Association between a microRNA binding site polymorphism in SLCO1A2 and the risk of delayed methotrexate elimination in Chinese children with acute lymphoblastic leukemia.

Organic anion-transporting polypeptide 1A2 (OATP1A2) is involved in the cellular uptake of methotrexate (MTX). Genetic variation in solute carrier organic anion transporter family member 1A2 (SLCO1A2, the coding gene of OATP1A2) has important implications for the elimination of MTX. We investigated the association between a microRNA (miRNA) binding site polymorphism (rs4149009 G > A) in the 3'-untranslated region (3'-UTR) of SLCO1A2 with the serum MTX concentrations in Chinese children with acute lymphoblastic leukemia (ALL). Genotyping for SLCO1A2 rs4149009 G > A in 141 children with ALL was performed using the Sequenom MassARRAY system. Serum MTX concentrations were determined by fluorescence polarization immunoassay. The percentages of MTX level ≥1 µmol/L at 42 h were compared among the AA, GA, and GG genotypes. The minor allele frequency observed in this study (33.0%) was significantly lower than that in the African samples reported in the 1000 Genomes Project (57.4%, P = 0.00). The incidence rate of delayed MTX elimination was significantly higher in patients with the GG genotype (23.1%) compared with the AA genotype (0.0%, P = 0.03). Bioinformatics tools predicted that the rs4149009 A allele would disrupt the putative binding sites of hsa-miR-324-3p and hsa-miR-1913. These results indicate that the rs4149009 G > A polymorphism might affect MTX pharmacokinetics by interfering with the function of miRNAs.

Association between MTHFR microRNA binding site polymorphisms and methotrexate concentrations in Chinese pediatric patients with acute lymphoblastic leukemia.

BACKGROUND: The pharmacokinetics and therapeutic response to methotrexate (MTX) display large variability in the treatment of acute lymphoblastic leukemia (ALL). The aim of the present study was to investigate the association of two microRNA (miRNA) binding site polymorphisms (rs3737966 G > A and rs35134728 DEL/TTC) in the 3'-untranslated region of MTHFR with serum MTX concentrations, in a Chinese pediatric population with ALL. METHODS: Genotyping for MTHFR rs3737966 and rs35134728 in 144 children with ALL was performed using the Sequenom MassArray system (Sequenom, San Diego, CA, USA). Serum MTX concentrations were measured by a fluorescence polarization immunoassay 24 h (C24h ) and 42 h (C42h ) after administration. The effects of the polymorphisms on concentration-to-dose (C/D) ratios of MTX were assessed. RESULTS: Complete linkage disequilibrium between rs3737966 and rs35134728 polymorphisms (r2  = 1) was found in the study population. The minor allele frequency observed in the present study (17.4%) was significantly lower than those in European and African samples reported in the 1000 Genomes Project (42.9% and 63.9%, respectively; p < 0.01). The C/D ratios of MTX at 24 and 42 h for the TTC/TTC-A/A haplotype carriers (11.74 and 0.07 µmol/l per g/m2 , respectively) were significantly lower than those in DEL/DEL-G/G or DEL/TTC-G/G haplotype carriers (12.49 and 0.09 µmol/l per g/m2 , respectively; p < 0.05). Computational predictions suggested that the two polymorphisms overlapped with putative binding sites of several miRNAs. CONCLUSIONS: The rs3737966 and rs35134728 polymorphisms in MTHFR were associated with serum MTX concentrations. The findings of the present study indicate that miRNAs might be involved in the post-transcriptional regulation of MTHFR.

Preparation of Rat Whole-kidney Acellular Matrix via Peristaltic Pump.

PURPOSE: To design a whole-kidney a cellular matrix scaffold using peristaltic pump perfusion and to ascertain the retention of extra cellular proteins by the scaffold. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats weighing 200-250 g were used. Intravenous catheters were inserted into the renal artery followed by perfusion of decellularization solution using a peristaltic pump. After decellularization, the acellular matrix was observed under a microscope after hematoxylin and eosin (H&E) staining and a fluorescence microscope after 4',6-diamidino-2-phenylindole (DAPI) staining. Immunohistochemistry was used to identify the composition of kidney acellular matrix. RESULTS: The result of H&E and DAPI staining demonstrate the removal of cellular material in kidney a cellular matrix. Immunohistochemistry confirmed the conservation of the natural expression of extra cellular matrix proteins including collagen types I and IV, fibrin and laminin. CONCLUSION: Peristaltic pump perfusion enables successful preparation of renal a cellular matrix, to retainthe criticalproteins of natural extra cellular matrix. The resulting kidney a cellular matrix represents an ideal natural scaffold for renal tissue engineering.

Effects of a microRNA binding site polymorphism in SLC19A1 on methotrexate concentrations in Chinese children with acute lymphoblastic leukemia.

MicroRNAs (miRNAs) are a class of short non-coding RNA that can specially bind to the 3'-untranslated region of target mRNAs and regulate gene expression at the posttranscriptional level. This study investigated the effects of a miRNA binding site polymorphism (rs1051296) in solute carrier family 19, member 1 (SLC19A1) on serum methotrexate (MTX) concentrations in Chinese children with acute lymphoblastic leukemia (ALL). Genotyping for SLC19A1 rs1051296 G>T in 131 children with ALL was performed using the Sequenom MassArray system. A total of 131 patients received high-dose MTX treatment, and serum MTX concentrations were measured by a fluorescence polarization immunoassay 24 (MTX C24h) and 42 h (MTX C42h) after administration. The frequency of the rs1051296 T allele observed in this study (46.2 %) was significantly lower than that previously observed in a European population (60.7 %, P = 0.002). There was significant association between rs1051296 G>T and MTX C24h (29.97, 32.34, and 39.01 µmol/L for GG, GT, and TT genotypes, respectively, P = 0.04). The percentage of patients with an MTX concentration above the therapeutic threshold (40 µmol/L) was significantly lower in GG carriers compared with that in GT and TT carriers (8.6 % for GG genotype vs. 26.8 and 40.0 % for CT and TT genotypes, respectively, P = 0.02). Delayed elimination of MTX (C42h > 1 µmol/L) was less frequent in GG carriers than in GT and TT carriers. Rs1051296 G>T was associated with MTX plasma concentration, suggesting that miRNAs might be involved in the post-transcriptional regulation of SLC19A1.

Influence of genetic polymorphisms of FPGS, GGH, and MTHFR on serum methotrexate levels in Chinese children with acute lymphoblastic leukemia.

PURPOSE: To investigate the correlation between common genetic polymorphisms of folylpolyglutamate synthase (FPGS), gamma-glutamyl hydrolase (GGH), and methylenetetrahydrofolate reductase (MTHFR) and serum levels of methotrexate (MTX) in Chinese children with acute lymphoblastic leukemia (ALL). METHODS: Ninety-one children with ALL who received high-dose MTX were recruited. The polymorphisms FPGS (rs1544105 G>A), GGH (rs3758149 C>T), and MTHFR (rs1801133 C>T) were genotyped through polymerase chain reaction-restriction fragment length polymorphism analysis. Serum MTX was measured by fluorescence polarization immunoassay. The association between targeted polymorphisms and MTX concentration-to-dose (C/D) ratios was assessed, and between targeted polymorphisms and the percent of MTX above the therapeutic threshold (40 µmol/L). RESULTS: The minor allele frequencies of rs1544105 G (34.1%), rs3758149 T (19.2%), and rs1801133 C (48.4%) observed in our population were significantly lower than those reported for European populations (64.2, 30.8, and 69.0%, respectively). The association between the GGH rs3758149 polymorphism and MTX C/D was gender-specific; in girls, the MTX C/D at 24 h of GGH rs3758149 CC carriers (12.09 µmol/L per g/m(2)) was significantly lower than that of CT or TT carriers (16.80 µmol/L per g/m(2)). The percent of serum MTX above the therapeutic threshold in GGH rs3758149 CC carriers (18.3%) was significantly lower than that of CT and TT carriers (38.7%). The MTX C/D ratios at 24 h and the percent of MTX >40 µmol/L for the A-T-T (three variant alleles) haplotype were significantly higher than those for other haplotypes combined (P < 0.05). CONCLUSIONS: These data indicate that FPGS rs1544105, GGH rs3758149, and MTHFR rs1801133 polymorphisms contribute to the variability of MTX pharmacokinetics, and their genotyping may be useful to reduce toxicities associated with MTX therapy.