Roger Gaedigk

Human carboxylesterases HCE1 and HCE2: Ontogenic expression, inter-individual variability and differential hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin

Carboxylesterases hydrolyze chemicals containing such functional groups as a carboxylic acid ester, amide and thioester. The liver contains the highest carboxylesterase activity and expresses two major carboxylesterases: HCE1 and HCE2. In this study, we analyzed 104 individual liver samples for the expression patterns of both carboxylesterases. These samples were divided into three age groups: adults (>or= 18 years of age), children (0 days-10 years) and fetuses (82-224 gestation days). In general, the adult group expressed significantly higher HCE1 and HCE2 than the child group, which expressed significantly higher than the fetal group. The age-related expression was confirmed by RT-qPCR and Western immunoblotting. To determine whether the expression patterns reflected the hydrolytic activity, liver microsomes were pooled from each group and tested for the hydrolysis of drugs such as oseltamivir and insecticides such as deltamethrin. Consistent with the expression patterns, adult microsomes were approximately 4 times as active as child microsomes and 10 times as active as fetal microsomes in hydrolyzing these chemicals. Within the same age group, particularly in the fetal and child groups, a large inter-individual variability was detected in mRNA (430-fold), protein (100-fold) and hydrolytic activity (127-fold). Carboxylesterases are recognized to play critical roles in drug metabolism and insecticide detoxication. The findings on the large variability among different age groups or even within the same age group have important pharmacological and toxicological implications, particularly in relation to pharmacokinetic alterations of ester drugs in children and vulnerability of fetuses and children to pyrethroid insecticides.

A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in Asthma

RATIONALE Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes. OBJECTIVES We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma. METHODS Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV(1), FVC, and FEV(1)/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study. MEASUREMENTS AND MAIN RESULTS Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7-16 wk) and the canalicular (gestational age: 17-26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV(1) (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV(1)/FVC (combined P = 0.003). CONCLUSIONS Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.

The effect of genotype on methotrexate polyglutamate variability in juvenile idiopathic arthritis and association with drug response

OBJECTIVE The response to and toxicity of methotrexate (MTX) are unpredictable in patients with juvenile idiopathic arthritis (JIA). Intracellular polyglutamation of MTX, assessed by measuring concentrations of MTX polyglutamates (MTXGlu), has been demonstrated to be a promising predictor of drug response. Therefore, this study was aimed at investigating the genetic predictors of MTXGlu variability and associations between MTXGlu and drug response in JIA. METHODS The study was designed as a single-center cross-sectional analysis of patients with JIA who were receiving stable doses of MTX at a tertiary care childrens hospital. After informed consent was obtained from the 104 patients with JIA, blood was withdrawn during routine MTX-screening laboratory testing. Clinical data were collected by chart review. Genotyping for 34 single-nucleotide polymorphisms (SNPs) in 18 genes within the MTX metabolic pathway was performed. An ion-pair chromatographic procedure with mass spectrometric detection was used to measure MTXGlu1-7. RESULTS Analysis and genotyping of MTXGlu was completed in the 104 patients. K-means clustering resulted in 3 distinct patterns of MTX polyglutamation. Cluster 1 had low red blood cell (RBC) MTXGlu concentrations, cluster 2 had moderately high RBC MTXGlu1+2 concentrations, and cluster 3 had high concentrations of MTXGlu, specifically MTXGlu3-5. SNPs in the purine and pyrimidine synthesis pathways, as well as the adenosine pathway, were significantly associated with cluster subtype. The cluster with high concentrations of MTXGlu3-5 was associated with elevated liver enzyme levels on liver function tests (LFTs), and there were higher concentrations of MTXGlu3-5 in children who reported gastrointestinal side effects and had abnormal findings on LFTs. No association was noted between MTXGlu and active arthritis. CONCLUSION MTXGlu remains a potentially useful tool for determining outcomes in patients with JIA being treated with MTX. The genetic predictors of MTXGlu variability may also contribute to a better understanding of the intracellular biotransformation of MTX in these patients.

Ontogeny of human hepatic and intestinal transporter gene expression during childhood: age matters.

Many drugs prescribed to children are drug transporter substrates. Drug transporters are membrane-bound proteins that mediate the cellular uptake or efflux of drugs and are important to drug absorption and elimination. Very limited data are available on the effect of age on transporter expression. Our study assessed age-related gene expression of hepatic and intestinal drug transporters. Multidrug resistance protein 2 (MRP2), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3 expression was determined in postmortem liver samples (fetal n = 6, neonatal n = 19, infant n = 7, child n = 2, adult n = 11) and multidrug resistance 1 (MDR1) expression in 61 pediatric liver samples. Intestinal expression of MDR1, MRP2, and OATP2B1 was determined in surgical small bowel samples (neonates n = 15, infants n = 3, adults n = 14). Using real-time reverse-transcription polymerase chain reaction, we measured fetal and pediatric gene expression relative to 18S rRNA (liver) and villin (intestines), and we compared it with adults using the 2−∆∆Ct method. Hepatic expression of MRP2, OATP1B1, and OATP1B3 in all pediatric age groups was significantly lower than in adults. Hepatic MDR1 mRNA expression in fetuses, neonates, and infants was significantly lower than in adults. Neonatal intestinal expressions of MDR1 and MRP2 were comparable to those in adults. Intestinal OATP2B1 expression in neonates was significantly higher than in adults. We provide new data that show organ- and transporter-dependent differences in hepatic and intestinal drug transporter expression in an age-dependent fashion. This suggests that substrate drug absorption mediated by these transporters may be subject to age-related variation in a transporter dependent pattern.

NUCLEAR RECEPTOR EXPRESSION IN FETAL AND PEDIATRIC LIVER: CORRELATION WITH CYP3A EXPRESSION

The mechanisms underlying interindividual variation and developmental changes in cytochrome P450 3A (CYP3A) expression and activity are not fully understood. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect, during human fetal and pediatric development, mRNA expression of nuclear receptors involved in the regulation of CYP3A genes. Quantitative RT-PCR was conducted on RNA extracted from prenatal (n = 60, 76 days to 32 weeks estimated gestational age) and pediatric (n = 20, 4 days to 18 years of age) liver tissue with primers for nuclear receptors implicated in regulating CYP3A gene expression. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) were expressed at low (and highly variable) levels in pre- and neonatal liver relative to liver tissue derived from older children. CAR was expressed at higher levels relative to PXR in prenatal liver (757 ± 480 molecules CAR/ng of RNA versus 271 ± 190 molecules PXR/ng of RNA after correction for 18S rRNA). In contrast, mRNA expression of the heterodimer partner RXRα was less variable (33-fold) and did not differ appreciably between pre- and postnatal liver samples (219 ± 101 molecules/ng of RNA prenatal versus 253 ± 232 molecules/ng of RNA postnatal). Expression of HNF4α1 mRNA was similar to that of RXRα. Log CYP3A7 mRNA expression was significantly correlated with PXR (r2 = 0.372) and CAR (r2 = 0.380) mRNA in fetal liver, but associations were weaker than those observed with CYP3A4 mRNA in postnatal liver (r2 = 0.610 and 0.723 for PXR and CAR, respectively). In conclusion, nuclear receptor mRNA expression demonstrates considerable interindividual variability in human fetal and pediatric liver and is significantly correlated with CYP3A expression.

PharmGKB summary: Methotrexate pathway

Methotrexate is a folate analog that is used in the treatment of cancers (e.g. acute lymphoblastic leukemia, non-Hodgkin lymphoma, osteosarcoma, and colon cancer) and autoimmune diseases (e.g. rheumatoid arthritis, Crohn’s disease, and psoriasis). In the treatment of autoimmune diseases, methotrexate is usually administrated orally or subcutaneously, whereas in the cancer treatment, it can be given orally, intramuscularly, as intrathecal injections, or as intravenous infusions (up to 12 g/m2) [1-3]. The pharmacokinetics and pharmacodynamics of methotrexate show large interpatient variability regardless of the route of administration or disease being treated [4-6]. The goal of this study is to provide an introduction to methotrexate pharmacogenomics, showing the candidate genes in the PharmGKB methotrexate pathway (Fig. 1), important variants (Tables ​(Tables11 and ​and2),2), discussing key knowledge, and pointing to more in-depth resources.

Expression analysis of asthma candidate genes during human and murine lung development

BackgroundLittle is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.ObjectiveTo investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.MethodsAsthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.ResultsIn total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.ConclusionsOur data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.

Analysis of intracellular methotrexate polyglutamates in patients with juvenile idiopathic arthritis: Effect of route of administration on variability in intracellular methotrexate polyglutamate concentrations

OBJECTIVE Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients. METHODS Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for >or=3 months. Clinical data were collected by chart review. Concentrations of MTXGlu(1-7) in red blood cell lysates were quantitated using an innovative ion-pairing chromatography procedure, with detection by mass spectrometry. RESULTS Patients with JIA from a single center (n = 99; mean +/- SD age 117.8 +/- 56.5 months, 69 female) were included in the analysis. The mean +/- SD dose of MTX was 0.51 +/- 0.25 mg/kg per week, with a median treatment duration of 18 months (interquartile range 3-156 months). MTX was administered subcutaneously in 66 patients (67%). Fifty-six patients (57%) had active arthritis at the time of the clinic visit. Total intracellular MTXGlu (MTXGlu(TOT)) concentrations varied 40-fold, with a mean +/- SD total concentration of 85.8 +/- 48.4 nmoles/liter. Concentrations of each MTXGlu subtype (MTXGlu(1-7)) were measured individually and as a percentage of MTXGlu(TOT) in each patient. MTXGlu(3) was the most prominent subtype identified, comprising 42% of MTXGlu(TOT), and the interindividual variability in the concentration of MTXGlu(3) was the most highly correlated with that of MTXGlu(TOT) (r = 0.96). The route of MTX administration was significantly associated with MTXGlu(1-5) subtypes; higher concentrations of MTXGlu(1 + 2) were observed in patients receiving oral doses of MTX, whereas higher concentrations of MTXGlu(3-5) were observed in patients receiving subcutaneous doses of MTX (P < 0.0001). CONCLUSION In this cohort of patients with JIA, the MTXGlu(TOT) concentration varied 40-fold. Individual MTXGlu metabolites (MTXGlu(1-7)), which have, until now, not been previously reported in patients with JIA, were detected. The route of MTX administration contributed to the variability in concentrations of MTXGlu(1-5).

Variability of CYP2J2 Expression in Human Fetal Tissues

CYP2J2 metabolizes arachidonic acid to 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids (EETs), which play a critical role in the regulation of renal, pulmonary, cardiac, and vascular function. However, the contribution of CYP2J2 to EET formation in the liver remains poorly characterized. Likewise, information is sparse regarding the extent and variability of CYP2J2 expression during human development. This investigation was undertaken to characterize the variability of CYP2J2 expression in fetal liver, heart, kidney, lung, intestine, and brain and in postnatal liver samples. CYP2J2 mRNA expression was measured using quantitative polymerase chain reaction, and immunoreactive CYP2J2 was examined using two anti-CYP2J2 antibodies. CYP2J2 mRNA was ubiquitously expressed in pre- and postnatal samples. Fetal hepatic mRNA expression varied 127-fold (1351 ± 717 transcripts/ng total RNA), but this variation was reduced to 8-fold after exclusion of four samples with extremely low levels of mRNA. Amounts of immunoreactive protein also varied substantially among samples without an apparent relationship with transcript number or genotype. Western blot analysis revealed a different protein pattern between prenatal and postnatal liver samples. DNA resequencing of selected subjects identified a single novel single-nucleotide polymorphism (CYP2J2*10), which was found in only one subject and therefore did not explain the large variability in CYP2J2 protein content. In vitro expression suggests that the protein product of CYP2J2*10 confers reduced enzymatic activity. Aberrant splicing produces three minor transcripts, which were present in all samples tested. Due to premature termination codons, none encodes functional protein. The mechanisms leading to variable amounts of immunoreactive protein and distinct pre- and postnatal CYP2J2 protein patterns warrant further investigation.

Genetic Heterogeneity in the rRNA Gene Locus of Trichophyton tonsurans

ABSTRACT Trichophyton tonsurans is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of T. tonsurans by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated T. tonsurans from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in T. tonsurans.