ALDH1L2 Knockout in U251 Glioblastoma Cells Reduces Tumor Sphere Formation by Increasing Oxidative Stress and Suppressing Methionine Dependency.

Previously, the in vitro growth of cancer stem cells in the form of tumor spheres from five different brain cancer cell lines was found to be methionine-dependent. As this earlier work indicated that ALDH1L2, a folate-dependent mitochondria aldehyde dehydrogenase gene, is upregulated in glioblastoma stem cells, we invalidated this gene using CRISPR-cas 9 technique in this present work. We reported here that this invalidation was effective in U251 glioblastoma cells, and no cas9 off target site could be detected by genome sequencing of the two independent knockout targeting either exon I or exon III. The knockout of ALDH1L2 gene in U251 cells rendered the growth of the cancer stem cells of U251 methionine independent. In addition, a much higher ROS (reactive oxygen radicals) level can be detected in the knockout cells compared to the wild type cells. Our evidence here linked the excessive ROS level of the knockout cells to reduced total cellular NADPH. Our evidence suggested also that the cause of the slower growth of the knockout turmor sphere may be related to its partial differentiation.

Could Cytochrome P450 2D6, 3A4 and 3A5 Polymorphisms Explain the Variability in Clinical Response to Clomiphene Citrate of Anovulatory PCOS Women?

Introduction: Cytochrome P450 2D6, 3A4 and 3A5 are involved in the metabolism of many drugs. These enzymes have a genetic polymorphism responsible for different metabolic phenotypes. They play a role in the metabolism of clomiphene citrate (CC), which is used to induce ovulation. Response to CC treatment is variable, and no predictive factors have thus far been identified. Objective: To study a possible link between the cytochrome P450 2D6, 3A4 and 3A5 polymorphisms and clinical response to CC. Study Design: Seventy-seven women with anovulatory Polycystic Ovarian Syndrome (PCOS) treated with CC were included which determined their cytochrome P450 2D6, 3A4 and 3A5 genotypes and used the results to predict ovarian response to this drug. Predicted responses based on the cytochrome genotypes were compared with the observed clinical responses using the calculation of a weighted Kappa coefficient. Main Outcome Measures: Number of dominant follicles assessed by ultrasound at the end of the follicular phase and confirmation of ovulation by blood progesterone assay in the luteal phase. Results: Concordance between the predicted and observed responses for the combination of the three cytochromes was 36.71%, with a negative Kappa coefficient (K = -0.0240), which corresponds to a major disagreement. Similarly, for predictions based on the cytochrome P450 2D6 genotype alone, only 39.24% of predictions were verified (coefficient K = -0.0609). Conclusion: The genetic polymorphism of cytochromes P450 2D6, 3A4 and 3A5 does not appear to influence clinical response to CC used to induce ovulation in anovulatory PCOS women.

Impact of Tacrolimus Daily Dose Limitation in Renal Transplant Recipients Expressing CYP3A5: A Retrospective Study.

The pharmacokinetic variability of tacrolimus can be partly explained by CYP3A5 activity. Our objective was to evaluate a tacrolimus sparing policy on renal graft outcome according to CYP3A5 6986A>G genetic polymorphism. This retrospective study included 1114 recipients with a median follow-up of 6.3 years. Genotyping of the 6986A>G allelic variant corresponding to CYP3A5*3 was systematically performed. One year after transplantation, tacrolimus blood trough concentration (C0) target range was 5-7 ng/mL. However, daily dose was capped to 0.10 mg/kg/day regardless of the CYP3A5 genotype. A total 208 CYP3A5*1/- patients were included. Despite a higher daily dose, CYP3A5*1/- recipients exhibited lower C0 during follow-up (p < 0.01). Multivariate analysis did not show any significant influence of CYP3A5*1/- genotype (HR = 0.70, 0.46-1.07, p = 0.10) on patient-graft survival. Glomerular Filtration Rate (GFR) decline was significantly lower for the CYP3A5*1/- group (p = 0.02). The CYP3A5*1/- genotype did not significantly impact the risk of biopsy-proven acute rejection (BPAR) (HR = 1.01, 0.68-1.49, p = 0.97) despite significantly lower C0. Based on our experience, a strategy of tacrolimus capping is associated with a better GFR evolution in CYP3A5*1/- recipients without any significant increase of BPAR incidence. Our study raised some issues about specific therapeutic tacrolimus C0 targets for CYP3A5*1/- patients and suggests to set up randomized control studies in this specific population.

Neonatal-onset Progressive Familial Intrahepatic Cholestasis (PFIC): first molecular study in Tunisian patients.

Progressive familial intrahepatic is a heterogeneous group of rare autosomal recessive liver disorders. Neonatal onset is characteristic of the PFIC 1 and PFIC 2, which result from mutations in genes respectivelyATP8B1 and ABCB11. Four Tunisian patients, three of them with PFIC 2 and one with PFIC1, were described. They all had typical clinical and biological features. However, they all had newly reported mutations. The same mutation was found in the patients with PFIC2, which could facilitate the diagnosis in Tunisian patients suspected in the future. The patient diagnosed with PFIC1 had also a newly described mutation, with a probable phenotypic particularity that is congenital hypothyroidism. Advances are being made to establish a molecular diagnosis in neonatal onset cholestasis. Indeed, next generation sequencing gene panels (NGSGP) potentially decrease the need for invasive procedures in these patients, enable early initiation of treatment and adequate genetic counseling.

Diagnostic utility of whole-genome sequencing for nephronophthisis.

Next-generation sequencing has revolutionized the molecular diagnosis of individuals affected by genetic kidney diseases. Indeed, rapid genetic testing in individuals with suspected inherited nephropathy has not only important implications for diagnosis and prognosis but also for genetic counseling. Nephronophthisis (NPHP) and related syndromes, a leading cause of end-stage renal failure, are autosomal recessive disorders characterized by the variable presentation and considerable locus heterogeneity with more than 90 genes described as single-gene causes. In this case report, we demonstrate the utility of whole-genome sequencing (WGS) for the molecular diagnosis of NPHP by identifying two putative disease-causing intronic mutations in the NPHP3 gene, including one deep intronic variant. We further show that both intronic variants, by affecting splicing, result in a truncated nephrocystin-3 protein. This study provides a framework for applying WGS as a first-line diagnostic tool for highly heterogeneous disease such as NPHP and further suggests that deep intronic variations are an important underestimated cause of monogenic disorders.

Caveolin-1 rs4730751 single-nucleotide polymorphism may not influence kidney transplant allograft survival.

Caveolin-1 is a protein (encoded by the CAV1 gene) supposedly harboring a protective effect against fibrosis. CAV1 rs4730751 single nucleotide polymorphism (SNP) AA genotype was initially associated with lower graft survival compared to non-AA. However, subsequent studies could not find the same effect. CAV1 rs4730751 SNP was investigated on 918 kidney donors. Multivariate Cox-model analyses were performed to evaluate risk factors for graft loss. Longitudinal changes on long-term estimated glomerular filtration rate (eGFRs) were evaluated with a linear mixed model. Histopathological findings from protocolled biopsies after 3 months post transplantation were also analyzed. Donor CAV1 rs4730751 genotyping proportions were 7.1% for AA, 41.6% for AC and 51.3% for CC. The AA genotype, compared to non-AA, was not associated with lower graft survival censored or not for death (multivariate analysis: HR = 1.23 [0.74-2.05] and HR = 1.27 [0.84-1.92]). Linear mixed model on long-term eGFRs revealed also no significant difference according to the genotype, yet we observed a trend. AA genotype was also not associated with a higher degree of fibrosis index on protocolled kidney biopsies at 3 months. To conclude, donor CAV1 rs4730751 SNP may impact on kidney transplantation outcomes, but this study could not confirm this hypothesis.

The Long Noncoding RNA DNM3OS Is a Reservoir of FibromiRs with Major Functions in Lung Fibroblast Response to TGF-ß and Pulmonary Fibrosis.

Rationale: Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. TGF-ß (transforming growth factor-ß) is the main profibrotic factor, but its inhibition is associated with severe side effects because of its pleiotropic role. Objectives: To determine if downstream noncoding effectors of TGF-ß in fibroblasts may represent new effective therapeutic targets whose modulation may be well tolerated. Methods: We investigated the whole noncoding fraction of TGF-ß-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblasts. Differential expression of the long noncoding RNA (lncRNA) DNM3OS (dynamin 3 opposite strand) and its associated microRNAs (miRNAs) was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. Measurements and Main Results: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-ß-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e., miR-199a-5p/3p and miR-214-3p), which influence SMAD and non-SMAD components of TGF-ß signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. Conclusions: Pharmacological approaches aiming at interfering with the lncRNA DNM3OS may represent new effective therapeutic strategies in IPF.

[Dihydropyrimidine déhydrogenase (DPD) deficiency screening and securing of fluoropyrimidine-based chemotherapies: Update and recommendations of the French GPCO-Unicancer and RNPGx networks]. / Dépistage du déficit en dihydropyrimidine déshydrogénase (DPD) et sécurisation des chimiothérapies à base de fluoropyrimidines : mise au point et recommandations nationales du GPCO-Unicancer et du RNPGx.

Fluoropyrimidines (FU) are still the most prescribed anticancer drugs for the treatment of solid cancers. However, fluoropyrimidines cause severe toxicities in 10 to 40% of patients and toxic deaths in 0.2 to 0.8% of patients, resulting in a real public health problem. The main origin of FU-related toxicities is a deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of 5-FU catabolism. DPD deficiency may be identified through pharmacogenetics testing including phenotyping (direct or indirect measurement of enzyme activity) or genotyping (detection of inactivating polymorphisms on the DPYD gene). Approximately 3 to 15% of patients exhibit a partial deficiency and 0.1 to 0.5% a complete DPD deficiency. Currently, there is no regulatory obligation for DPD deficiency screening in patients scheduled to receive a fluoropyrimidine-based chemotherapy. Based on the levels of evidence from the literature data and considering current French practices, the Group of Clinical Pharmacology in Oncology (GPCO)-UNICANCER and the French Network of Pharmacogenetics (RNPGx) recommend the following: (1) to screen DPD deficiency before initiating any chemotherapy containing 5-FU or capecitabine; (2) to perform DPD phenotyping by measuring plasma uracil (U) concentrations (possibly associated with dihydrouracil/U ratio), and DPYD genotyping (variants *2A, *13, p.D949V, HapB3); (3) to reduce the initial FU dose (first cycle) according to DPD status, if needed, and further, to consider increasing the dose at subsequent cycles according to treatment tolerance. In France, 17 public laboratories currently undertake routine screening of DPD deficiency.

Chronic dialysis, NAT2 polymorphisms, and the risk of isoniazid-induced encephalopathy - case report and literature review.

BACKGROUND: Isoniazid is the most widely used anti-tuberculosis agent, yet it may lead to life-threatening complications. CASE PRESENTATION: Here we report the case of a chronic hemodialysis patient who developed severe encephalopathy after the start of isoniazid. Blood levels of isoniazid were elevated, and acetyl-isoniazid over isoniazid ratio was decreased 3 h after intake of the medication, suggesting that a slow acetylator phenotype may have contributed to drug toxicity, in addition to pyridoxal phosphate removal by dialysis. This hypothesis was confirmed by sequencing of NAT2, the gene responsible for isoniazid elimination, and identification of NAT2 polymorphisms compatible with a slow acetylator phenotype. Isoniazid withdrawal along with supplementation using high doses of pyridoxine successfully reversed the drug toxicity. Isoniazid toxicity occurs in populations at risk, including patients with chronic kidney failure or NAT2 polymorphisms, who have a disturbed metabolism of pyridoxine or isoniazid, respectively, and those on renal replacement therapies, in whom pyridoxal phosphate - the active metabolite of pyridoxine - is inadvertently removed by dialysis. CONCLUSIONS: Physicians should be aware of the increased risk of isoniazid toxicity in patients on dialysis and in those with a slow acetylator phenotype conferred by NAT2 polymorphisms. Adaptation of prescription - either with higher doses of pyridoxine or decreased doses of isoniazid, respectively - has been suggested to reduce the risk of potentially life-threatening toxicity of isoniazid.

Pediatric neurofibromatosis type 2: clinical and molecular presentation, management of vestibular schwannomas, and hearing rehabilitation.

OBJECTIVE: This study aims to describe the clinical and molecular presentation of pediatric neurofibromatosis type 2 (NF2) and the subsequent management of vestibular schwannomas (VS) and hearing rehabilitation. METHODS: This is a single-center retrospective study of neurofibromatosis type 2 diagnosed before the age of 18 years old from 1997. Natural history of vestibular schwannomas and surgical outcomes were evaluated using volumetric MRI, hearing, and facial nerve assessment. Patients included in chemotherapy protocols were excluded. RESULTS: From a database of 80 patients followed up for NF2 on a regular basis, 25 patients were eligible (11 sporadic cases, 14 inherited in five families). The mean age at diagnosis was 11.6 years old. The average clinical follow-up was 6.5 years. NF2 mutation was identified in 81 % of the probands. The average growth rate based on the maximum linear diameter (DGR) was 1.68 mm/year (n = 33, average follow-up 4.22 years) and 545 mm3/year in volumetric assessment (VGR) for VS larger than 1 cm (n = 21, average follow-up 3.4 years). In unoperated ears, hearing was stable in about 50 % of ears. The mean change in dB HL was 9.5 dB/year for pure-tone average and 3.5 for speech-recognition threshold (n = 34, 5.5 years 1-12). Eight children required removal through a translabyrinthine approach (mean follow-up was 4.5 years), six patients were operated on for hearing preservation (mean postoperative follow-up 4.3 years). Six patients were eligible for hearing rehabilitation with cochlear implantation (I), and five received placement of an auditory brainstem implant. CONCLUSION: Early diagnosis and treatment of small growing VS should be carefully discussed considering familial history and possible rehabilitation with a CI.

Applicability and Efficiency of NGS in Routine Diagnosis: In-Depth Performance Analysis of a Complete Workflow for CFTR Mutation Analysis.

BACKGROUND: Actually, about 2000 sequence variations have been documented in the CFTR gene requiring extensive and multi-step genetic testing in the diagnosis of cystic fibrosis and CFTR-related disorders. We present a two phases study, with validation and performance monitoring, of a single experiment methodology based on multiplex PCR and high throughput sequencing that allows detection of all variants, including large rearrangements, affecting the coding regions plus three deep intronic loci. METHODS: A total of 340 samples, including 257 patients and 83 previously characterized control samples, were sequenced in 17 MiSeq runs and analyzed with two bioinformatic pipelines in routine diagnostic conditions. We obtained 100% coverage for all the target regions in every tested sample. RESULTS: We correctly identified all the 87 known variants in the control samples and successfully confirmed the 62 variants identified among the patients without observing false positive results. Large rearrangements were identified in 18/18 control samples. Only 17 patient samples showed false positive signals (6.6%), 12 of which showed a borderline result for a single amplicon. We also demonstrated the ability of the assay to detect allele specific dropout of amplicons when a sequence variation occurs at a primer binding site thus limiting the risk for false negative results. CONCLUSIONS: We described here the first NGS workflow for CFTR routine analysis that demonstrated equivalent diagnostic performances compared to Sanger sequencing and multiplex ligation-dependent probe amplification. This study illustrates the advantages of NGS in term of scalability, workload reduction and cost-effectiveness in combination with an improvement of the overall data quality due to the simultaneous detection of SNVs and large rearrangements.

Donor caveolin 1 (CAV1) genetic polymorphism influences graft function after renal transplantation.

BACKGROUND: Identification of the culprit genes underlying multifactorial diseases is one of the most important current challenges of molecular genetics. While recent advances in genomics research have accelerated the discovery of susceptibility genes, much remains to be learned about the functions of disease-associated genetic variants. Recently, Moore and co-workers identified, in the donor genome, an association between a common genetic variant (rs4730751) in the gene encoding caveolin-1 (CAV1), a major structural component of caveolae, and long-term allograft survival. METHODS: Four hundred seventy-five renal recipients consecutively transplanted were included in this study. Donor genomic DNA was extracted and used to genotype CAV1 rs4730751 Single Nucleotide Polymorphism. RESULTS: Patients receiving a graft carrying CAV1 rs4730751 AA genotype displayed a significant decrease in estimated glomerular filtration rate and a significant increase in serum creatinine in both univariate and multivariate analyzes. Moreover, patients receiving a graft with CAV1 AA genotype significantly developed more interstitial fibrosis lesions on systematic biopsies performed 3 months post-transplantation. CONCLUSIONS: Genotyping of CAV1 may be relevant to identify patients at risk of adverse renal transplant outcome.

UGT1A1 genotype and irinotecan therapy: general review and implementation in routine practice.

Irinotecan is a major drug in the treatment of advanced colorectal cancer. Its active form is the SN38 metabolite, which is cleared by the biliary route after glucuronidation by uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1). UGT1A1 activity exhibits a wide intersubject variability, in part related to UGT1A1 gene polymorphisms. The present review on the impact of the deficient UGT1A1*28 variant on irinotecan efficacy and toxicity was produced by a French joint workgroup comprising the Group of Clinical Onco-pharmacology (GPCO-Unicancer) and the National Pharmacogenetics Network (RNPGx). It clearly emerges that for irinotecan doses at least equal to 180 mg/m(2) , patients homozygous for the UGT1A1*28 allele are at increased risk of developing hematological and/or digestive toxicities. Irinotecan dose reduction is thus recommended in homozygous *28/*28 patients. In addition, this personalized medicine strategy aims to secure high-dose irinotecan administration (≥240 mg/m(2) ) that have proven to be safe in homozygous *1/*1 patients only. The clinical relevance of this test is discussed in terms of treatment efficacy improvement, as increasing the irinotecan dose appears to be safe in patients not bearing a deficient allele. Best execution practices, cost-effectiveness, and result interpretation are discussed with the aim of facilitating the implementation of this analysis in clinical practice. The existence of networks of laboratories performing this test in routine hospital treatment, as in France, offers the prospect of widespread screening, thus guaranteeing equal access to safe treatment and optimized therapy for patients receiving irinotecan-based therapy in advanced colorectal cancer.

A case of respiratory depression in a child with ultrarapid CYP2D6 metabolism after tramadol.

We discuss a case of severe respiratory depression in a child, with ultrarapid CYP2D6 genotype and obstructive sleep apnea syndrome, after taking tramadol for pain relief related to a day-case tonsillectomy.

Donor ABCB1 genetic polymorphisms influence epithelial-to-mesenchyme transition in tacrolimus-treated kidney recipients.

AIM: The contribution of epithelial-mesenchymal transition (EMT) has been suggested in renal transplant recipients receiving calcineurin inhibitors and developing nephrotoxicity. MATERIALS & METHODS: We assessed whether interindividual variability in tacrolimus pharmacokinetics is associated with the occurrence in tubular cells of two EMT markers (vimentin, ß-catenin) detected at 3-month in 140 allograft biopsies. We investigated whether genetic polymorphisms affecting CYP3A5 and ABCB1 influence EMT and kidney fibrosis. RESULTS: In univariate analysis, the donor CYP3A5*1 allele was significantly associated with a lower vimentin expression. In multivariate analysis, grafts carrying ABCB1 3435T allele(s) developed significantly less EMT and less interstitial fibrosis. CONCLUSION: Donor SNPs significantly influence the epithelial program in the context of kidney transplantation, and the epithelial metabolism of tacrolimus is one key to understand graft fibrogenesis.

Expression profiles of genes involved in xenobiotic metabolism and disposition in human renal tissues and renal cell models.

Numerous xenobiotics have been shown to be harmful for the kidney. Thus, to improve our knowledge of the cellular processing of these nephrotoxic compounds, we evaluated, by real-time PCR, the mRNA expression level of 377 genes encoding xenobiotic-metabolizing enzymes (XMEs), transporters, as well as nuclear receptors and transcription factors that coordinate their expression in eight normal human renal cortical tissues. Additionally, since several renal in vitro models are commonly used in pharmacological and toxicological studies, we investigated their metabolic capacities and compared them with those of renal tissues. The same set of genes was thus investigated in HEK293 and HK2 immortalized cell lines in commercial primary cultures of epithelial renal cells and in proximal tubular cell primary cultures. Altogether, our data offers a comprehensive description of kidney ability to process xenobiotics. Moreover, by hierarchical clustering, we observed large variations in gene expression profiles between renal cell lines and renal tissues. Primary cultures of proximal tubular epithelial cells exhibited the highest similarities with renal tissue in terms of transcript profiling. Moreover, compared to other renal cell models, Tacrolimus dose dependent toxic effects were lower in proximal tubular cell primary cultures that display the highest metabolism and disposition capacity. Therefore, primary cultures appear to be the most relevant in vitro model for investigating the metabolism and bioactivation of nephrotoxic compounds and for toxicological and pharmacological studies.

[Interest of UGT1A1 genotyping within digestive cancers treatment by irinotecan]. / Intérêt du génotypage de l'UGT1A1 dans le cadre du traitement des cancers digestifs par irinotécan.

Irinotecan is a cytotoxic agent administered by IV infusion in the treatment of advanced colorectal cancer. Its anticancer activity results from its bioactivation into SN-38 metabolite, which is cleared through glucuronidation by the hepatic enzyme uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1). In the general population, there is wide inter-subject variability in UGT1A1 enzyme activity related to UGT1A1 gene polymorphisms. The French joint workgroup coming from the National Pharmacogenetic Network (RNPGx) and the Group of Clinical Oncologic Pharmacology (GPCO) herein presents an updated review dealing with efficacy and toxicity clinical studies related to UGT1A1 genetic variants. From a critical analysis of this review it clearly emerges that, for doses higher than 180 mg/m(2), hematologic and digestive irinotecan-induced toxicities could be prevented in daily clinical practice by generalizing the use of a simple pharmacogenetic test before starting treatment. The clinical relevance of this test is also discussed in terms of treatment efficacy improvement, with the possibility of increasing the irinotecan dose in patients not bearing the deleterious allele. This test involves using a blood sample to analyze the promoter region of the UGT1A1 gene (UGT1A1*28 allele). Best execution practices, laboratory costs, as well as results interpretation are described with the aim of facilitating the implementation of this analysis in clinical routine. The existence of a French laboratories network performing this test in clinical routine makes it possible to generalize UGT1A1 deficiency screening, so as to guarantee equal access to safe treatment and optimized irinorecan-based therapy for the many patients receiving irinotecan-based therapy in advanced colorectal cancer.

Use of dried blood spots and inductively coupled plasma mass spectrometry for multi-element determination in blood.

The paper describes the development of an inductively coupled plasma mass spectrometry (ICP MS) method for multitrace element determination in dried blood spots (DBSs). The analytical conditions were optimized using Seronorm™ L-3 and L-1 Certified Reference Materials. The best results were obtained by sampling blood drops on a decontaminated PVDF filter membrane. After drying under metal-free conditions, the DBSs underwent acidic digestion and were analyzed with ICP MS. The method was then validated for As, Cd, Cu, Pb, Mo, Se and Zn. Using a matrix-matched calibration curve, the recovery levels ranged from 96% to 117%. The repeatability and reproducibility were generally below 15%. Limits of quantification ranging from 0.5 to 50 µg/L. In order to investigate the analytical procedure under real sampling conditions, the results obtained from DBSs and liquid blood aliquots (less subject to contamination) from two adult subjects were compared.

Influence of delta-aminolevulinic acid dehydratase gene polymorphism on selected lead exposure biomarkers in a cohort of ex-smelter workers.

Lead (Pb) body burden and toxicity may be influenced by genetic polymorphisms. The aim of this study was to investigate the influence of G177C delta-aminolevulinic acid dehydratase (ALAD) polymorphism (rs1800435) on selected Pb exposure biomarkers in a population of workers highly exposed to this metal in the past. A cross-sectional survey was conducted between 2007 and 2009 within the cohort of ex-employees of a smelter in the north of France that closed down in 2003. A questionnaire was completed by each participant and blood samples enabled determination of Pb levels and ALAD polymorphism. Five parameters estimating the Pb body burden and its variations were studied: last blood lead level (BLL) during activity, cumulative blood Pb index, BLL at the time of the study, and absolute and percent changes in BLL after cessation of metal exposure. Multiple regression models were used to evaluate links between ALAD polymorphism and the selected Pb exposure biomarkers. Two hundred and four men were included. At the time of inclusion, the median age was 53.5 yr. The median duration of Pb exposure was 25 yr and the median latency since end of exposure was 5.6 yr. The frequency of ALAD-2 allele was 9.3%, with 34 subjects being heterozygous (ALAD1-2) and 2 homozygous (ALAD2-2). According to genotype, there was no significant difference for any of the five selected Pb exposure biomarkers. These results lend support to the notion that ALAD polymorphism exerts no marked impact on Pb body burden.

Characterization of functional polymorphisms and glucocorticoid-responsive elements in the promoter of TDO2, a candidate gene for ethanol-induced behavioural disorders.

AIMS: In response to acute ethanol consumption, tryptophan 2,3-dioxygenase (TDO) induces the kynurenine pathway (KP) through a glucocorticoid-mediated mechanism, which could lead to a dramatic accumulation of neurotoxic metabolites in association with serotonin depletion. As a result, interindividual variability in ethanol-induced behavioural disorders, such as black-outs and violent impulsive behaviours (BOVIBs) following binge drinking, could be partly explained by genetic polymorphisms affecting the KP. The aim of this study was to identify polymorphisms on the promoter of the TDO2 gene that could affect expression and/or activity of TDO through glucocorticoid induction. METHODS: Polymorphisms were screened using a PCR-sequencing strategy applied to 31 alcohol-dependent patients and 49 unrelated healthy volunteers, and functionally analysed with bioinformatic prediction tools and gene reporter assays in HepG2 and A549 cell lines. RESULTS: We identified 12 polymorphisms in the human TDO2 promoter region, 2 of them corresponding to previously unknown single-nucleotide polymorphisms (SNPs) and 3 of them located in putative glucocorticoid-responsive elements (GREs). Gene reporter assays using HepG2 and A549 cell lines confirmed the presence of several functional GREs in the promoter region of TDO2 and revealed that some of the identified polymorphisms affect the promoter activity under glucocorticoid receptor over-expression and dexamethasone exposure conditions. CONCLUSIONS: Correlational studies in larger samples could help to determine whether these polymorphisms are responsible for variations of expression and/or activity of TDO, in particular under conditions where release of glucocorticoids is increased, such as acute ethanol intake. If confirmed, such results would be of major interest in explaining part of the interindividual variability observed in behavioural responses to acute ethanol consumption.