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Human liver gene regulatory (Bayesian) network analysis was previously used to identify a cytochrome P450 (P450) gene subnetwork with Aldo-keto reductase 1D1 (AKR1D1) as a key regulatory driver of this subnetwork. This study assessed the biologic importance of AKR1D1 [a key enzyme in the synthesis of bile acids, ligand activators of farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), known transcriptional regulators of P450s] to hepatic P450 expression. Overexpression of AKR1D1 in primary human hepatocytes led to increased expression of CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6. Conversely, AKR1D1 knockdown decreased expression of these P450s. We resequenced AKR1D1 from 98 donor livers and identified a 3'-untranslated region (UTR) (rs1872930) single nucleotide polymorphism (SNP) significantly associated with higher AKR1D1 mRNA expression. AKR1D1 3'-UTR-luciferase reporter studies showed that the variant allele resulted in higher luciferase activity, suggesting that the SNP increases AKR1D1 mRNA stability and/or translation efficiency. Consistent with AKR1D1's putative role as a driver of the P450 subnetwork, the AKR1D1 3'-UTR SNP was significantly associated with increased hepatic mRNA expression of multiple P450s (CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2B6) and CYP3A4, CYP2C8, CYP2C19, and CYP2B6 activities. After adjusting for multiple testing, the association remained significant for AKR1D1, CYP2C9, and CYP2C8 mRNA expression and CYP2C8 activity. These results provide new insights into the variation in expression and activity of P450s that can account for interindividual differences in drug metabolism/efficacy and adverse drug events. In conclusion, we provide the first experimental evidence supporting a role for AKR1D1 as a key genetic regulator of the P450 network.

The subarachnoid space, where cerebrospinal fluid (CSF) flows over the brain and spinal cord, is lined on one side by arachnoid barrier (AB) cells that form part of the blood-CSF barrier. However, despite the fact that drugs are administered into the CSF and CSF drug concentrations are used as a surrogate for brain drug concentration following systemic drug administration, the tight-junctioned AB cells have never been examined for whether they express drug transporters that would influence CSF and central nervous system drug disposition. Hence, we characterized drug transporter expression and function in AB cells. Immunohistochemical analysis showed P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in mouse AB cells but not other meningeal tissue. The Gene Expression Nervous System Atlas (GENSAT) database and the Allen Mouse Brain Atlas confirmed these observations. Microarray analysis of mouse and human arachnoidal tissue revealed expression of many drug transporters and some drug-metabolizing enzymes. Immortalized mouse AB cells express functional P-gp on the apical (dura-facing) membrane and BCRP on both apical and basal (CSF-facing) membranes. Thus, like blood-brain barrier cells and choroid plexus cells, AB cells highly express drug transport proteins and likely contribute to the blood-CSF drug permeation barrier.

The effects of sex, ethnicity, and genetic polymorphism on hepatic CYP2B6 (cytochrome P450 2B6) expression and activity were previously demonstrated in vitro. Race/ethnic differences in CYP2B6 genotype and phenotype were observed only in women. To identify important covariates associated with interindividual variation in CYP2B6 activity in vivo, we evaluated these effects in healthy volunteers using bupropion (Wellbutrin SR GlaxoSmithKline, Research Triangle Park, NC) as a CYP2B6 probe substrate. A fixed 150-mg oral sustained-release dose of bupropion was administered to 100 healthy volunteers comprising four sex/ethnicity cohorts (n = 25 each): Caucasian men and Caucasian, African American, and Hispanic women. Blood samples were obtained at 0 and 6 hours postdose for the measurement of serum bupropion (BU) and hydroxybupropion (HB) concentrations. Whole blood was obtained at baseline for CYP2B6 genotyping. To characterize the relationship between CYP2B6 activity and ethnicity, sex, and genotype when accounting for serum BU concentrations (dose-adjusted log(10)-transformed), analysis of covariance model was fitted in which the dependent variable was CYP2B6 activity represented as the log(10)-transformed, metabolic ratio of HB to BU concentrations. Several CYP2B6 polymorphisms were associated with CYP2B6 activity. Evidence of dependence of CYP2B6 activity on ethnicity or genotype-by-ethnicity interactions was not detected in women. These results suggest that CYP2B6 genotype is the most important patient variable for predicting the level of CYP2B6 activity in women, when measured by the metabolism of bupropion. The bupropion metabolic ratio appears to detect known differences in CYP2B6 activity associated with genetic polymorphism, across different ethnic groups. Prospective studies will be needed to validate the use of bupropion as a probe substrate for clinical use.

Variation within the melanocortin receptor 1 (MC1R) gene, that influences phenotypic traits and susceptibility to melanoma, is abundant across the populations. We assessed and compared the risk of melanoma in 2 European populations, German and Spanish, by genotyping MC1R variants through direct DNA sequencing from 1,185 melanoma cases and 1,582 controls. The presence of any variant in both populations was associated with a significantly increased risk of melanoma (odds ratio OR = 1.67, 95% confidence interval CI 1.40-1.99). The population attributable fractions (PAF) associated with the MC1R variants in both populations was over 25%. However, the results showed a statistically significant (p < 0.0001) higher frequency of MC1R variants in the German (70%) than in the Spanish population (60%). The red-hair colour (RHC) variants, though associated with increased risk in both populations, were more common in the German than in the Spanish population (p < 0.0001). Interestingly, non-RHC variants increased the disease risk in the Spanish (OR = 1.60, 95% CI 1.20-2.14) but not in the German population (OR = 1.07, 95% CI 0.80-1.44). Although RHC variants explained a major proportion of the observed PAF in the German population, in the Spanish population the major contributor to the PAF was the non-RHC V60L variant. We also observed reduced historic linkage disequilibrium between the variants V92M and T314T in the gene in German melanoma cases. In conclusion, our data underscored the unambiguous importance of the MC1R variants towards the population burden of melanoma. However, the variants that are associated with the disease differ between the investigated populations.

Single nucleotide polymorphisms, besides influencing susceptibility can potentially alter progression and survival in melanoma patients. In this study we evaluated the association of polymorphisms in the base-excision repair genes XRCC1 and APEX1 with overall survival (OS), metastasis-free survival (MFS) and survival following the first metastasis (SFM) in patients with cutaneous melanoma. We genotyped the D148E APEX1, -77 T>C XRCC1, R280H XRCC1, and R399Q XRCC1 polymorphisms in 400 German melanoma patients (Tx, N0, M0) using an allelic discrimination method. The results were correlated with the patient follow-up parameters. The significant association detected between the R399Q XRCC1 polymorphism and MFS was also evaluated in 529 Spanish melanoma patients. In a Kaplan-Meier survival model the AA genotype of the polymorphism showed a median OS of 24.4 years compared to 11.5 years for two other genotypes. Similarly patients with the AA genotype showed median MFS of 20.9 years compared to 5.3 years for two other genotypes. In a multivariate Cox proportional hazard model, the AA genotype was associated with a hazard ratio (HR) of 0.40, 95% (CI 0.21-0.78, p=0.007) for MFS and 0.32 (95% CI 0.11-0.90, p=0.03) for OS in 400 German melanoma patients. The decreased risk of metastasis was confirmed by adding 529 Spanish melanoma patients with a combined HR of 0.40 (95% CI 0.24-0.68, p=0.0006). A significant association with SFM was also found for -77 T>C XRCC1 (HR 1.73, 95% CI 1.02-2.94, p=0.04). Our results show that non-synonymous variants or those located in potential regulatory regions of DNA repair genes probably influence the disease outcome in melanoma patients and have potentially significant implications for patient surveillance and tailored treatment.

This manuscript provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

Purpose The results of a study of variant cytochrome P-450 (CYP) alleles and associated risks of drug–drug interactions (DDIs) and altered drug metabolism are reported. Methods The records of a pharmacogenetic testing laboratory were retrospectively analyzed to identify patients tested for polymorphisms of genes coding for five CYP isozymes important in drug metabolism (CYP2D6, CYP2C9, CYP2C19, CYP3A4, and CYP3A5) over a 16-month period. Based on the results of phenotyping, the patients were categorized by expected CYP isozyme activity (e.g., normal or poor metabolizer, expresser or nonexpresser). Using proprietary Web-based software, researchers analyzed phenotyping data and medication lists submitted by patients to determine the potential for DDIs, drug–gene interactions (DGIs), and drug–drug–gene interactions (DDGIs). Results In the mixed-race study population of more than 22,000 male and female patients (age range, 1–108 years; mean, 60 years), phenotypes associated with alterations of CYP metabolic pathways were common. Among patients in whom phenotypes for all five isozymes of interest were determined ( n = 14,578), about 93% were not categorized as normal metabolizers of all five proteins. In many cases, potential interaction threats were rated by clinicians as severe enough to warrant implementation or consideration of a medication regimen change or dose adjustment. Analysis of patient-provided medication lists indicated frequent use of medications posing DDI, DGI, or DDGI risks. Conclusion In a mixed-race population of over 20,000 U.S. patients, CYP gene polymorphisms associated with DDIs and other interaction threats were prevalent, and most individuals were not categorized as normal metabolizers of all five CYP isozymes of interest.

We studied gene expression in 18 melanocytic nevi with and four nevi without the V600E mutation in the BRAF gene using HG-U133A 2.0 microarray with 22,277 transcripts. Data analysis revealed 92 genes up-regulated and 105 genes down-regulated in nevi with the mutation compared to nevi without mutation. Pathway analysis showed that differentially regulated genes mapped to 10 genetic networks. The major network included genes involved in cell death, cell cycle, and cellular growth and proliferation. Up-regulated genes in nevi with the mutation included CDKN2A, CDKN1C, and MITF; whereas down-regulated genes included those involved in apoptotic and other pathways. Principal component analysis identified 22 probe sets (20 genes) that caused separate segregation of nevi with and without mutations. In conclusion, our data showed differences in gene expression between nevi with and without the V600E BRAF mutation. Moreover, nevi with mutations showed over-expression of genes involved in melanocytic senescence and cell cycle inhibition. © 2007 Wiley-Liss, Inc.

Biomarkers of individual susceptibility: field studiesBiomarker (including alleles if genetic): XPC(A>C;K939Q), XPD(A>C;K715Q), XPG(G>C;D1104H), APEX1(T>G;D148E), XRCC1(G>A;R399Q), XRCC3(C>T;T241M), NBS1(G>C;E185Q)Effect studied (phenotype/pathology):modulation of BCC riskTissue/biological material/sample size: BLOODMethod of analysis: PCRStudy design: case-controlStudy size: 529 subjects with BCC, 533 controlsImpact on outcome (including dose-response): XRCC3(C>T;T241M) decreased risk for BCC, PR 0.66, CI95%, 0.51-0.86, P=0.002NBS1(G>C;E185Q) increased risk for BCC in men OR1.44, CI95%, 1.1-1.88, P=0.008, not in women OR0.96, CI95%, 0.74-1024, P=0.76All other polymorphisms no correlation found with risk. KEYWORDS CLASSIFICATION: adverse effects;Adolescent;Adult;Aged;Aged,80 and over;biomarkers of individual susceptibility: field studies;cancer epidemiology;Carcinoma,Basal Cell;Case-Control Studies;Cell Cycle;Cell Cycle Proteins;Child;Child,Preschool;DNA Repair;epidemiology;Female;genetics;Genetic Predisposition to Disease;Genotype;Germany;Humans;Hungary;Male;Middle Aged;Neoplasm Proteins;Nuclear Proteins;Odds Ratio;Polymorphism,Single Nucleotide;Proteins;Research;Risk Factors;Romania;Skin Neoplasms;Slovakia;Ultraviolet Rays. In addition to environmental exposures like UV radiation and, in some cases, arsenic contamination of drinking water, genetic factors may also influence the individual susceptibility to basal cell carcinoma of skin (BCC). In the present study, 529 cases diagnosed with BCC and 533 controls from Hungary, Romania and Slovakia were genotyped for one polymorphism in each of seven DNA repair genes. The variant allele for T241M (C>T) polymorphism in the XRCC3 gene was associated with a decreased cancer risk [odds ratio (OR), 0.73; 95% confidence interval (CI), 0.61-0.88; P = 0.0007, multiple testing corrected P = 0.004]. The risk of multiple BCC was significantly lower among variant allele carriers than in non-carriers (P = 0.04). Men homozygous for the C-allele for E185Q (G>C) polymorphism in the NBS1 gene showed an increased BCC risk (OR, 2.19; 95% CI, 1.23-3.91), but not women (OR, 0.84; 95% CI, 0.49-1.47). In men, the age and nationality adjusted OR for the genotype CC (XRCC3)/CC (NBS1) was 8.79 (95% CI, 2.10-36.8), compared with the genotype TT (XRCC3)/GG (NBS1). The data from this study show overall risk modulation of BCC by variant allele for T241M polymorphism in XRCC3 and gender-specific effect by E185Q polymorphism in NBS1.

Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan® probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However, this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

Background In polypharmacy patients under home health management, pharmacogenetic testing coupled with guidance from a clinical decision support tool (CDST) on reducing drug, gene, and cumulative interaction risk may provide valuable insights in prescription drug treatment, reducing re-hospitalization and emergency department (ED) visits. We assessed the clinical impact of pharmacogenetic profiling integrating binary and cumulative drug and gene interaction warnings on home health polypharmacy patients. Methods and findings This prospective, open-label, randomized controlled trial was conducted at one hospital-based home health agency between February 2015 and February 2016. Recruitment came from patient referrals to home health at hospital discharge. Eligible patients were aged 50 years and older and taking or initiating treatment with medications with potential or significant drug-gene-based interactions. Subjects (n = 110) were randomized to pharmacogenetic profiling (n = 57). The study pharmacist reviewed drug-drug, drug-gene, and cumulative drug and/or gene interactions using the YouScript® CDST to provide drug therapy recommendations to clinicians. The control group (n = 53) received treatment as usual including pharmacist guided medication management using a standard drug information resource. The primary outcome measure was the number of re-hospitalizations and ED visits at 30 and 60 days after discharge from the hospital. The mean number of re-hospitalizations per patient in the tested vs. untested group was 0.25 vs. 0.38 at 30 days (relative risk (RR), 0.65; 95% confidence interval (CI), 0.32–1.28; P = 0.21) and 0.33 vs. 0.70 at 60 days following enrollment (RR, 0.48; 95% CI, 0.27–0.82; P = 0.007). The mean number of ED visits per patient in the tested vs. untested group was 0.25 vs. 0.40 at 30 days (RR, 0.62; 95% CI, 0.31–1.21; P = 0.16) and 0.39 vs. 0.66 at 60 days (RR, 0.58; 95% CI, 0.34–0.99; P = 0.045). Differences in composite outcomes at 60 days (exploratory endpoints) were also found. Of the total 124 drug therapy recommendations passed on to clinicians, 96 (77%) were followed. These findings should be verified with additional prospective confirmatory studies involving real-world applications in larger populations to broaden acceptance in routine clinical practice. Conclusions Pharmacogenetic testing of polypharmacy patients aged 50 and older, supported by an appropriate CDST, considerably reduced re-hospitalizations and ED visits at 60 days following enrollment resulting in potential health resource utilization savings and improved healthcare. Trial registration ClinicalTrials.gov NCT02378220

Aim: Genetic polymorphisms have the potential to influence drug metabolism and vary among ethnic groups. This study evaluated the correlation of genetic polymorphisms with nevirapine pharmacokinetics exposure in Malawians. Materials & methods: CYP450 2B6, 2D6, 3A4 and 3A5, ABCB1 and constitutive androstane receptor and pregnane X receptor, were analyzed for polymorphisms in 26 subjects. Results: Allele frequencies (variant) were: CYP2B6 514G>T (0.31) CYP2D6*4 (0.02); CYP2D6*17 (0.35); CYP3A4*1B (0.77); CYP3A5*3 (0.25); ABCB1 2677G>T (0.0), ABCB1 3435C>T (0.21), NR1I3 13711152T>C (0.02), NR1I2 44477T>C (0.10), NR1I2 63396C>T (0.33), NR1I2 6-bp indel (del: 0.17). CYP2B6 516G>T (non-wild-type/wild-type) correlated with nevirapine pharmacokinetic parameters; geometric mean ratios (95% CI): 1.75 (1.27–2.40) for area under the concentration time curve (AUC)0–12 h, 1.58 (1.03–2.42) for C0, and 0.53 (0.31–0.91) for clearance. In a multivariable model, nevirapine AUC increased by 1.5% per year of age (p < 0.0001), CYP2B6 516 T allele increased AUC by 92% (p < 0.0001), and CYP3A5*3 decreased AUC by 31% (p = 0.0027). Conclusion: Allele frequencies were similar to other sub-Saharan African populations. The T allele for CYP2B6 516 was significantly associated with nevirapine exposure. Original submitted 15 June 2010; Revision submitted 5 July 2011

Single-nucleotide polymorphisms in different DNA-repair genes are reported to modulate risk of various cancers including melanoma. We genotyped DNA from 1186 melanoma patients and 1280 healthy controls for 13 different polymorphisms in eight DNA-repair genes. Data analyses showed that none of the polymorphisms except T241M XRCC3 was associated with an increased risk for cutaneous melanoma. Carriers of the variant alleles were associated with a decreased risk (OR 0.83; 95% CI, 0.79-0.98). Three additional polymorphisms together with T241M XRCC3 that tagged the entire gene region and the neighbouring genes KLC1, ZFYVE21 and PPP1R13B were not associated with the disease risk; neither were the inferred haplotypes. Imputation showed association of comparable magnitude with 11 non-genotyped neighbouring polymorphisms. Finally, the combination of results for all polymorphisms genotyped in the present study with published data suggests that none of the investigated polymorphisms was associated with melanoma susceptibility. We conclude that 13 non-synonymous polymorphisms in eight DNA-repair genes that are frequently investigated with respect to modulation of cancer risk in populations are not associated with susceptibility to cutaneous melanoma. (C) 2010 Elsevier B.V. All rights reserved.

The CYP3A locus encodes hepatic enzymes that metabolize many clinically used drugs. However, there is marked interindividual variability in enzyme expression and clearance of drugs metabolized by these enzymes. We utilized comparative genomics and computational prediction of transcriptional factor binding sites to evaluate regions within CYP3A that were most likely to contribute to this variation. We then used a haplotype tagging single-nucleotide polymorphisms (htSNPs) approach to evaluate the entire locus with the fewest number of maximally informative SNPs. We investigated the association between these htSNPs and in vivo CYP3A enzyme activity using a single-point IV midazolam clearance assay. We found associations between the midazolam phenotype and age, diagnosis of hypertension and one htSNP (141689) located upstream of CYP3A4. 141689 lies near the xenobiotic responsive enhancer module (XREM) regulatory region of CYP3A4. Cell-based studies show increased transcriptional activation with the minor allele at 141689, in agreement with the in vivo association study findings. This study marks the first systematic evaluation of coding and noncoding variation that may contribute to CYP3A phenotypic variability.

A 19-base pair germline deletion in exon 2 of the CDKN2A (cyclin-dependent kinase inhibitor 2A) gene (Leiden mutation) has been detected in Dutch families with familial melanomas. The penetrance of CDKN2A mutations varies widely and is influenced by environmental and unrelated genetic factors such as variants in the MC1R gene.We describe a 25-year-old German woman who developed 8 invasive melanomas and 6 in situ melanomas after radiation therapy and polychemotherapy for Hodgkin lymphoma. Genetic testing revealed a constitutional CDKN2A Leiden mutation in the proband and her sister, mother, and mother's sister. The proband also carried high-risk MC1R variant alleles R151C and R160W, which she had inherited from her father and her mother, respectively. The less affected mutation carrier sister did not have high-risk MC1R variant alleles. Analysis of DNA from paraffin-embedded tissues showed loss of heterozygosity at CDKN2A loci in all 3 melanomas studied but not in Hodgkin lymphoma. The pedigree revealed several types of cancers on both sides of the family, but no Dutch ancestors were found. No mutations in the CDK4, B-raf, and N-ras genes were detected either in the germline or in tumors from the patient.This study shows the variability of the penetrance of the CDKN2A Leiden mutation within the same family, which could be due to genetic or exogenous factors.

BACKGROUND: There is a wide variation in susceptibility to health effects of arsenic, which, in part, may be due to differences in arsenic metabolism. Arsenic is metabolized by reduction and methylation reactions, catalyzed by reductases and methyltransferases. OBJECTIVES: Our goal in this study was to elucidate the influence of various demographic and genetic factors on the metabolism of arsenic. METHODS: We studied 415 individuals from Hungary, Romania, and Slovakia by measuring arsenic metabolites in urine using liquid chromatography with hydride generation and inductively coupled plasma mass spectrometry (HPLC-HG-ICPMS). We performed genotyping of arsenic (+III) methyltransferase (AS3MT), glutathione S-transferase omega 1 (GSTO1), and methylene-tetrahydrofolate reductase (MTHFR). RESULTS: The results show that the M287T (T-->C) polymorphism in the AS3MT gene, the A222V (C-->T) polymorphism in the MTHFR gene, body mass index, and sex are major factors that influence arsenic metabolism in this population, with a median of 8.0 microg/L arsenic in urine. Females < 60 years of age had, in general, higher methylation efficiency than males, indicating an influence of sex steroids. That might also explain the observed better methylation in overweight or obese women, compared with normal weight men. The influence of the M287T (T-->C) polymorphism in the AS3MT gene on the methylation capacity was much more pronounced in men than in women. CONCLUSIONS: The factors investigated explained almost 20% of the variation seen in the metabolism of arsenic among men and only around 4% of the variation among women. The rest of the variation is probably explained by other methyltransferases backing up the methylation of arsenic.

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients.

We studied differential global gene expression in four melanoma cell lines with three cell lines without homozygous deletion of the CDKN2A locus using HG-U133A microarrays with 22 277 transcripts. None of the cell lines carried mutations in the B-RAF and N-RAS genes. Data analysis using stringent criteria showed specific upregulation of 70 genes and downregulation of 86 genes in cell lines with homozygous deletion of the CDKN2A gene. A comparison with previous expression data showed overlapping of upregulation and downregulation of seven and 23 genes, respectively, in melanoma cell lines with homozygous deletion of the CDKN2A locus or mutations in the B-RAF and N-RAS genes. Microarray data for eight selected genes were validated with an extended number of cell lines using quantitative real-time polymerase chain reaction. The upregulated genes in cell lines with the deletion besides others included MAGE A2 [fold change 128, 95% confidence interval (CI) 82.8-172.2; t-test P=0.004], MAGE A6 (fold change 623, 95% CI 473.4-772.1; t-test P=0.001), MAGE A12 (fold change 90, 95% CI 65.1-115.5; t-test P=0.001) and dopachrome tautomerase (fold change 42, 95% CI 32.5-51.8; t-test P=0.001). Downregulated genes included interleukin 18 (fold change 489, 95% CI 146.4-831.2; t-test P=0.04), ID2 (fold change 3, 95% CI 2.2-4.9; t-test P=0.001), KLF4 (fold change 9, 95% CI 4.3-14.7; P=0.01) and CD24 antigen (fold change 1308, 95% CI 766.0-1850.8; t-test P=0.01). The upregulated genes common to cell lines with homozygous deletion of the CDKN2A gene and mutations in B-RAF and N-RAS gene included those that are involved in RAS/RAF/MEK/ERK pathways. Our results highlight effects of homozygous deletion of the CDKN2A locus on global gene expression.

Summary This case–control study examines the association between lung cancer and genetic polymorphisms in two base excision repair (BER) genes, XRCC1 and APEX1 and two genes involved in homologous recombination repair (HR), XRCC3 and NBS1. Never-smoking lung cancer patients were recruited, and also the next diagnosed ever-smoking case of the same gender and age group. Controls were recruited from the regional population register, frequency matched to cases by hospital catchment area, gender, age group and smoking category. As a result more than 70% of the study population were women. A total of 331 individuals were analysed. Presence of the XRCC1 399Gln allele was associated with a significantly decreased risk for lung cancer among non-smoking women (odds ratio (OR) 0.4, 95% confidence interval (CI) 0.2–0.9). No significant effect was seen with the APEX1 polymorphism. Women smokers carrying the XRCC3 241Met allele showed a significantly decreased risk for lung cancer (OR 0.3, CI 0.2–0.7). The NBS1 185Gln allele was significantly associated with an increased risk for lung cancer among non-smoking women (OR 2.2, CI 1.0–4.8) and low-dose smoking women (OR 4.8, CI 1.5–15.7). The protective effect of the variant XRCC3 241Met allele was strengthened when combined with the low-risk Glu185 allele of the NBS1 gene. Smokers (OR 0.38, CI 0.16–0.90) and women (OR 0.42, CI 0.21–0.85) with at least three low-risk alleles in these two HR genes showed a significantly decreased risk for lung cancer. Thus, in spite of a relatively small study population, this study, including a comparatively large number of never-smokers and women, presents several novel aspects on genetic susceptibility to lung cancer. Our results show that the genetic variation in XRCC1, XRCC3 and NBS1 influence lung cancer susceptibility among women, and that combinations of risk alleles in the two HR genes can enhance the effects.

The endothelin signaling pathway plays a crucial role in melanocyte differentiation and migration. In this study, we investigated whether germline mutations of endothelin receptor B (EDNRB), a gene involved in Hirschsprung disease (HSCR), could also predispose for malignant melanoma (MM). The coding region of EDNRB was sequenced in 137 MM patients and in 130 ethnically matched Caucasian control subjects. Six nonsynonymous EDNRB variants were found in 15 patients (11%), but only two were found in four control subjects (3%, odds ratio [OR] = 3.87, 95% confidence interval [CI] = 1.25 to 12; P = .012). Overall, 14 out of 15 MM patients carried EDNRB mutations reported in HSCR, some of which had previously been shown to lead to loss of function. In multivariable logistic regression analysis including skin type, eye and hair color, number of nevi, and dorsal lentigines (freckles), the association between EDNRB mutations and MM risk remained statistically significant (OR = 19.9, 95% CI = 1.34 to 296.2; P = .03). Our data strongly suggest that EDNRB is involved in predisposition for two different multigenic disorders, HSCR and melanoma.