Can we identify patients carrying targeted deleterious DPYD variants with plasma uracil and dihydrouracil? A GPCO-RNPGx retrospective analysis.

OBJECTIVES: Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe fluoropyrimidine-related toxicities. The best strategy for identifying DPD-deficient patients is still not defined. The EMA recommends targeted DPYD genotyping or uracilemia (U) testing. We analyzed the concordance between both approaches. METHODS: This study included 19,376 consecutive French patients with pre-treatment plasma U, UH2 and targeted DPYD genotyping (*2A, *13, D949V, *7) analyzed at Eurofins Biomnis (2015-2022). RESULTS: Mean U was 9.9 ± 10.1 ng/mL (median 8.7, range 1.6-856). According to French recommendations, 7.3 % of patients were partially deficient (U 16-150 ng/mL) and 0.02 % completely deficient (U≥150 ng/mL). DPYD variant frequencies were *2A: 0.83 %, *13: 0.17 %, D949V: 1.16 %, *7: 0.05 % (2 homozygous patients with U at 22 and 856 ng/mL). Variant carriers exhibited higher U (median 13.8 vs. 8.6 ng/mL), and lower UH2/U (median 7.2 vs. 11.8) and UH2/U2 (median 0.54 vs. 1.37) relative to wild-type patients (p<0.00001). Sixty-six% of variant carriers exhibited uracilemia <16 ng/mL, challenging correct identification of DPD deficiency based on U. The sensitivity (% patients with a deficient phenotype among variant carriers) of U threshold at 16 ng/mL was 34 %. The best discriminant marker for identifying variant carriers was UH2/U2. UH2/U2<0.942 (29.7 % of patients) showed enhanced sensitivity (81 %) in identifying deleterious genotypes across different variants compared to 16 ng/mL U. CONCLUSIONS: These results reaffirm the poor concordance between DPD phenotyping and genotyping, suggesting that both approaches may be complementary and that targeted DPYD genotyping is not sufficiently reliable to identify all patients with complete deficiency.

Corrected speciation and gyromitrin content of false morels linked to ALS patients with mostly slow-acetylator phenotypes.

A case-control study of sporadic amyotrophic lateral sclerosis (ALS) in a mountainous village in the French Alps discovered an association of cases with a history of eating wild fungi (false morels) collected locally and initially identified and erroneously reported as Gyromitra gigas. Specialist re-examination of dried specimens of the ALS-associated fungi demonstrated they were members of the G. esculenta group, namely G. venenata and G. esculenta, species that have been reported to contain substantially higher concentrations of gyromitrin than present in G. gigas. Gyromitrin is metabolized to monomethylhydrazine, which is responsible not only for the acute oral toxic and neurotoxic properties of false morels but also has genotoxic potential with proposed mechanistic relevance to the etiology of neurodegenerative disease. Most ALS patients had a slow- or intermediate-acetylator phenotype predicted by N-acetyltransferase-2 (NAT2) genotyping, which would increase the risk for neurotoxic and genotoxic effects of gyromitrin metabolites.

Dihydropyrimidine dehydrogenase gene variants for predicting grade 4-5 fluoropyrimidine-induced toxicity: FUSAFE individual patient data meta-analysis.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) deficiency is the main known cause of life-threatening fluoropyrimidine (FP)-induced toxicities. We conducted a meta-analysis on individual patient data to assess the contribution of deleterious DPYD variants *2A/D949V/*13/HapB3 (recommended by EMA) and clinical factors, for predicting G4-5 toxicity. METHODS: Study eligibility criteria included recruitment of Caucasian patients without DPD-based FP-dose adjustment. Main endpoint was 12-week haematological or digestive G4-5 toxicity. The value of DPYD variants *2A/p.D949V/*13 merged, HapB3, and MIR27A rs895819 was evaluated using multivariable logistic models (AUC). RESULTS: Among 25 eligible studies, complete clinical variables and primary endpoint were available in 15 studies (8733 patients). Twelve-week G4-5 toxicity prevalence was 7.3% (641 events). The clinical model included age, sex, body mass index, schedule of FP-administration, concomitant anticancer drugs. Adding *2A/p.D949V/*13 variants (at least one allele, prevalence 2.2%, OR 9.5 [95%CI 6.7-13.5]) significantly improved the model (p < 0.0001). The addition of HapB3 (prevalence 4.0%, 98.6% heterozygous), in spite of significant association with toxicity (OR 1.8 [95%CI 1.2-2.7]), did not improve the model. MIR27A rs895819 was not associated with toxicity, irrespective of DPYD variants. CONCLUSIONS: FUSAFE meta-analysis highlights the major relevance of DPYD *2A/p.D949V/*13 combined with clinical variables to identify patients at risk of very severe FP-related toxicity.

Plasma lipidomic analysis to investigate putative biomarkers of P-glycoprotein activity in healthy volunteers.

P-glycoprotein (P-gp) is an efflux transporter involved in the bioavailability of many drugs currently on the market. P-gp is responsible for several drug-drug interactions encountered in clinical practice leading to iatrogenic hospital admissions, especially in polypharmacy situations. ABCB1 genotyping only reflects an indirect estimate of P-gp activity. Therefore, it would be useful to identify endogenous biomarkers to determine the P-gp phenotype to predict in vivo activity prior to the initiation of treatment and to assess the effects of drugs on P-gp activity. The objective of this study was to assess changes in plasma lipidome composition among healthy volunteers selected on the basis of their ABCB1 genotype and who received clarithromycin, a known inhibitor of P-gp. Untargeted lipidomic analysis based on liquid chromatography-tandem mass spectrometry was performed before and after clarithromycin administration. Our results revealed changes in plasma levels of some ceramides (Cers) {Cer(d18:1/22:0), Cer(d18:1/22:1), and Cer(d18:1/20:0) by ~38% (p < 0.0001), 13% (p < 0.0001), and 13% (p < 0.0001), respectively} and phosphatidylcholines (PCs) {PC(17:0/14:1), PC(16:0/18:3), and PC(14:0/18:3) by ~24% (p < 0.001), 10% (p < 0.001), and 23.6% (p < 0.001)} associated with both ABCB1 genotype and clarithromycin intake. Through the examination of plasma lipids, our results highlight the relevance of untargeted lipidomics for studying in vivo P-gp activity and, more generally, to safely phenotyping transporters.

Azathioprine-induced vanishing bile duct syndrome: The value of early thiopurine metabolism assessment.

About 15% to 28% of patients treated with thiopurines experienced adverse drug reactions, such as haematological and hepatic toxicities. Some of these related to the polymorphic activity of the thiopurine S-methyltransferase (TPMT), the key detoxifying enzyme of thiopurine metabolism. We report here a case of thiopurine-induced ductopenia with a comprehensive pharmacological analysis on thiopurine metabolism. A 34-year-old woman, with a medical history of severe systemic lupus erythematosus with recent introduction of azathioprine therapy, presented with mild fluctuating transaminase blood levels consistent with a hepatocellular pattern, which evolved to a cholestatic pattern over the next weeks. A blood thiopurine metabolite assay revealed low 6-thioguanine nucleotides (6-TGN) level and a dramatically increased 6-methylmercaptopurine ribonucleotides (6-MMPN) level, together with an unfavourable [6-MMPN:6-TGN] metabolite ratio and a high TPMT activity. After a total of about 6 months of thiopurine therapy, a transjugular liver biopsy revealed a ductopenia, and azathioprine discontinuation led to further clinical improvement. In line with previous reports from the literature, our case supports the fact that ductopenia is a rare adverse drug reaction of azathioprine. The mechanism of reaction is unknown but may involve high 6-MMPN blood level, due to unusual thiopurine metabolism (switched metabolism). Early therapeutic drug monitoring with measurement of 6-TGN and 6-MMPN blood levels may help physicians to identify patients at risk of similar duct injury.

Screening for dihydropyrimidine dehydrogenase deficiency by measuring uracilemia in chronic kidney disease patients is associated with a high rate of false positives.

BACKGROUND: Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency based on the measurement of plasma uracil ([U]) is recommended prior to the administration of fluoropyrimidine-based chemotherapy. Cancer patients frequently have impaired kidney function, but the extent to which kidney function decline impacts [U] levels has not been comprehensively investigated. METHODS: We assessed the relationship between DPD phenotypes and estimated glomerular filtration rate (eGFR) in 1751 patients who benefited on the same day from a screening for DPD deficiency by measuring [U] and [UH2]:[U], and an evaluation of eGFR. The impact of a kidney function decline on [U] levels and [UH2]:[U] ratio was evaluated. RESULTS: We observed that [U] was negatively correlated with eGFR, indicating that [U] levels increase as eGFR declines. For each ml/min of eGFR decrease, [U] value increased in average by 0.035 ng/ml. Using the KDIGO classification of chronic kidney disease (CKD), we observed that [U] values >16 ng/ml (DPD deficiency) were measured in 3.6 % and 4.4 % of stage 1 and 2 CKD (normal-high eGFR, >60 ml/min/1.73 m2) patients, but in 6.7 % of stage 3A CKD patients (45 to 59 ml/min/1.73 m2), 25% of stage 3B CKD patients (30 to 44 ml/min/1.73 m2), 22.7% of stage 4 CKD patients (15 to 29 ml/min/1.73 m2 and 26.7% of stage 5 CKD patients (<15 ml/min/1.73 m2). [UH2]:[U] ratios were not impacted by kidney function. CONCLUSION: DPD phenotyping based on the measurement of plasma [U] in patients with decreased eGFR is associated with an exceedingly high rate of false positives when kidney function decline reaches 45 ml/minute/1.73 m2 of eGFR or lower. In this population, an alternative strategy that remain to be evaluated would be to measure the [UH2]:[U] ratio in addition to [U].

Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency.

Fluoropyrimidine drugs (FP) are the backbone of many chemotherapy protocols for treating solid tumours. The rate-limiting step of fluoropyrimidine catabolism is dihydropyrimidine dehydrogenase (DPD), and deficiency in DPD activity can result in severe and even fatal toxicity. In this review, we survey the evidence-based pharmacogenetics and therapeutic recommendations regarding DPYD (the gene encoding DPD) genotyping and DPD phenotyping to prevent toxicity and optimize dosing adaptation before FP administration. The French experience of mandatory DPD-deficiency screening prior to initiating FP is discussed.

Imbalance between alpha-1-antitrypsin and interleukin 6 is associated with in-hospital mortality and thrombosis during COVID-19.

Thrombosis is a hallmark of severe COVID-19. Alpha-1-antitrypsin (AAT), an inflammation-inducible serpin with anti-inflammatory, tissue protective and anticoagulant properties may be involved in severe COVID-19 pathophysiology including thrombosis onset. In this study, we examined AAT ability to predict occurrence of thrombosis and in-hospital mortality during COVID-19. To do so, we performed a monocentric cross-sectional study of 137 hospitalized patients with COVID-19 of whom 56 (41%) were critically ill and 33 (22.4%) suffered from thrombosis during hospitalization. We measured AAT and IL-6 plasma levels in all patients and phenotyped AAT in a subset of patients with or without thrombosis paired for age, sex and COVID-19 severity. We observed that AAT levels at admission were higher in both non-survivors and thrombosis patients than in survivors and non-thrombosis patients. AAT: IL-6 ratio was lower in non-survivors and thrombosis patients. In a logistic regression multivariable analysis model adjusted on age, BMI and D-dimer levels, a higher AAT: IL-6 was a protective factor of both in-hospital mortality (Odds ratio, OR: 0.07 95%CI [0.02-0.25], p < 0.001) and thrombosis (OR 0.36 95%CI [0.14-0.82], p = 0.02). AAT phenotyping did not show a higher proportion of AAT abnormal variants in thrombosis patients.Our findings suggest an insufficient production of AAT regarding inflammation intensity during severe COVID-19. AAT appeared as a powerful predictive marker of severity, mortality and thrombosis mirroring the imbalance between harmful inflammation and protective counter-balancing mechanism in COVID-19. Restoring the balance between AAT and inflammation could offer therapeutic opportunities in severe COVID-19.

Validation of a liquid chromatography coupled to tandem mass spectrometry method for simultaneous quantification of tryptophan and 10 key metabolites of the kynurenine pathway in plasma and urine: Application to a cohort of acute kidney injury patients.

A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of tryptophan (Trp) and ten metabolites of kynurenine pathway, including kynurenine (Kyn), 3-hydroxy-kynurenine (3-HK), kynurenic acid (KA), xanthurenic acid (XA), 3-Hydroxy-anthranilic acid (3-HANA), quinolinic acid (QA), nicotinic acid mononucleotide (NaMN), picolinic acid (Pic), nicotinamide (NAM) and nicotinic acid (NA) in both plasma and urine. This LC-MS/MS method was used to predict the occurrence of acute kidney injury (AKI) in a cohort of patients with cardiac surgery under cardiopulmonary bypass (CPB). Urinary concentrations of Pic, as well as Pic to Trp and Pic to 3-HANA ratios were highly predictive of an AKI episode the week after CPB, indicating that Pic could be a predictive biomarker of AKI. Thus, monitoring the kynurenine pathway activity with this LC-MS/MS method is a clinically relevant tool to identify new biomarkers of kidney injury.

Machine learning-driven identification of drugs inhibiting cytochrome P450 2C9.

Cytochrome P450 2C9 (CYP2C9) is a major drug-metabolizing enzyme that represents 20% of the hepatic CYPs and is responsible for the metabolism of 15% of drugs. A general concern in drug discovery is to avoid the inhibition of CYP leading to toxic drug accumulation and adverse drug-drug interactions. However, the prediction of CYP inhibition remains challenging due to its complexity. We developed an original machine learning approach for the prediction of drug-like molecules inhibiting CYP2C9. We created new predictive models by integrating CYP2C9 protein structure and dynamics knowledge, an original selection of physicochemical properties of CYP2C9 inhibitors, and machine learning modeling. We tested the machine learning models on publicly available data and demonstrated that our models successfully predicted CYP2C9 inhibitors with an accuracy, sensitivity and specificity of approximately 80%. We experimentally validated the developed approach and provided the first identification of the drugs vatalanib, piriqualone, ticagrelor and cloperidone as strong inhibitors of CYP2C9 with IC values <18 µM and sertindole, asapiprant, duvelisib and dasatinib as moderate inhibitors with IC50 values between 40 and 85 µM. Vatalanib was identified as the strongest inhibitor with an IC50 value of 0.067 µM. Metabolism assays allowed the characterization of specific metabolites of abemaciclib, cloperidone, vatalanib and tarafenacin produced by CYP2C9. The obtained results demonstrate that such a strategy could improve the prediction of drug-drug interactions in clinical practice and could be utilized to prioritize drug candidates in drug discovery pipelines.

Pretherapeutic screening for Dihydropyrimidine deshydrogenase deficiency in measuring uracilemia in dialysis patients leads to a high rate of falsely positive results.

BACKGROUND: Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD deficiency in End Stage Renal Disease (ESRD) patients is unknown. METHODS: We assessed the characteristics of both DPD phenotypes and DPYD genotypes in 20 dialyzed patients before and after dialysis session. The extent to which the concentrations of uracil [U] and dihydrouracil [UH2] were affected by dialysis was evaluated. RESULTS: Mean [U] was 14 ± 3.3 ng/ml before the dialysis session, and 7.9 ± 2.7 ng/ml after. Notably, mean [U] in 119 non-ESRD patients during the same timeline was 8.7 ± 3.9 ng/ml, which is similar to [U] values after dialysis session (p = 0.38). [U] values > 16 ng/ml were measured in 4 ESRD patients (20%), whereas the rate was 3.3% in the non-ESRD cohort. Whole gene sequencing did not reveal DPYD deleterious allelic variants in the 4 ESRD patients with [U] values > 16 ng/ml. The profile of [UH2] values during dialysis was similar to that of [U]: 385 ± 86 ng/ml before, and 185 ± 62 ng/ml after (mean reduction rate 42.5%). Thus, [UH2]:[U] ratio remained unaffected by dialysis, and was similar to the values in non-ESRD patients (22.4 ± 7.1). CONCLUSION: Phenotyping based on measuring plasma [U] before a dialysis sessions in ESRD patients is associated with an unacceptable high rate of false positives. The optimal strategy for the identification of patients with DPD deficiency in this population would be the monitor the [UH2]:[U] ratio, which remains unaffected.

Identification of rare defective allelic variants in cases of thiopurine S-methyltransferase deficient activity.

Aim: To assess rare TPMT variants in patients carrying a deficient phenotype not predicted by the four more frequent genotypes (*2, *3A, *3B and *3C). Materials & methods: Next-generation sequencing of TPMT in 39 patients with a discordant genotype. Results: None of the variants identified explained the discordances assuming that they are of uncertain significance according to the Clinical Pharmacogenetics Implementation Consortium classification. Two unknown variants were detected and predicted to result in a splicing defect. We show that TPMT*16 and TMPT*21 are defective alleles, and TPMT*8 and TPMT*24 are associated with a normal activity. Conclusion: Whole-exon sequencing for rare TPMT mutations has a low diagnostic yield. A reassessment of the functional impact of rare variants of uncertain significance is a critical issue.

A comprehensive population-based study comparing the phenotype and genotype in a pretherapeutic screen of dihydropyrimidine dehydrogenase deficiency.

BACKGROUND: Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive. METHODS: Among a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated. RESULTS: When [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a "wild-type" genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant. CONCLUSIONS: Frequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Cost-effectiveness analysis of pretreatment screening for NUDT15 defective alleles.

BACKGROUND: Nucleotide triphosphate diphosphatase (NUDT15) genetic testing in addition to thiopurine methyl transferase (TPMT) is recommended to reduce the incidence of adverse severe myelotoxicity episodes induced by thiopurines. OBJECTIVE: We assessed the cost-effectiveness ratio of combined screening for TMPT and NUDT15 defective alleles by genotyping or next-generation sequencing (NGS) using TPMT genotyping as the reference. Because of the genetic differences in thiopurine toxicity, we tested the screening strategies on individuals of Caucasian and Asian descent. METHODS: A decision tree compared conventional TPMT genotyping with combined TPMT/NUDT15 genotyping or NGS using a Monte-Carlo microsimulation model of patients with inflammatory bowel disease. The main outcome was the incremental cost-effectiveness ratios (ICER) with effectiveness being one averted severe myelotoxicity requiring hospitalization. RESULTS: The mean estimated cost of the TPMT genotyping for one year is twice in Asian compared with Caucasian patients (980 euro/patient versus 488 euro/patient), and the effectiveness of TPMT genotyping in Caucasian avoided 43 severe myelosuppressions per 10 000 patients over a year compared with 3.6 per 10 000 patients in Asian. Combined TPMT/NUDT15 genotyping compared with TPMT genotyping had an ICER of 7 491 281 euro per severe myelotoxicity averted in Caucasian, compared to 619 euro in Asian. The ICER of the NGS-based screening strategy is disproportionally high compared with genotyping, irrespective of ethnic descent. CONCLUSION: With a low cost-effectiveness threshold, combined screening for NUDT15 and TPMT defective alleles is cost-effective compared to TMPT screening alone in patients of Asian descent, but is unrealistic from a cost-effectiveness point of view in Caucasians.

[Pharmacogenetics for patient care in France: A discipline that evolves!] / La pharmacogénétique au service du soin en France : une discipline qui évolue !

Pharmacogenetics, which concepts are known for a long time, is entering a new period at least as far as its practical applications for patients are concerned. In recent years there have been more and more initiatives to promote widespread dissemination, and health authorities are increasingly incorporating these concepts into drug labels. In France, the national network of pharmacogenetics (RNPGx) works to promote these activities, both with health actors (biologists, clinicians) and health authorities. This article reviews the current situation in France and the milestones of the year 2018. It highlights recent advances in this field, in terms of currently recommended analyses, sharing of information or technological developments, and the prospects for future developments in the near future from targeted pharmacogenetics to eventually preemptive approaches.