DNA methylation of the TPMT gene and azathioprine pharmacokinetics in children with very early onset inflammatory bowel disease.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a crucial enzyme for azathioprine biotransformation and its activity is higher in very early onset inflammatory bowel disease (VEO-IBD) patients than in adolescents with IBD (aIBD). AIMS: The aims of this pharmacoepigenetic study were to evaluate differences in peripheral blood DNA methylation of the TPMT gene and in azathioprine pharmacokinetics in patients with VEO-IBD compared to aIBD. METHODS: The association of age with whole genome DNA methylation profile was evaluated in a pilot group of patients and confirmed by a meta-analysis on 3 cohorts of patients available on the public functional genomics data repository. Effects of candidate CpG sites in the TPMT gene were validated in a larger cohort using pyrosequencing. TPMT activity and azathioprine metabolites (TGN) were measured in patients' erythrocytes by HPLC and associated with patients' age group and TPMT DNA methylation. RESULTS: Whole genome DNA methylation pilot analysis, combined with the meta-analysis revealed cg22736354, located on TPMT downstream neighboring region, as the only statistically significant CpG whose methylation increases with age, resulting lower in VEO-IBD patients compared to aIBD (median 9.6% vs 12%, p = 0.029). Pyrosequencing confirmed lower cg22736354 methylation in VEO-IBD patients (median 4.0% vs 6.0%, p = 4.6 ×10-5). No differences in TPMT promoter methylation were found. Reduced cg22736354 methylation was associated with lower TGN concentrations (rho = 0.31, p = 0.01) in patients with VEO-IBD and aIBD. CONCLUSION: Methylation of cg22736354 in TPMT gene neighborhood is lower in patients with VEO-IBD and is associated with reduced azathioprine inactivation and increased TGN concentrations.

Responses of patients with juvenile idiopathic arthritis to methotrexate: a genomic outlook.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is a chronic disease characterized by persistent joint inflammation. JIA is the most common pediatric chronic rheumatic disease and no curative therapy is currently available. Methotrexate (MTX) is an important treatment for JIA even though a high inter-individual variability in response is observed in patients. Among the factors of this variability, genetics and epigenetics might play an important role. AREAS COVERED: This review summarizes the results of pharmacogenetic and pharmacoepigenetic studies regarding MTX response in JIA. Studies considering epigenetic factors in JIA patients are still very limited, therefore this review includes also studies performed in adult patients with rheumatoid arthritis. Moreover, the relevance of biomarkers measured in blood or urine of JIA patients in relation to MTX treatment is discussed. EXPERT OPINION: Nowadays, even though many pharmacogenomics studies have been published, a specific genetic marker predictor of MTX efficacy or adverse events has not yet been identified. Encouraging results are available and great expectations rely on the study of epigenetics. Future studies are needed in order to identify genetic and epigenetic biomarkers that can be implemented in the clinical practice.

Gender May Influence the Immunosuppressive Actions of Prednisone in Young Patients With Inflammatory Bowel Disease.

Although the use of glucocorticoids (GC) is well established, the therapeutic response to these agents often shows important interindividual differences, in particular among young patients with inflammatory bowel diseases (IBD). Currently, GC resistance or dependence cannot be predicted by clinical or laboratory findings. The aim of this study was to investigate the association of gender and age with GC efficacy and with the expression of Glucocorticoid-Induced Leucine Zipper (GILZ). One hundred thirty patients (mean age at enrolment 12.6 years, 53 Crohn's disease, 70 males) were enrolled in this retrospective study. IBD patients with active disease despite prednisone at a daily dose of up to 2 mg/kg over a period of 4 weeks were defined as steroid resistant. Patients who initially responded but relapsed upon dose reduction were considered steroid-dependent. Total RNA was extracted from biopsies of 14 patients (9 males) and the levels of GILZ mRNA were evaluated by real-time PCR. Association between clinical response to prednisone and the considered demographic variables was evaluated using logistic regression models. After 4 weeks of treatment, 112 patients were responders to prednisone and 18 were resistant; at this time-point, resistant patients were older than responders (p=0.032). After 12 weeks, 42, 71 and 12 patients were sensitive, dependent and resistant respectively; at this time-point, females were more prone than males to develop prednisone dependence vs a good response (p=0.028) while age had no effect. Age was associated with response both at 4 and 12 weeks in the subgroups of females: resistant patients were older than sensitive ones at 4 weeks (p=0.02). Likewise, at 12 weeks of therapy, dependent patients resulted older than sensitive ones (p=0.05). No association of age with prednisone response was found in males. In a subgroup of 14 patients (5 females), GILZ mRNA expression in intestinal biopsies was higher in males (p=0.0031). Patients with unfavorable response (7) presented lower GILZ expression at disease onset in comparison to the responder group (p=0.017). Older females with IBD have a higher incidence of prednisone unfavorable response and reduced intestinal expression of the GC pharmacodynamic marker GILZ.

Hypomethylation of NLRP3 gene promoter discriminates glucocorticoid-resistant from glucocorticoid-sensitive idiopathic nephrotic syndrome patients.

To assess whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid (GC)-resistant from GC-sensitive idiopathic nephrotic syndrome (INS), patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS), we measured the methylation level of NLRP3 promoter in DNA from peripheral blood cells of 10 adult patients with GC-resistant FSGS already in hemodialysis and 18 patients with GC-sensitive INS (13 MCD/5 FSGS) and in 21 pediatric patients with INS with MCD/FSGS before starting any treatment. Association of NLRP3 inflammasome with GC resistance was recapitulated in vitro in monocytic cell lines (THP-1 and U937). In both adults and pediatric patients, NLRP3 promoter methylation was significantly reduced in GC-resistant compared with GC-sensitive patients. Indeed, NLRP3 methylation distinguished GC-resistant and GC-sensitive patients (area under the receiver operating characteristic curve [AUROC] 86.7% in adults, p = 0.00019, and 73.5% in children, p = 0.00097). NLRP3 knock-down augmented sensitivity to GCs in THP-1 cells, whereas NLRP3 inflammasome activation lowered GC receptor concentration, increasing GC resistance in U937 cells. Our results uncovered a new biological mechanism by which patients with INS may acquire GC resistance, that could be used in future as a novel noninvasive diagnostic tool. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ☑ Approximately 80% of patients with idiopathic nephrotic syndrome (INS) respond to glucocorticoids, with the remaining 20% being steroid-resistant. WHAT QUESTION DID THIS STUDY ADDRESS? ☑ Whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid-resistant from glucocorticoid (GC)-sensitive INS. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ☑ In both adults and children, NLRP3 promoter methylation was significantly reduced in leukocytes of patients with GC-resistant compared with GC-sensitive INS. NLRP3 inflammasome activation lowered GC receptor concentration and augmented GC resistance, whereas NLRP3 knockdown increased sensitivity to GCs in cell lines representative of monocytes (U937 and THP1). HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? ☑ Our findings uncovered a new biological mechanism whereby patients with INS may develop resistance to GCs that could be used in the future as a novel noninvasive diagnostic tool.

Pharmacogenetics of treatments for inflammatory bowel disease.

INTRODUCTION: Inflammatory bowel disease is a chronic inflammation of the gut whose pathogenesis is still unclear. Although no curative therapy is currently available, a number of drugs are used in induction and maintenance therapy; however, for most of these drugs, a high inter-individual variability in response is observed. Among the factors of this variability, genetics plays an important role. Areas covered: This review summarizes the results of pharmacogenetic studies, considering the most important drugs used and in particular aminosalycilates, glucocorticoids, thiopurines, monoclonal antibodies and thalidomide. Most studies used a candidate gene approach, even if significant breakthroughs have been obtained recently from applying genome-wide studies. When available, also investigations considering epigenetics and pharmacogenetic dosing guidelines have been included. Expert opinion: Only for thiopurines, genetic markers identified as predictors of efficacy or adverse events have allowed the development of dosing guidelines. For the other drugs, encouraging results are available and great expectations rely on the study of epigenetics and integration with pharmacokinetic information, especially useful for biologics. However, to improve therapy of IBD patients with these drugs, for implementation in the clinics of pharmacogenetics, informatic clinical decision support systems and training about pharmacogenetics of health providers are needed.