Single-nucleotide polymorphisms in the genes of CES2, CDA and enzymatic activity of CDA for prediction of the efficacy of capecitabine-containing chemotherapy in patients with metastatic breast cancer.

We examined whether genetic polymorphisms (SNPs) in the capecitabine activation pathway and CDA enzymatic activity were associated with prognosis, benefit from capecitabine-containing treatment or capecitabine-related toxicities. The study population comprised 188 metastatic breast cancer patients of the ATX trial (EudraCT 2006-006058-83) randomized for first-line paclitaxel and bevacizumab with (ATX) or without capecitabine (AT). Cumulative capecitabine dose until grade ≥2 hand-foot syndrome or until first dose reduction were toxicity endpoints. We genotyped CDA c.-451C>T (rs532545), CDA c.-33delC (rs3215400) and CES2 c.-806C>G (rs11075646). CDA activity in baseline serum was measured with a spectrophotometric assay and values were analyzed using a median cut-off or as continuous variable. CDA c.-33delC was prognostic for overall survival (OS) independent of hormone receptor status. For the predictive analysis, progression-free survival benefit from ATX over AT was observed in patients with a CDA c.-33del/del or del/insC genotype, a CDA c.-451CC or CT genotype, and a CES2 c.-806CC genotype compared with their counterparts. There was a higher response rate for ATX over AT in patients with a CDA c.-451CT or TT genotype. Patients with high CDA enzymatic activity had more benefit from capecitabine, while this was marginally observed in the CDA low group. Toxicity endpoints were not associated with any candidate markers. In conclusion, CDA c.-33delC was associated with OS. Since particular SNPs in CDA and CES2 were associated with benefit from the addition of capecitabine to AT, their predictive value should be explored in a higher number of patients.

Flexible and Scalable Full-Length CYP2D6 Long Amplicon PacBio Sequencing.

Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.

Insulin-like growth factor 1 receptor expression and IGF1R 3129G > T polymorphism are associated with response to neoadjuvant chemotherapy in breast cancer patients: results from the NEOZOTAC trial (BOOG 2010-01).

BACKGROUND: The insulin-like growth factor 1 (IGF-1) pathway is involved in cell growth and proliferation and is associated with tumorigenesis and therapy resistance. This study aims to elucidate whether variation in the IGF-1 pathway is predictive for pathologic response in early HER2 negative breast cancer (BC) patients, taking part in the phase III NEOZOTAC trial, randomizing between 6 cycles of neoadjuvant TAC chemotherapy with or without zoledronic acid. METHODS: Formalin-fixed paraffin-embedded tissue samples of pre-chemotherapy biopsies and operation specimens were collected for analysis of IGF-1 receptor (IGF-1R) expression (n = 216) and for analysis of 8 candidate single nucleotide polymorphisms (SNPs) in genes of the IGF-1 pathway (n = 184) using OpenArray® RealTime PCR. Associations with patient and tumor characteristics and chemotherapy response according to Miller and Payne pathologic response were performed using chi-square and regression analysis. RESULTS: During chemotherapy, a significant number of tumors (47.2 %) showed a decrease in IGF-1R expression, while in a small number of tumors an upregulation was seen (15.1 %). IGF-1R expression before treatment was not associated with pathological response, however, absence of IGF-1R expression after treatment was associated with a better response in multivariate analysis (P = 0.006) and patients with a decrease in expression during treatment showed a better response to chemotherapy as well (P = 0.020). Moreover, the variant T allele of 3129G > T in IGF1R (rs2016347) was associated with a better pathological response in multivariate analysis (P = 0.032). CONCLUSIONS: Absent or diminished expression of IGF-1R after neoadjuvant chemotherapy was associated with a better pathological response. Additionally, we found a SNP (rs2016347) in IGF1R as a potential predictive marker for chemotherapy efficacy in BC patients treated with TAC. TRIAL REGISTRATION: ClinicalTrials.gov NCT01099436 . Registered April 6, 2010.

Genotypes of CYP2C8 and FGD4 and their association with peripheral neuropathy or early dose reduction in paclitaxel-treated breast cancer patients.

BACKGROUND: The purpose of this study was to evaluate single-nucleotide polymorphisms (SNPs) in genes encoding key metabolising enzymes or involved in pharmacodynamics for possible associations with paclitaxel-induced peripheral neuropathy. METHODS: The study population consists of 188 women from the multicenter, randomised, phase II ATX trial (BOOG2006-06; EudraCT number 2006-006058-83) that received paclitaxel and bevacizumab without or with capecitabine as first-line palliative therapy of HER2-negative metastatic breast cancer. Genotyping of CYP2C8*3 (c.416G>A), CYP3A4*22 (c.522-191C>T), TUBB2A (c.-101T>C), FGD4 (c.2044-236G>A) and EPHA5 (c.2895G>A) was performed by real-time PCR. Toxicity endpoints were cumulative dose (1) until first onset of grade ⩾1 peripheral neuropathy and (2) until first paclitaxel dose reduction from related toxicity (NCI-CTCAE version 3.0). SNPs were evaluated using the Kaplan-Meier method, the Gehan-Breslow-Wilcoxon test and the multivariate Cox regression analysis. RESULTS: The rate of grade ⩾1 peripheral neuropathy was 67% (n=126). The rate of dose reduction was 46% (n=87). Age ⩾65 years was a risk factor for peripheral neuropathy (HR=1.87, P<0.008), but not for dose reduction. When adjusted for age, body surface area and total cumulative paclitaxel dose, CYP2C8*3 carriers had an increased risk of peripheral neuropathy (HR=1.59, P=0.045). FGD4 c.2044-236 A-allele carriers had an increased risk of paclitaxel dose reduction (HR per A-allele=1.38, P=0.036) when adjusted for total cumulative paclitaxel dose. CONCLUSIONS: These findings may point towards clinically useful indicators of early toxicity, but warrant further investigation.

Exploring genetic and non-genetic risk factors for delayed graft function, acute and subclinical rejection in renal transplant recipients.

AIMS: This study aimed at identifying pharmacological factors such as pharmacogenetics and drug exposure as new predictive biomarkers for delayed graft function (DGF), acute rejection (AR) and/or subclinical rejection (SCR). METHODS: Adult renal transplant recipients (n = 361) on cyclosporine-based immunosuppression were followed for the first 6 months after transplantation. The incidence of DGF and AR were documented as well as the prevalence of SCR at 6 months in surveillance biopsies. Demographic, transplant-related factors, pharmacological and pharmacogenetic factors (ABCB1, CYP3A5, CYP3A4, CYP2C8, NR1I2, PPP3CA and PPP3CB) were analysed in a combined approach in relation to the occurrence of DGF, AR and prevalence of SCR at month 6 using a proportional odds model and time to event model. RESULTS: Fourteen per cent of the patients experienced at least one clinical rejection episode and only DGF showed a significant effect on the time to AR. The incidence of DGF correlated with a deceased donor kidney transplant (27% vs. 0.6% of living donors). Pharmacogenetic factors were not associated with risk for DGF, AR or SCR. A deceased donor kidney and acute rejection history were the most important determinants for SCR, resulting in a 52% risk of SCR at 6 months (vs. 11% average). In a sub-analysis of the patients with AR, those treated with rejection treatment including ATG, significantly less frequent SCR was found in the 6-month biopsy (13% vs. 50%). CONCLUSIONS: Transplant-related factors remain the most important determinants of DGF, AR and SCR. Furthermore, rejection treatment with depleting antibodies effectively prevented SCR in 6-month surveillance biopsies.

A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

CYP2D6 genotype in relation to hot flashes as tamoxifen side effect in a Dutch cohort of the tamoxifen exemestane adjuvant multinational (TEAM) trial.

In tamoxifen-treated breast cancer patients the occurrence of hot flashes may be associated with effective estrogen receptor antagonism dependent on genetic variations of metabolic enzymes and the estrogen receptor. Early breast cancer patients who were randomized to receive tamoxifen, followed by exemestane within the tamoxifen exemestane adjuvant multinational trial were genotyped for five CYP2D6 alleles. CYP2D6 genotypes and phenotypes were related to the occurrence of hot flashes as adverse event during the first year of tamoxifen use (primary aim) and the time to the occurrence of hot flashes as AE during the complete time on tamoxifen (secondary aim). In addition, exploratory analyses on 22 genetic variants of other metabolic enzymes and two common polymorphisms in the estrogen receptor-1 were performed. No association was found between the CYP2D6 genotype/phenotype or any other genetic variant and hot flashes during the first year. Only higher age was related to a lower incidence of hot flashes in the first year (adjusted odds ratio 0.94, 95 % CI 0.92-0.96; p < 0.001). The ESR1 PvuII XbaI CG haplotype was associated with the time to the occurrence of hot flashes during the complete time on tamoxifen (CG/CG vs. CG/other + other/other: adjusted hazard ratio 0.49, 95 % CI 0.25-0.97; p = 0.04). In conclusion, the CYP2D6 genotypes and phenotypes were not associated with the occurrence of hot flashes. Common polymorphisms in the estrogen receptor-1 might predict hot flashes as common tamoxifen side effect, although this finding needs replication.

Germline variants in the CYP19A1 gene are related to specific adverse events in aromatase inhibitor users: a substudy of Dutch patients in the TEAM trial.

Musculoskeletal adverse events (MSAEs) and vasomotor symptoms (VMSs) are known side-effects of aromatase inhibitors, and may be related to genetic variations of the aromatase gene (CYP19A1). We investigated the relationship between these specific AEs and single nucleotide polymorphisms (SNPs) in the CYP19A1 gene in postmenopausal, hormone receptor-positive early breast cancer (BC) patients treated with adjuvant exemestane for 5 years. Dutch patients who were randomized to receive 5 years of exemestane in the Tamoxifen Exemestane Adjuvant Multinational (TEAM) trial were included. A tagging-SNP approach was performed, covering 80 % of variations of the CYP19A1 gene with 30 SNPs. Logistic regression analyses were used to assess the risk of reporting VMSs or MSAEs in relation to genotypes within selected SNPs. Of 737 included patients, 281 patients reported at least one MSAE (n = 210) or VMS (n = 163). Homozygous AA genotype of rs934635 was associated with a significantly higher odds of MSAEs (multivariate odds ratio (OR) 4.66, p = 0.008) and VMSs (multivariate OR 2.78, p = 0.044). Regarding both rs1694189 and rs7176005, the homozygous variant genotypes (TT) were associated with a higher odds of VMSs, but not MSAEs (OR 1.758, p = 0.025 and OR 6.361, p = 0.021, respectively). Our exploratory analysis demonstrated that some CYP19A1 gene variations may be associated with MSAEs and/or VMSs. Specifically, patients with the homozygous variant rs934635 genotype reported more MSAEs and VMSs. Although further confirmatory studies are warranted, genomic profiling can help identify patients at an increased risk of reporting these specific AEs, potentiating further personalized BC treatment.

Excision Repair Cross-Complementation group 1 (ERCC1) C118T SNP does not affect cellular response to oxaliplatin.

AIMS: ERCC1 is involved in the repair of oxaliplatin-induced DNA damage. Studies for the association of the C118T SNP with clinical response to treatment with platinum drugs have rendered inconsistent results. We investigated the ERCC1 C118T SNP with respect to overall and progression-free survival in patients with advanced colorectal cancer (ACC) treated with oxaliplatin and in vitro DNA repair capacity after oxaliplatin exposure. In addition we discuss discrepancies from other studies concerning ERCC1 C118T. MATERIALS AND METHODS: Progression-free survival was determined in 145 ACC patients treated with oxaliplatin-based chemotherapy in a phase 3 trial. For the in vitro studies regarding ERCC1 functionality, we transfected an ERCC1 negative cell line with 118C or 118T ERCC1. Cellular sensitivity and DNA repair capacity after exposure to oxaliplatin was examined by Sulphorodamine B growth inhibition assay, COMET assay and Rad51 foci staining. RESULTS: We found no association between ERCC1 C118T and progression-free or overall survival. In addition, transfection of either 118C or 118T restores DNA-repair capacity of UV20 cells to the same level and chemosensitivity to oxaliplatin was similar in ERCC1 118C and 118T transfected cells. CONCLUSION: This study shows that the ERCC1 C118T variants are not associated with survival in ACC patients treated with oxaliplatin or the in vitro sensitivity and DNA-repair capacity in 118C and 118T transfected cell lines. Therefore, ERCC1 C118T genotyping seems of no value in individualizing oxaliplatin based chemotherapy in ACC.

A novel specific pyrosequencing method for genotyping FCGR3A rs396991 without coamplification of homologous gene FCGR3B.

Monoclonal antibodies, such as rituximab, trastuzumab, and cetuximab, mediate immune response by binding to Fcγ receptors. The frequently occurring Phe158Val variant of the FCGR3A gene has increased binding affinity and consequently may affect immune response. Several pharmacogenetic association studies have genotyped this variant (FCGR3A rs396991), but with disconcordant results. In addition, in some of these studies genotype distribution was not in Hardy-Weinberg equilibrium, and samples were excluded from analysis because of genotype inconsistency. Genotyping problems of FCGR3A rs396991 are most likely due to sequence homology with the FCGR3B gene. For that reason, we developed a novel pyrosequencing method specifically for genotyping FCGR3A rs396991 and confirmed that the FCGR3B gene is not coamplified.

Exploratory analysis of 1936 SNPs in ADME genes for association with busulfan clearance in adult hematopoietic stem cell recipients.

BACKGROUND: Busulfan is used in preparative regimens before stem cell transplantation. There is significant interpatient variability in busulfan pharmacokinetics (PK) and exposure is related to outcome. Polymorphisms in genes encoding glutathione-S-transferases have been associated with busulfan PK but only explain a limited portion of the observed variability. AIM: The aim of this study is to identify additional genetic variants associated with busulfan PK by interrogating 1936 variants in 225 genes involved in drug absorption, distribution, metabolism, and excretion (ADME). MATERIALS AND METHODS: In an exploratory cohort (n=65), patients who received busulfan were genotyped with the DMET array. Top SNPs and haplotypes associated with busulfan clearance were validated in an independent validation cohort (n=78). RESULTS: In the exploratory cohort, seven variants were identified to be associated with busulfan clearance (P<0.001). In the validation cohort, only GSTA5 (rs4715354 and rs7746993) remained significantly associated with busulfan clearance (P=0.025). CONCLUSION: This is the first study using an exploratory pharmacogenetic approach to explain the interindividual variability in busulfan PK. The role of glutathione-S-transferases was confirmed, but no additional genetic markers involved in drug ADME appear to be associated with busulfan PK.

Excision repair of BPDE-adducts in human lymphocytes: diminished capacity associated with ERCC1 C8092A (rs3212986) polymorphism.

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a metabolite of Benzo[a]pyrene (B[a]P), is a high-risk factor for development of a number of cancers. DNA damage caused by BPDE is normally repaired by Nucleotide Excision Repair system of which ERCC1 exerts an important role. We investigated whether two single nucleotide polymorphisms in ERCC1 (C19007T; rs11615 and C8092A; rs3213986) affected the repair efficacy of BPDE-DNA adducts. We collected peripheral blood of 780 healthy individuals from the northeast of China and detected the genotypes of rs11615 and rs3213986. The amount of induced BPDE-DNA adducts in lymphocytes from 117 randomly selected participants was assessed by HPLC. Presence of BPDE-DNA adducts in nucleus of lymphocytes was visualized using the modified comet assay. ERCC1 and CAST (3' adjacent gene of ERCC1) mRNA expression levels were quantified after in vitro exposure to BPDE. We found that the minor A allele in rs3212986 was related to higher levels of BPDE-DNA adducts and holistic marking DNA damage (P < 0.01). Haplotype CA (rs11615 and rs3213986) was also associated with an elevated risk of high BPDE-DNA adduct levels (OR = 1.801, 95 % CI of OR 1.191-2.724). Interestingly, in participants with AA genotype for rs3213986, CAST mRNA level was decreased compared to individuals with the homozygous CC genotype. Our findings suggests that ERCC1 C8092A (rs3213986) is associated with a diminished capacity of repairing BPDE-DNA adducts and may be used as a valid biomarker to predict an individual's risk to develop cancer upon exposure to environmental carcinogens.

ERCC1 and XPD/ERCC2 polymorphisms' predictive value of oxaliplatin-based chemotherapies in advanced colorectal cancer has an ethnic discrepancy: a meta-analysis.

Our purpose is to evaluate the predictive value of the genetic polymorphisms of Excision repair cross-complementing group 1 (ERCC1) and xeroderma pigmentosum group D/excision repair cross-complementing group 2 (XPD/ERCC2) in patients with advanced colorectal cancer receiving oxaliplatin-based chemotherapy, and we performed a meta-analysis in order to obtain a more precise estimation for a more optimizing individual chemotherapy. The relevant cohort studies were identified by searching the electronic databases of MEDLINE, EMBASE, and CNKI. We used ''colorectal,'' ''cancer,'' ''carcinoma,'' ''ERCC1,'' ''XPD or ERCC2,'' ''polymorphism,'' ''oxaliplatin,'' ''treatment,'' or ''chemotherapy'' as key words. Inclusion criteria were patients with advanced colorectal cancer receiving oxaliplatin-based chemotherapy, evaluation of polymorphism of ERCC1 and XPD/ ERCC2, and overall response rate (ORR). In this meta-analysis, a total of seven studies were selected according to the inclusion criteria. Five studies investigated ERCC1 codon 118 polymorphisms and three studies evaluated XPD/ERCC2 codon 751 polymorphisms. For ERCC1 codon C118T polymorphism, the ORR to oxaliplatin-based chemotherapy in patients with C/C wild genotype was 77.27% and it was 69.30% for C/T and T/T variant genotype. The pooled odds ratio (OR) for C/C wild-type vs. C/T and T/T genotype was 1.11 (95% CI, 0.86-1.42; P = 0.42). For XPD/ERCC2 Lys751Gln polymorphism, the response rate was 86.58 and 67.57% in patients with the A/A and either one or two C alleles (A/C or C/C) respectively, and the pooled OR was 1.15 (95% CI, 1.01-1.30; P = 0.03). Furthermore, we chose subgroup analysis in order to find the difference between the Caucasian and Asian ethnicity. The results indicated that Oxaliplatin sensitivity was significantly associated with ERCC1 C118T polymorphism in Asian people. XPD/ERCC2 Lys751Gln polymorphism had the predictive value especially for the patients from the America and Europe.

Influence of pharmacogenetic variability on the pharmacokinetics and toxicity of the aurora kinase inhibitor danusertib.

OBJECTIVES: Danusertib is a serine/threonine kinase inhibitor of multiple kinases, including aurora-A, B, and C. This explorative study aims to identify possible relationships between single nucleotide polymorphisms in genes coding for drug metabolizing enzymes and transporter proteins and clearance of danusertib, to clarify the interpatient variability in exposure. In addition, this study explores the relationship between target receptor polymorphisms and toxicity of danusertib. METHODS: For associations with clearance, 48 cancer patients treated in a phase I study were analyzed for ABCB1, ABCG2 and FMO3 polymorphisms. Association analyses between neutropenia and drug target receptors, including KDR, RET, FLT3, FLT4, AURKB and AURKA, were performed in 30 patients treated at recommended phase II dose-levels in three danusertib phase I or phase II trials. RESULTS: No relationships between danusertib clearance and drug metabolizing enzymes and transporter protein polymorphisms were found. Only, for the one patient with FMO3 18281AA polymorphism, a significantly higher clearance was noticed, compared to patients carrying at least 1 wild type allele. No effect of target receptor genotypes or haplotypes on neutropenia was observed. CONCLUSIONS: As we did not find any major correlations between pharmacogenetic variability in the studied enzymes and transporters and pharmacokinetics nor toxicity, it is unlikely that danusertib is highly susceptible for pharmacogenetic variation. Therefore, no dosing alterations of danusertib are expected in the future, based on the polymorphisms studied. However, the relationship between FMO3 polymorphisms and clearance of danusertib warrants further research, as we could study only a small group of patients.

Alternative methods to a TaqMan assay to detect a tri-allelic single nucleotide polymorphism rs757210 in the HNF1ß gene.

BACKGROUND: Several studies report difficulties in genotyping HNF1ß rs757210 using TaqMan probes. This is possibly due to the tri-allelic nature of this single nucleotide polymorphism (SNP). The aim of the present research was to develop alternative methods for genotyping rs757210. METHODS: Pyrosequencing and high resolution melting analysis of small amplicons (HRM) were developed and tested in panels of type 2 diabetes mellitus patients (n=258) and healthy blood donors (n=183). Results were confirmed by Sanger sequencing. RESULTS: With pyrosequencing, allele frequencies for the A, G and C allele of 0.42, 0.56, 0.02 and 0.37, 0.62, 0.01 were established in the panel of type 2 diabetes mellitus patients and healthy blood donors, respectively. Similar results were found using the more routinely available HRM method. Results for pyrosequencing and HRM were in 99.6% concordance. CONCLUSIONS: Pyrosequencing and HRM can be used to genotype the tri-allelic SNP rs757210 in the HNF1ß gene and have the advantage over the commercially available TaqMan analysis that they can determine the rare C-allele variant.

Evidence for qualified quantitative mRNA analysis in formalin-fixed and paraffin-embedded colorectal carcinoma cells and tissue.

Analysis of gene expression can provide important information related to prognostic or predictive factors in clinical work. Analysis of DNA or RNA isolated from formalin fixed and paraffin-embedded tissue is generally accepted to be difficult due to degradation and crosslinking. Factors affecting mRNA quality such as formalin fixation length and autolysis period before fixation/freezing have not been systematically addressed. In this study, we analyzed the effect of formalin fixation length and autolysis period before fixation/freezing on the expression of several reference genes in formalin-fixed paraffin-embedded (FFPE) colorectal carcinoma cells and tissue. We also used a factorial experimental design to further analyze the interaction of both variables. It was found that mRNA levels can be reproducibly quantified, independent of the tested variables. Our findings confirm that clinical tumor specimens removed by routine surgical procedures and combined with real-time RT-PCR can be used for the analysis of gene expression in routine FFPE materials and provide some useful information related to the achieved tissues largely stored in histopathology departments.

Gene-gene interactions in folate and adenosine biosynthesis pathways affect methotrexate efficacy and tolerability in rheumatoid arthritis.

OBJECTIVE: As no single nucleotide polymorphism has emerged as pivotal to predict the lack of efficacy and dose-limiting toxicities to methotrexate (MTX), we evaluated the contribution of gene-gene interactions to the effects of this prodrug in rheumatoid arthritis. METHODS: A total of 255 patients treated with MTX for at least 3 months were evaluated with efficacy assessed using the European League Against Rheumatism response criteria or a physician's assessment of patient's response to MTX visual analog scale. Gastrointestinal and neurological idiosyncrasies were recorded in 158 patients. Fourteen single nucleotide polymorphisms in folate and adenosine biosynthesis pathways were measured and detection of gene-gene interactions was performed using multifactor-dimensionality reduction, a method that reduces high-dimensional genetic data into a single dimension of predisposing or risk-genotype combinations. RESULTS: Efficacy to MTX (53% responders) was associated with high-order epistasis among variants in inosine-triphosphate pyrophosphatase, aminoimidazole-carboxamide ribonucleotide transformylase, and reduced folate carrier genes. In the absence of predisposing genotype combinations, a 3.8-fold lower likelihood of efficacy was observed (vs. in their presence, 95% confidence interval: 2.2-6.4; P<0.001). Increasing MTX polyglutamate concentrations tended to partially overcome this selective disadvantage. Idiosyncrasies occurred in 29% of patients. In the presence of risk-genotype combinations among variants in methylene tetrahydrofolate reductase, γ-glutamyl-hydrolase, thymidylate synthase, serine hydroxymethyltransferase, and inosine-triphosphate pyrophosphatase genes, an 8.9-fold higher likelihood to exhibit toxicities was observed (vs. in their absence, 95% confidence interval: 3.6-21.9; P<0.001). False-positive report probabilities were below 0.2, thereby indicating that true signals were likely detected in this cohort. CONCLUSION: These data indicate that gene-gene interactions impact MTX efficacy and tolerability in rheumatoid arthritis.

Explaining variability in tacrolimus pharmacokinetics to optimize early exposure in adult kidney transplant recipients.

To prevent acute rejection episodes, it is important to reach adequate tacrolimus (TRL) exposure early after kidney transplantation. With a better understanding of the high variability in the pharmacokinetics of TRL, the starting dose can be individualized, resulting in a reduction in dose adjustments to obtain the target exposure. A population pharmacokinetic analysis was performed to estimate the effects of demographic factors, hematocrit, serum albumin concentration, prednisolone dose, TRL dose interval, polymorphisms in genes coding for ABCB1, CYP3A5, CYP3A4, and the pregnane X receptor on TRL pharmacokinetics. Pharmacokinetic data were prospectively obtained in 31 de novo kidney transplant patients randomized to receive TRL once or twice daily, and subsequently, the data were analyzed by means of nonlinear mixed-effects modeling. TRL clearance was 1.5-fold higher for patients with the CYP3A5*1/*3 genotype compared with the CYP3A5*3/*3 genotype (5.5 +/- 0.5 L/h versus 3.7 +/- 0.3 L/h, respectively). This factor explained 30% of the interindividual variability in apparent clearance (exposure). Also, a relationship between the pregnane X receptor A+7635G genotype and TRL clearance was identified with a clearance of 3.9 +/- 0.3 L/h in the A allele carriers versus 5.4 +/- 0.6 L/h in the GG genotype. Finally, a concomitant prednisolone dose of more than 10 mg/d increased the TRL apparent clearance by 15%. In contrast, body weight was not related to TRL clearance in this population. Because patients are typically dosed per kilogram body weight, this might result in underexposure and overexposure in patients, with a low and high body weight, respectively. This integrated analysis shows that adult renal transplant recipients with the CYP3A5*1/*3 genotype require a 1.5 times higher, fixed, starting dose compared with CYP3A5*3/*3 to reach the predefined target exposure early after transplantation.

Influence of the d3-growth hormone (GH) receptor isoform on short-term and long-term treatment response to GH replacement in GH-deficient adults.

OBJECTIVE: Recombinant human GH (rhGH) replacement in adults is aimed at improving signs and symptoms of the adult GH deficiency (GHD) syndrome. In children, a common polymorphism of the GH receptor (exon-3 deletion, d3GHR) increases the response to rhGH replacement. The aim of this study was to assess the effects of this polymorphism on the response to rhGH replacement in adults. DESIGN: Prospective intervention with rhGH during 1 yr (n = 99) and in a subset during 5 yr (n = 53). PATIENTS AND METHODS: The presence of the d3GHR variant was established in GHD patients and linked to short-term and long-term effects of rhGH replacement on IGF-I, lipid metabolism, anthropometric parameters, and bone mineral density. RESULTS: Fifty-five patients had two wild-type alleles (56%), whereas 38 patients (38%) had one allele and six patients (6%) had two alleles coding the d3GHR isoform. During short-term rhGH replacement, the increase in IGF-I was higher in patients bearing at least one d3GHR allele, compared with those with two wild-type alleles (at an identical mean dose of rhGH). The decrease in total cholesterol and low-density lipoprotein cholesterol was lower in the group bearing at least one d3GHR allele, whereas the increase in high-density lipoprotein cholesterol was higher, compared with patients with the wild-type genotype. In contrast, these differential responses of GHR genotype could not be demonstrated during long-term rhGH replacement. CONCLUSION: The d3GHR genotype contributes, at least for some parameters, to the interindividual differences in efficacy of short-term, but not long-term, rhGH replacement in adults with GHD.

ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway.

Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5ERCC2 (AA) as compared to UV5ERCC2 (CC) after cisplatin treatment. The DNA damage level of UV5ERCC2 (AA) was significant decreased compared to UV5ERCC2 (CC) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5ERCC2 (AA) alleviated the apoptosis compared to UV5ERCC2 (CC), meanwhile P53mRNA levels in UVERCC2 (AA) was also lower when compared UV5ERCC2 (CC). It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism.