Clinical, structural and functional implications of mutations and polymorphisms in human NADPH P450 oxidoreductase

Cytochrome P450 proteins are involved in metabolism of drugs and xenobiotics. In the endoplasmic reticulum a single nicotinamide adenine dinucleotide phosphate (NADPH) P450 oxidoreductase (POR) supplies electrons to all microsomal P450s for catalytic activity. POR is a flavoprotein that contains both flavin mononucleotide and flavin adenine dinucleotide as cofactors and uses NADPH as the source of electrons. We have recently reported a number of POR mutations in the patients with disordered steroidogenesis. In the first report we had described missense mutations (A287P, R457H, V492E, C569Y, and V608F) identified in four patients with defects in steroid production. Each POR variant was produced as recombinant N-27 form of the enzyme in bacteria and as full-length form in yeast. Membranes from bacteria or yeast expressing normal or variant POR were purified and their activities were characterized in cytochrome c and CYP17A1 assays. Later we have published a larger study that described a whole range of POR mutations and characterized the mutants/polymorphisms A115V, T142A, M263V, Y459H, A503V, G539R, L565P, R616X, V631I, and F646del from the sequencing of patient DNA. We also studied POR variants Y181D, P228L, R316W, G413S, and G504R that were available in public databases or published literature. Three-dimensional structure of rat POR is known and we have used this structure to deduce the structure-function correlation of POR mutations in human. The missense mutations found in patients with disordered steroidogenesis are generally in the co-factor binding and functionally important domains of POR and the apparent polymorphisms are found in regions with lesser structural importance. A variation in POR can alter the activity of all microsomal P450s, and therefore, can affect the metabolism of drugs and xenobiotics even when the P450s involved are otherwise normal. It is important to study the genetic and biochemical basis of POR variants in human population to gain information about possible differences in P450 mediated reactions among the individuals carrying a variant or polymorphic form of POR that could impact their metabolism.

Mutations and polymorphisms in the human argininosuccinate lyase (ASL) gene

Argininosuccinate lyase deficiency (ASLD) is caused by a defect of the urea cycle enzyme argininosuccinate lyase (ASL) encoded by the ASL gene. Patients often present early after birth with hyperammonemia but can also manifest outside the neonatal period mainly triggered by excessive protein catabolism. Clinical courses comprise asymptomatic individuals who only excrete the biochemical marker, argininosuccinic acid, in urine, and patients who succumb to their first hyperammonemic decompensation. Some patients without any hyperammonemia develop severe neurological disease. Here, we are providing an update on the molecular basis of ASLD by collecting all published (n = 67) as well as novel mutations (n = 67) of the ASL gene. We compile data on all 160 different genotypes ever identified in 223 ASLD patients, including clinical courses whenever available. Finally, we are presenting structural considerations focusing on the relevance of mutations for ASL homotetramer formation. ASLD can be considered as a panethnic disease with only single founder mutations identified in the Finnish (c.299T>C, p.Ile100Thr) and Arab (c.1060C>T, p.Gln354*) population. Most mutations are private with only few genotypes recurring in unrelated patients. The majority of mutations are missense changes including some with more frequent occurrence such as p.Arg12Gln, p.Ile100Thr, p.Val178Met, p.Arg186Trp, p.Glu189Gly, p.Gln286Arg, and p.Arg385Cys.

A retrospective comparative exploratory study on two methylentetrahydrofolate reductase (MTHFR) polymorphisms in esophagogastric cancer: the A1298C MTHFR polymorphism is an independent prognostic factor only in neoadjuvantly treated gastric cancer patients.

BACKGROUND Methylentetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism and consequently could be an important factor for the efficacy of a treatment with 5-fluorouracil. Our aim was to evaluate the prognostic and predictive value of two well characterized constitutional MTHFR gene polymorphisms for primarily resected and neoadjuvantly treated esophagogastric adenocarcinomas. METHODS 569 patients from two centers were analyzed (gastric cancer: 218, carcinoma of the esophagogastric junction (AEG II, III): 208 and esophagus (AEG I): 143). 369 patients received neoadjuvant chemotherapy followed by surgery, 200 patients were resected without preoperative treatment. The MTHFR C677T and A1298C polymorphisms were determined in DNA from peripheral blood lymphozytes. Associations with prognosis, response and clinicopathological factors were analyzed retrospectively within a prospective database (chi-square, log-rank, cox regression). RESULTS Only the MTHFR A1298C polymorphisms had prognostic relevance in neoadjuvantly treated patients but it was not a predictor for response to neoadjuvant chemotherapy. The AC genotype of the MTHFR A1298C polymorphisms was significantly associated with worse outcome (p = 0.02, HR 1.47 (1.06-2.04). If neoadjuvantly treated patients were analyzed based on their tumor localization, the AC genotype of the MTHFR A1298C polymorphisms was a significant negative prognostic factor in patients with gastric cancer according to UICC 6th edition (gastric cancer including AEG type II, III: HR 2.0, 95% CI 1.3-2.0, p = 0.001) and 7th edition (gastric cancer without AEG II, III: HR 2.8, 95% CI 1.5-5.7, p = 0.003), not for AEG I. For both definitions of gastric cancer the AC genotype was confirmed as an independent negative prognostic factor in cox regression analysis. In primarily resected patients neither the MTHFR A1298C nor the MTHFR C677T polymorphisms had prognostic impact. CONCLUSIONS The MTHFR A1298C polymorphisms was an independent prognostic factor in patients with neoadjuvantly treated gastric adenocarcinomas (according to both UICC 6th or 7th definitions for gastric cancer) but not in AEG I nor in primarily resected patients, which confirms the impact of this enzyme on chemotherapy associated outcome.

Polymorphisms in MIR27A Associated with Early-Onset Toxicity in Fluoropyrimidine-Based Chemotherapy

PURPOSE The microRNA miR-27a was recently shown to directly regulate dihydropyrimidine dehydrogenase (DPD), the key enzyme in fluoropyrimidine catabolism. A common polymorphism (rs895819A>G) in the miR-27a genomic region (MIR27A) was associated with reduced DPD activity in healthy volunteers, but the clinical relevance of this effect is still unknown. Here, we assessed the association of MIR27A germline variants with early-onset fluoropyrimidine toxicity. EXPERIMENTAL DESIGN MIR27A was sequenced in 514 patients with cancer receiving fluoropyrimidine-based chemotherapy. Associations of MIR27A polymorphisms with early-onset (cycles 1-2) fluoropyrimidine toxicity were assessed in the context of known risk variants in the DPD gene (DPYD) and additional covariates associated with toxicity. RESULTS The association of rs895819A>G with early-onset fluoropyrimidine toxicity was strongly dependent on DPYD risk variant carrier status (Pinteraction = 0.0025). In patients carrying DPYD risk variants, rs895819G was associated with a strongly increased toxicity risk [OR, 7.6; 95% confidence interval (CI), 1.7-34.7; P = 0.0085]. Overall, 71% (12/17) of patients who carried both rs895819G and a DPYD risk variant experienced severe toxicity. In patients without DPYD risk variants, rs895819G was associated with a modest decrease in toxicity risk (OR, 0.62; 95% CI, 0.43-0.9; P = 0.012). CONCLUSIONS These results indicate that miR-27a and rs895819A>G may be clinically relevant for further toxicity risk stratification in carriers of DPYD risk variants. Our data suggest that direct suppression of DPD by miR-27a is primarily relevant in the context of fluoropyrimidine toxicity in patients with reduced DPD activity. However, miR-27a regulation of additional targets may outweigh its effect on DPD in patients without DPYD risk variants.

The impact of ABCC11 polymorphisms on the risk of early-onset fluoropyrimidine toxicity

A missense variant (c.1637C>T, T546M) in ABCC11 encoding the MRP8 (multidrug resistance protein 8), a transporter of 5-fluorodeoxyuridine monophosphate, has been associated with an increased risk of 5-fluorouracil-related severe leukopenia. To validate this association, we investigated the impact of the ABCC11 variants c.1637C>T, c.538G>A and c.395+1087C>T on the risk of early-onset fluoropyrimidine-related toxicity in 514 cancer patients. The ABCC11 variant c.1637C>T was strongly associated with severe leukopenia in patients carrying risk variants in DPYD, encoding the key fluoropyrimidine-metabolizing enzyme dihydropyrimidine dehydrogenase (odds ratio (OR): 71.0; 95% confidence interval (CI): 2.5-2004.8; Pc.1637C>T*DPYD=0.013). In contrast, in patients without DPYD risk variants, no association with leukopenia (OR: 0.95; 95% CI: 0.34-2.6) or overall fluoropyrimidine-related toxicity (OR: 1.02; 95% CI: 0.5-2.1) was observed. Our study thus suggests that c.1637C>T affects fluoropyrimidine toxicity to leukocytes particularly in patients with high drug exposure, for example, because of reduced fluoropyrimidine catabolism.