CYP2D6 genotyping strategy based on gene copy number determination by TaqMan real-time PCR

The genetic polymorphism of the cytochrome P450 monooxygenase, CYP2D6, comprises at least 43 alleles giving rise to distinct drug metabolism phenotypes termed ultrarapid, extensive, intermediate, and poor metabolizers. As a consequence, drug side effects or lack of drug effect may occur if standard doses are applied. Genetic prediction of drug oxidation phenotype as a basis for dose selection requires analysis of single nucleotide polymorphisms and of alleles with duplicated or deleted genes. Here we developed a novel method to determine the CYP2D6 gene dose per genome. A TaqMan real-time PCR assay to specifically amplify genomic CYP2D6 was established by using a specific set of amplification primers and probe, located in exon 9, which effectively prevent amplification of CYP2D7 and CYP2D8 pseudogenes. Quantitative CYP2D6 amplification data were normalized to albumin as an internal reference gene which was coamplified simultaneously in a single-tube biplex assay. The assay was validated with a selection of previously genotyped DNA samples containing none, one, two, or three CYP2D6 gene copies. The results were highly reproducible and closely matched the number of genes with no overlap between the groups. Analysis of DNA samples comprising all major alleles and genotypes revealed high sensitivity and specificity of the assay, as demonstrated by agreement of the determined gene dose with the presence of CYP2D6(*)2 x 2 (gene duplication) and CYP2D6(*) 5 (gene deletion) alleles. The predictability of the new strategy was systematically evaluated. The semiautomatic TaqMan assay allows high sample throughput and will be useful for pharmacogenetic studies and in the clinical setting.     

Isoniazid Mediates the CYP2B6*6 Genotype-Dependent Interaction between Efavirenz and Antituberculosis Drug Therapy through Mechanism-Based Inactivation of CYP2A6

Efavirenz is commonly used to treat patients coinfected with human immunodeficiency virus and tuberculosis. Previous clinical studies have observed paradoxically elevated efavirenz plasma concentrations in patients with the CYP2B6*6/*6 genotype (but not the CYP2B6*1/*1 genotype) during coadministration with the commonly used four-drug antituberculosis therapy. This study sought to elucidate the mechanism underlying this genotype-dependent drug-drug interaction. In vitro studies were conducted to determine whether one or more of the antituberculosis drugs (rifampin, isoniazid, pyrazinamide, or ethambutol) potently inhibit efavirenz 8-hydroxylation by CYP2B6 or efavirenz 7-hydroxylation by CYP2A6, the main mechanisms of efavirenz clearance. Time- and concentration-dependent kinetics of inhibition by the antituberculosis drugs were determined using genotyped human liver microsomes (HLMs) and recombinant CYP2A6, CYP2B6.1, and CYP2B6.6 enzymes. Although none of the antituberculosis drugs evaluated at up to 10 times clinical plasma concentrations were found to inhibit efavirenz 8-hydroxylation by HLMs, both rifampin (apparent inhibition constant [K_i] = 368 μM) and pyrazinamide (K_i = 637 μM) showed relatively weak inhibition of efavirenz 7-hydroxylation. Importantly, isoniazid demonstrated potent time-dependent inhibition of efavirenz 7-hydroxylation in both HLMs (inhibitor concentration required for half-maximal inactivation [K_I] = 30 μM; maximal rate constant of inactivation [k_inact] = 0.023 min⁻¹) and recombinant CYP2A6 (K_I = 15 μM; k_inact = 0.024 min⁻¹) and also formed a metabolite intermediate complex consistent with mechanism-based inhibition. Selective inhibition of the CYP2B6.6 allozyme could not be demonstrated for any of the antituberculosis drugs using either recombinant enzymes or CYP2B6*6 genotype HLMs. In conclusion, the results of this study identify isoniazid as the most likely perpetrator of this clinically important drug-drug interaction through mechanism-based inactivation of CYP2A6.     

<Emphasis Type="Italic">GSTP1</Emphasis> and <Emphasis Type="Italic">MDR1</Emphasis> Genotypes and Central Nervous System Relapse in Childhood Acute Lymphoblastic Leukemia

The probability of event-free survival of childhood acute lymphoblastic leukemia (ALL) approaches 80% or more with the use of modern multiagent chemotherapeutic regimens. One major contribution to this success has been reduction of the rate of central nervous system (CNS) relapses to less than 5%. However, heterogeneity is observed with regard to the incidence of CNS relapse in homogenously treated patient populations. One potential explanation for this heterogeneity is variation in the genetic background of these populations. Glutathione S-transferase P1 and P-glycoprotein are implicated in resistance to a variety of chemotherapeutic agents and have been localized to the blood-brain barrier. In a matched case-control study, we investigated the associations between CNS relapse in childhood ALL and the presence of phenotypically relevant single nucleotide polymorphisms within the GSTP1 (codon 105 and 114) and MDR1 genes (ABCB1; coding for Pgp; exon 26, C3435T). Significant reductions in risk of CNS relapse were observed for patients homozygous for the GSTP1 Val105 allele as well as for patients with the MDR1 3435T/T or C/T genotype. For both genotypes, the effect was restricted to patients at intermediate or high risk of treatment failure. These results suggested a modulating role for host genetic variation in the development of CNS relapse in childhood ALL treated according to Berlin-Frankfurt-Münster protocols.     

Cellular and subcellular distribution of injected lipopolysaccharide in rat liver and its inactivation by bile salts

The cellular and subcellular distribution of biologically tritiated Salmonella abortus equi lipopolysaccharide (LPS) was studied at different time intervals after intravenous injection in rats. At 1 min after injection of LPS via the portal vein label was present over Kupffer cell phagosomes. Between 30 min and 7 days after injection, silver grains were mainly associated with phagosomes and lysosomes and occasionally with the membrane of Kupffer cells. A few parenchymal cells were labeled at 5 min in their mitochondria, cell membrane and the periphery of the cell. Radioactivity was also present in the rough endoplasmic reticulum (from 15 min), fat droplets and the nucleus (from 3 h) up to 7 days. Sinusoidal endothelial and fat-storing cells were never labeled. In conclusion, both Kupffer cells and parenchymal cells play a role in the uptake of LPS by the liver. The uptake and processing of endotoxin is rapid, since label is found early after administration and radioactivity is detected in the bile within 1 h. This radioactivity represents non-detoxified LPS, since it is lethal for galactosamine-sensitised mice after extraction with hot phenol/water. However, in the presence of bile salts, the LPS is non-lethal and not capable of clotting the limulus amebocyte lysate. LPS injection causes bile flow reduction within 45 min.     

Increase in the prevalence of Panton–Valentine leukocidin and clonal shift in community-onset methicillin-resistant Staphylococcus aureus causing skin and soft-tissue infections in the Rhine-Neckar Region,Germany, 2012–2016

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major challenge for patient care. Community-associated (CA)-MRSA often have a fitness and virulence advantage compared with their nosocomial counterparts. Increased mobility, travel activities and migration accelerate the intercontinental spread of virulent CA-MRSA strains. Outpatient clinics are the most important route of entry for CA-MRSA into hospitals. However, systematic data on CA-MRSA in Germany are limited. In this study, community-onset (CO)-MRSA skin and soft-tissue infection (SSTI) isolates in the Rhine-Neckar Region from 2012–2016 were characterised to gain an insight into their molecular epidemiology and to monitor potential introduction of virulent and dominant MRSA strains into our hospital. A total of 2475 patients with S. aureus SSTI were identified in the outpatient departments of our hospital, of which 94 (3.8%) were MRSA. In addition, 40.4% of the CO-MRSA harboured the virulence factor Panton–Valentine leukocidin (PVL). ST8-t008-MRSA-IVa/c (23.7%; 9/39) and ST80-t044-MRSA-IVc (15.8%; 6/38) were the predominant PVL-positive MRSA. Molecular typing and epidemiological data revealed that 42.6% (40/94) of strains could be traced back to a local origin and 44.7% (42/94) were endemic outside of Europe. Resistance to quinolones, clindamycin and macrolides was common, whilst resistance to trimethoprim/sulfamethoxazole, tetracycline, mupirocin, chlorhexidine and fusidic acid was low. No resistance to rifampicin, fosfomycin or linezolid was observed. This study provides insight into the clonal composition of CO-MRSA in the Rhine-Neckar Region. The increase of PVL-positive MRSA and the introduction of imported strains may affect the local MRSA landscape in the near future and should be monitored closely.     

Probiotic Therapy Reduces Periodontal Tissue Destruction and Improves the Intestinal Morphology in Rats With Ligature‐Induced Periodontitis

Background: With increase in the incidence of resistance to antibiotics, probiotics are emerging as a promising adjunctive periodontal therapy. The authors of this study evaluate the influence of probiotic (PROB) supplementation on ligature‐induced periodontitis (LIP) and intestinal morphology in rats. Methods: Thirty‐two rats were randomly divided into four groups: control (C), LIP, PROB, and LIP/PROB. In groups PROB and LIP/PROB, the PROB was administered orally by addition to the drinking water of the animals for 44 days. In groups LIP and LIP/PROB, the mandibular right first molar of the animals received a cotton ligature that was left in the same position for 14 days. All animals were euthanized 44 days after the start of the PROB supplementation. The jaws were resected and histomorphometric analyses were performed. The measurements included evaluation of attachment loss (AL) and alveolar bone level (ABL) on the distal root of the mandibular first molar. Samples of the duodenum, jejunum, and ileum were also dissected from each animal to evaluate the villous height (VH) and crypt depth (CD). The data obtained were subjected to statistical analyses (analysis of variance, Tukey; P <0.05). Results: Mean values of AL and ABL were significantly higher in group LIP compared with group LIP/PROB (AL: 3.05 ± 0.57 mm and 1.78 ± 0.63 mm, respectively; ABL: 4.21 ± 0.42 mm and 3.38 ± 0.17 mm, respectively). In group LIP/PROB, the mean values of VH and CD of the jejunum were significantly higher than the ones from group LIP (VH: 672.1 ± 83.3 µm and 528.0 ± 51.7 µm, respectively; CD: 463.8 ± 100.9 µm and 269.0 ± 48.4 µm, respectively). Conclusions: It can be concluded that PROB supplementation 1) reduces AL and alveolar bone loss in rats with LIP and 2) can protect the small intestine from reactive changes induced by LIP.     

Safe treatment of thiopurine S-methyltransferase deficient Crohn's disease patients with azathioprine

Thiopurine S-methyltransferase (TPMT) deficient patients develop life threatening haematotoxicity (for example, pancytopenia) when treated with a standard dose of azathioprine (AZA) and 6-mercaptopurine (6-MP) due to excessive accumulation of cytotoxic metabolites. At present, it is generally recommended that these patients should not receive AZA or 6-MP treatment for inflammatory bowel disease. We report for the first time that Crohn's disease patients with TPMT deficiency can be successfully treated with AZA. We illustrate this with three cases where treatment has been successful and toxicity has been avoided by carefully titrating the drug dose. Thus very low TPMT activity demands pharmacogenetically guided dosing.