Mara L. Becker

Development of consensus treatment plans for juvenile localized scleroderma: A roadmap toward comparative effectiveness studies in juvenile localized scleroderma

Juvenile localized scleroderma (LS) is a chronic inflammatory skin disorder associated with substantial morbidity and disability. Although a wide range of therapeutic strategies has been reported in the literature, a lack of agreement on treatment specifics and accepted methods for clinical assessment has made it difficult to compare approaches and identify optimal therapy. Our objective was to develop standardized treatment plans, clinical assessments, and response criteria for active, moderate to high severity juvenile LS.

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 After informed consent was obtained from the 104 patients with JIA, blood was withdrawn during routine MTX-screening laboratory testing.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.

Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases.

Genome-wide association studies (GWASs) have identified hundreds of susceptibility genes, including shared associations across clinically distinct autoimmune diseases. We performed an inverse χ2 meta-analysis across ten pediatric-age-of-onset autoimmune diseases (pAIDs) in a case-control study including more than 6,035 cases and 10,718 shared population-based controls. We identified 27 genome-wide significant loci associated with one or more pAIDs, mapping to in silico–replicated autoimmune-associated genes (including IL2RA) and new candidate loci with established immunoregulatory functions such as ADGRL2, TENM3, ANKRD30A, ADCY7 and CD40LG. The pAID-associated single-nucleotide polymorphisms (SNPs) were functionally enriched for deoxyribonuclease (DNase)-hypersensitivity sites, expression quantitative trait loci (eQTLs), microRNA (miRNA)-binding sites and coding variants. We also identified biologically correlated, pAID-associated candidate gene sets on the basis of immune cell expression profiling and found evidence of genetic sharing. Network and protein-interaction analyses demonstrated converging roles for the signaling pathways of type 1, 2 and 17 helper T cells (TH1, TH2 and TH17), JAK-STAT, interferon and interleukin in multiple autoimmune diseases.

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.

Clinical Characteristics of Children With Juvenile Dermatomyositis: The Childhood Arthritis and Rheumatology Research Alliance Registry

To investigate aspects of juvenile dermatomyositis (DM), including disease characteristics and treatment, through a national multicenter registry.

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).

Effectiveness and Toxicity of Methotrexate in Juvenile Idiopathic Arthritis: Comparison of 2 Initial Dosing Regimens

Objective. To compare the incidence of liver toxicity and clinical response between 2 initial dosing regimens of methotrexate (MTX) for treatment of juvenile idiopathic arthritis (JIA). Methods. Clinical and laboratory data were abstracted from the medical records of 220 children newly prescribed MTX from the same geographic region. One cohort received initial doses of MTX > 0.5 mg/kg/week (“high-dose”) and one cohort received initial doses of MTX ≤ 0.5 mg/kg/week (“low-dose”). Toxicity was defined as aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) above the normal range, and positive clinical response was defined as a reduction in active joint count during the first 6 months of MTX therapy. Results. One hundred twenty-six children were in the high-dose MTX group, 94 in the low-dose MTX group. At 6 months, the high-dose group was more likely to have an elevated AST or ALT (adjusted OR 3.89, 95% CI 1.82–8.29, p < 0.0001). Subjects receiving both MTX and nonsteroidal antiinflammatory drugs (NSAID) had no significant difference between groups in change of active joint count, while subjects in the high-dose group but not taking NSAID had more active joints (p = 0.036) at 6 months compared to the low-dose group. Conclusion. Initial high-dose MTX was associated with an increased risk of at least one liver enzyme abnormality with no significant improvement in active joint count. This suggests that there is no apparent benefit, while the potential for liver toxicity is increased, when using higher doses of MTX at treatment inception in patients with JIA.

A novel high‐performance liquid chromatography/mass spectrometry method for improved selective and sensitive measurement of methotrexate polyglutamation status in human red blood cells

The folate antagonist methotrexate is commonly used in low dose for treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Therapeutic effects are attributed to intracellular levels of various methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion-pairing reversed-phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of methotrexate and its polyglutamates(2-7), in human red blood cells. Sample preparation consists of perchloric acid protein precipitation followed by solid-phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient solvent program obtained from blending acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 microL injection volumes, limits of detection were 2.5 nM for individual methotrexate polyglutamates, while large volume (100 microL) injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of methotrexate and its polyglutamates in red blood cells obtained from patients being treated for juvenile idiopathic arthritis with methotrexate. Significantly, the methodology proved suitable for determination of long-chain methotrexate polyglutamates(5-7) and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches.

Genetic sharing and heritability of paediatric age of onset autoimmune diseases

Autoimmune diseases (AIDs) are polygenic diseases affecting 7–10% of the population in the Western Hemisphere with few effective therapies. Here, we quantify the heritability of paediatric AIDs (pAIDs), including JIA, SLE, CEL, T1D, UC, CD, PS, SPA and CVID, attributable to common genomic variations (SNP-h2). SNP-h2 estimates are most significant for T1D (0.863±s.e. 0.07) and JIA (0.727±s.e. 0.037), more modest for UC (0.386±s.e. 0.04) and CD (0.454±0.025), largely consistent with population estimates and are generally greater than that previously reported by adult GWAS. On pairwise analysis, we observed that the diseases UC-CD (0.69±s.e. 0.07) and JIA-CVID (0.343±s.e. 0.13) are the most strongly correlated. Variations across the MHC strongly contribute to SNP-h2 in T1D and JIA, but does not significantly contribute to the pairwise rG. Together, our results partition contributions of shared versus disease-specific genomic variations to pAID heritability, identifying pAIDs with unexpected risk sharing, while recapitulating known associations between autoimmune diseases previously reported in adult cohorts.

Identifying genomic and developmental causes of adverse drug reactions in children.

Adverse drug reactions are a concern for all clinicians who utilize medications to treat adults and children; however, the frequency of adult and pediatric adverse drug reactions is likely to be under-reported. In this age of genomics and personalized medicine, identifying genetic variation that results in differences in drug biotransformation and response has contributed to significant advances in the utilization of several commonly used medications in adults. In order to better understand the variability of drug response in children however, we must not only consider differences in genotype, but also variation in gene expression during growth and development, namely ontogeny. In this article, recommendations for systematically approaching pharmacogenomic studies in children are discussed, and several examples of studies that investigate the genomic and developmental contribution to adverse drug reactions in children are reviewed.