, it should be possible to identify the mutation in 94-99% of the diseased alleles in any investigated Middle European population. In patients with a mild form of the disease and no detectable mutation CYP21 gene polymorphisms should be considered as a plausible disease-causing mutation.

Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.

Adrenal 21-hydroxylase gene mutations in Slovenian hyperandrogenic women: evaluation of corticotrophin stimulation and HLA polymorphisms in screening for carrier status.

OBJECTIVE: To study the incidence of 21-hydroxylase deficiency in Slovenian hyperandrogenic women, at the gene level. Previous endocrine studies indicated large differences in the incidence of 21-hydroxylase deficiency in hyperandrogenic women. The predictive values of the 17-hydroxyprogesterone (17-OHP) response to ACTH stimulation and of HLA typing in screening for carrier status were re-evaluated. DESIGN: Molecular analysis of CYP21 gene, ACTH stimulation and human leucocyte antigen (HLA) typing were performed in 83 consecutive Slovenian hyperandrogenic women. MEASUREMENTS: Cortisol and 17-OHP concentrations were measured at baseline and 60 min after ACTH stimulation. Basal adrenal androgen concentrations were also measured. RESULTS: None of 83 hyperandrogenic patients was affected with non-classical 21-hydroxylase deficiency, but 12 of 81 patients (14.8%) had high concentrations of 17-OHP after stimulation, indicative of carrier status. The increase in 17-OHP concentrations could be explained by a carrier status for CYP21 gene mutations in only three of 12 patients (25%), whereas seven of 69 patients (10. 1%) with normal concentrations of 17-OHP after stimulation were found to be carriers of CYP21 gene mutations, indicating low positive predictive values of ACTH stimulation as a screening test for carriers of 21-hydroxylase deficiency. In total, 11 carriers were identified among 83 patients: seven CYP21 gene deletions/conversions, two Gln(318)Stop and one Val(281)Leu mutation and one gene conversion extending from exon 4 to exon 7 were found. The association between Val(281)Leu mutation and HLA-B14 antigen was confirmed in this Slovenian population. CONCLUSIONS: Basal or ACTH-stimulated 17-OHP concentrations are not a good indicator of the carrier status for 21-hydroxylase deficiency among Slovenian hyperandrogenic patients. Reliable screening for carriers of 21-hydroxylase deficiency is possible only by molecular analysis of the CYP21 gene.

Mutational spectrum of congenital adrenal hyperplasia in Slovenian patients: a novel Ala15Thr mutation and Pro30Leu within a larger gene conversion associated with a severe form of the disease.

OBJECTIVE: To analyse the mutational spectrum, the associated haplotypes and the genotype-phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. DESIGN: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. METHODS: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. RESULTS: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. CONCLUSIONS: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

Polymorphisms in CYP1A1, GSTM1, GSTT1 and lung cancer below the age of 45 years.

BACKGROUND: A genetic component of early-onset lung cancer has been suggested. The role of metabolic gene polymorphisms has never been studied in young lung cancer cases. Phase 1 and Phase 2 gene polymorphisms are involved in tobacco carcinogens' metabolism and therefore in lung cancer risk. METHODS: The effect of metabolic gene polymorphisms on lung cancer at young ages was studied by pooling data from the Genetic Susceptibility to Environmental Carcinogens (GSEC) database. All primary lung cancer cases of both sexes who were Caucasian and

The influence of gene-gene and gene-environment interactions on the risk of asbestosis.

This study investigated the influence of gene-gene and gene-environment interactions on the risk of developing asbestosis. The study comprised 262 cases with asbestosis and 265 controls with no asbestos-related disease previously studied for MnSOD, ECSOD, CAT, GSTT1, GSTM1, GSTP1, and iNOS polymorphisms. Data on cumulative asbestos and smoking were available for all subjects. To assess gene-gene and gene-environmental interactions, logistic regression was used. The associations between MnSOD Ala -9Val polymorphism and the risk of asbestosis and between iNOS genotypes and asbestosis were modified by CAT -262 C > T polymorphism (P = 0.038; P = 0.031). A strong interaction was found between GSTM1-null polymorphism and smoking (P = 0.007), iNOS (CCTTT) n polymorphism and smoking (P = 0.054), and between iNOS (CCTTT) n polymorphism and cumulative asbestos exposure (P = 0.037). The findings of this study suggest that the interactions between different genotypes, genotypes and smoking, and between genotypes and asbestos exposure have an important influence on the development of asbestosis and should be seriously considered in future research on occupational/environmental asbestos-related diseases.

Genetic susceptibility to environmental carcinogenesis in Slovenian melanoma patients.

The skin acts as the first defence barrier against external environmental pollutants, including chemicals and UV radiation. Cytochrome P450 CYP1A1 and glutathione S-transferases (GSTs) found in melanocytes and skin basal layers were shown to participate both in the metabolism of xenobiotics and in detoxification of reactive oxygen species (ROS). In our study we analysed the distribution of single and combined CYP1A1, GSTM1, GSTT1 and GSTP1 genotypes contributing to inter-individual differences in metabolism of xenobiotics and ROS in 125 Slovenian healthy individuals and in 140 patients with sporadic malignant melanoma. Our results showed no statistically significant differences between melanoma patients and healthy controls in the frequency of polymorphic CYP1A1 and GST genotypes. The risk of developing melanoma was not significantly increased in individuals homo- or heterozygous for the CYP1A1*2A allele combined with GSTM1*0 genotype (OR: 1.86; 95% CI: 0.36-7.71), but increased slightly in carriers of CYP1A1*2A combined with both GSTM1*0 and GSTT1*0 genotypes (OR: 3.42; 95% CI: 0.36-29.6). Our results indicate that factors other than the polymorphic genes coding xenobiotic metabolising enzymes play a major role in protection against environmental carcinogenesis in human skin.

Meta- and pooled analysis of GSTT1 and lung cancer: a HuGE-GSEC review.

Lung cancer is the most common malignancy in the Western world, and the main risk factor is tobacco smoking. Polymorphisms in metabolic genes may modulate the risk associated with environmental factors. The glutathione S-transferase theta 1 gene (GSTT1) is a particularly attractive candidate for lung cancer susceptibility because of its involvement in the metabolism of polycyclic aromatic hydrocarbons found in tobacco smoke and of other chemicals, pesticides, and industrial solvents. The frequency of the GSTT1 null genotype is lower among Caucasians (10-20%) than among Asians (50-60%). The authors present a meta- and a pooled analysis of case-control, genotype-based studies that examined the association between GSTT1 and lung cancer (34 studies, 7,629 cases and 10,087 controls for the meta-analysis; 34 studies, 7,044 cases and 10,000 controls for the pooled analysis). No association was observed between GSTT1 deletion and lung cancer for Caucasians (odds ratio (OR) = 0.99, 95% confidence interval (CI): 0.87, 1.12); for Asians, a positive association was found (OR = 1.28, 95% CI: 1.10, 1.49). In the pooled analysis, the odds ratios were not significant for either Asians (OR = 0.97, 95% CI: 0.83, 1.13) or Caucasians (OR = 1.09, 95% CI: 0.99, 1.21). No significant interaction was observed between GSTT1 and smoking on lung cancer, whereas GSTT1 appeared to modulate occupational-related lung cancer.

Influence of CYP2C9 polymorphisms, demographic factors and concomitant drug therapy on warfarin metabolism and maintenance dose.

Warfarin is an anticoagulant drug with narrow therapeutic index and high interindividual variability in dose requirement. S-warfarin is metabolized mainly by polymorphic cytochrome P450 (CYP) 2C9. We systematically quantified the influence of CYP2C9 genotype, demographic factors and concomitant drug treatment on warfarin metabolism and maintenance dose. The mean warfarin doses were lower in carriers of one (2.71 mg/day, 59 patients) and two polymorphic alleles (1.64 mg/day, 11 patients) than in carriers of two wild-type alleles (4.88 mg/day, 118 patients). Multiple regression analysis demonstrated that CYP2C9 genotype, age, concomitant treatment with warfarin metabolism inducers and lean body weight contributed significantly to interindividual variability in warfarin dose requirement (adjusted R(2)=0.37). The same factors, except for age, significantly influenced S-warfarin clearance (adjusted R(2)=0.42). These results can serve as a starting point for designing prospective studies in patients in the initiation phase of genotype-based warfarin therapy.

Human CYP2D6 gene polymorphism in Slovene cancer patients and healthy controls.

The polymorphic CYP2D6 gene encoding debrisoquine hydroxylase has attracted much interest for its possible role in human pulmonary carcinogenesis. The purpose of this work was to determine the frequency of poor metabolizers (PM) and extensive metabolizers (EM) of debrisoquine in Slovene population of healthy individuals (n = 107), lung cancer patients (200) and melanoma patients (121). Polymorphism of CYP2D6 gene was studied by genotyping based on PCR analysis of the intron 3 exon 4 junction containing G to A mutation and one base pair deletion in exon 5, which are responsible for approximately 95% of poor metabolizer phenotype in Caucasians. In the healthy Slovene population 62.5% of individuals were identified as extensive metabolizers, 31% as extensive-heterozygous metabolizers and 6.5% as poor metabolizers of debrisoquine. The frequency of EM individuals was 70.5% in lung cancer patients and 64% in melanoma patients, the frequency of extensive-heterozygous subjects was 27% in lung cancer patients and 31% in melanoma patients. The frequency of PM individuals in the lung cancer patients was 2.5% and in melanoma patients 5%. The decrease in PM genotype in the group of Slovene lung cancer patients is similar to the decrease published for some other ethnic groups. Our results support the hypothesis that polymorphic CYP2D6 gene probably plays some, though not a prevalent role in chemical carcinogenesis. Poor metabolizer individuals appear to be less susceptible to lung cancer than EM individuals.

Genetic polymorphism of xenobiotic metabolising enzymes in Slovenian lung cancer patients.

Most carcinogenic substances require metabolic activation in order to become ultimate carcinogens. Genetic polymorphism of xenobiotic metabolising enzymes cytochromes P450 may therefore influence human cancer susceptibility. The aim of our study was to investigate if CYP1A1 gene polymorphism contributes to lung cancer susceptibility in Slovenian patients. Two polymorphic sites in CYP1A1 gene were analysed in DNA samples from 100 healthy controls and 199 lung cancer patients using genotyping approach. Our results indicate that CYP1A1 may be one of the factors determining susceptibility to squamous cell carcinoma of lung in Slovenian population. However the frequency of CYP1A1 polymorphisms is too low to be a potentially useful marker of increased lung cancer risk.

The contribution of HLA-DQB1 coding and QBP promoter alleles to anti-Ro alone autoantibody response in systemic lupus erythematosus.

OBJECTIVE: To analyse the influence of HLA-DR, DQ and corresponding DQA1 and DQB1 promoter alleles (QAP and QBP) on the anti-Ro alone autoantibody response in systemic lupus erythematosus (SLE). METHODS: Sixty-five unrelated anti-La antibody-negative SLE patients, 37 of them with and 28 without anti-Ro antibodies, were included. Anti-Ro antibodies were determined by both counter-immunoelectrophoresis and enzyme-linked immunosorbent assay. Seventy-four healthy individuals were selected as controls. The patients and controls were analysed for HLA-DRB1, QAP, DQA1, QBP and DQB1 alleles by DNA typing. The allelic frequencies of anti-Ro alone-positive and anti-Ro-negative SLE patients and healthy controls were compared using the chi(2) test or Fisher's exact test as appropriate. RESULTS: The DQB1*0202 allele showed a significant positive correlation with anti-Ro alone antibodies [odds ratio (OR)=16.949, P=0.0015, corrected P=0.018], while the QBP5.11 allele and the combination of DQB1*0301 and its promoter QBP3.1 were under-represented in anti-Ro-alone-positive SLE patients (P=0.01, corrected P=0.048 and corrected P=0.048 respectively). CONCLUSIONS: The above-mentioned alleles may contribute to the presence or absence of anti-Ro alone autoantibodies in SLE patients.