Association Of Polymorphisms And Transcripts Of The GR Gene With Glucocorticoid Resistance In Childhood Acute Lymphoblastic Leukemia

Background:Glucocorticoids(GCs) have been used for the treatment of pediatric acute lymphoblastic leukemia(ALL) in combination with chemotherapy due to their potency in inducing apoptosis of leukemic cells and/or cell arrest. GC resistant is the most common problem in the treatment of pediatric acute lymphoblastic leukemia, which is also one of the main reasons for treatment failure. Little is known about the causes of GC resistance in ALL, although multiple factors may be involved. GCs exert their effects by binding to the GC receptor(GR), which, as a transcription factor, subsequently triggers transactivation as well as transrepression of specific target genes. Thus, GR has become the focus in the mechanism of GC resistance. Recently, polymorphisms of the coding region in the GR gene have been found to be associated with altered GC sensitivity in several diseases. In addition, several studies showed that the differential expression of 5’ and 3’ transcripts in the GR gene were associated with GC sensitivity in multiple lymphoblastic leukemia cell lines in vitro. However, few studies have investigated the association of polymorphisms in the non-coding region of the GR gene with GC response in pediatric patients with ALL. Moreover, the association of differential expression of the transcripts in the GR gene with GC resistance in ALL patient’s blasts would be worthwhile explored. So in this study, we sought to determine whether the noncoding-region ploymorphisms and differential expression of the 5’ and 3’ transcripts in the GR gene were related to GC resistance in pediatric acute lymphoblastic leukemia.Part one: Association of single nucleotide polymorphisms of the glucocorticoid receptor gene with glucocorticoid response in vivo in childhood acute lymphoblastic leukemiaObjective:To investigate the polymorphisms in both the coding and non-coding region of the GR gene, and analyze their association with prednisone responsiveness in vivo in childhood ALL patients.Methods:1. 63 pediatric ALL patients and 33 healthy control children were recruited in the present study. According to the absolute number of the blasts/m L in peripheral blood on day 8 of treatment, the patients were divided into two groups(prednisone good response, PGR and prednisone poor response, PPR). In the present study, we adopt case-control method to investigate the association of polymorphisms and haplotypes in the GR gene with GC resistance of the pediatric ALL patients in Han descent in China.2. All samples were collected under protocols approved by the Institutional Review Board at the authors’ affiliated institution after informed consent was obtained from the parents or legal guardians of the study participants. One milliliter bone marrow fluid was collected from the newly diagnosed ALL children. And 200 ul peripheral blood was collected from the healthy control children and the ALL patients at complete remission. Genomic DNA of patients with ALL and normal control children was extracted. Five polymorphisms(rs7701443, rs10052957, rs41423247, rs6189/6190 and rs6198) were detected by the polymerase chain reaction(PCR) and gene sequencing technique.3. Hardy–Weinberg equilibrium for genotypic distributions was examined using the χ2 goodness-of-fit test. Analysis for the incidence and distribution of the genotype, allele and derived haplotype was performed with the χ2 test using SPSS statistical software version 16.0. Differences were considered to be significant at p<0.05. A logistic regression analysis, the χ2 test for trend and a test with correction for tied ranks were used to determine the relationship between different alleles and GC resistance in vivo. On the basis of the observed frequencies of three SNPs, we used the SHEsis analysis platform to infer haplotype frequencies.Results:1. The allele incidence of the investigated five SNPsMAF(minor allele frequency) of the three polymorphisms(rs7701443,rs10052957,rs41423247) in the non-coding region of the GR gene were respectively 0.404, 0.103, 0.246 in the ALL population, and 0.454, 0.09, 0.287 in the healthy control children. While rs6189/6190 and rs6198 polymorphisms were not observed in all study participants.2. The association of the polymorphisms(rs41423247, rs7701443 and rs10052957) with prednisone response in vivo in pediatric ALL patients.There was a significant difference in frequency distribution of the alleles for the rs41423247 polymorphism(odds ratio [OR]=9.58; 95% confidence interval [CI]: 1.23–74.21; p=0.01) between prednisone good and poor responders. Although there was no significant difference in the frequency distributions of the genotypes for polymorphism rs41423247 between prednisone good and poor responders(p>0.05), when we combined GG and CG(frequency of the GG genotype was 9.5%), there was a significant difference between prednisone good and poor responders(OR= 9.778, 95% CI: 1.174–81.433, p= 0.032). There was a significant difference in frequency distributions of the alleles for rs7701443 polymorphism(OR= 3.12; 95% CI: 1.08–9; p= 0.02) between prednisone good and poor responders. For the rs7701443 genotypes, even if we combined TT and CT, there was no significant difference between prednisone good and poor responders(p> 0.05). There was no significant difference between prednisone good and poor responders for the polymorphism rs10052957, both at the allelic level and genotypic level(p>0.05).3. The association of haplotype(derived from rs7701443, rs10052957, rs41423247) with prednisone response in vivo in pediatric ALL patients.There were four haplotypes(CCC, TCC, TCG and TTG) derived from the three polymorphisms(rs7701443, rs10052957 and rs41423247), whose frequencies were more than 3%. The CCC haplotype was significantly associated with prednisone poor response in ALL patients(p = 0.013), while the TCG haplotype was associated with prednisone good response(p =0.028).Conclusion:Our results suggested that the polymorphisms in the non-coding region of the GR gene and the derived haplotypes were probably associated with prednisone response in vivo in pediatric ALL in northeast Han Chinese.Part II Association of transcripts of the GR gene with glucocorticoid resistance in vitro in childhood ALL patient’s blastsObjective:To investigate the transcripts in both the 5’UTR and 3’ region of the GR gene, and analyze their association with prednisolone responsiveness in vitro in childhood ALL patient’s blasts.Methods:1. All samples were collected under protocols approved by the Institutional Review Board at the authors’ affiliated institution after informed consent was obtained from the parents or legal guardians of the study participants. One milliliter bone marrow fluid was collected from the newly diagnosed ALL children.2. Patient’s blasts were cultured with prednisolone in 96-cell plate. In vitro drug cytotoxicity was assessed using the CCK-8 assay. After 72 hours, CCK-8 was added, the value of IC50 was calculated. According to the value of IC50, the patient’s blasts were divided into GC sensitive group and resistant group. Eighteen ALL children were recruited in the present study.3. Patient’s blasts were cultured with or without prednisolone in 24-cell plate in vitro. The leukemic blasts were incubated and collected respectively at H0,H3,H8,H24 timepoint. Total RNA was extracted using the Trizol method and c DNA was synthesized. The expression of the m RNA levels of 5’ and 3’ transcripts in GR gene were detected by relative Quantitative real-time RT-PCR.4. The data were analyzed by SPSS 16.0 software. All P values were calculated based on two-sided tests, with a statistical significance defined as P value < 0.05.Results:1. The association of the change of GC-induced GR-1A, 1B, 1C m RNA expression with GC sensitivity in vitro in patient’s blasts.We found the m RNA levels of GR- 1A, 1B, 1C were up-regulated upon GC exposure for 3, 8, and 24 hours compared with the control samples, incubated without prednisolone(P<0.001). The degree of up-regulation of GC-induced GR-1A, 1B, 1C m RNA expression was not related to GC sensitivity(P>0.05). There was no significant difference for the differential expression of GC-induced GR-1A, 1B, 1C m RNA both in GC sensitive and resistant group(P>0.05).2. The association of the change of GC-induced GRα, GRβ m RNA expression with GC sensitivity in vitro in patient’s blasts.We found the m RNA levels of GRα, GRβ were up-regulated upon GC exposure for 3, 8, and 24 hours compared with the control samples, incubated without prednisolone(P<0.001). The degree of up-regulation of GC-induced GRαm RNA expression was related to GC sensitivity(P=0.026). The degree of up-regulation of GC-induced GRβm RNA expression was not related to GC sensitivity(P>0.05). The expression of GC-induced GRα was greater than GRβ both in GC sensitive and GC resistant group(P<0.05).Conclusion:Up-regulations of GR-1A, 1B, 1C, GRα and GRβ m RNA expression were observed when ALL blasts were exposured to prednisolone. The expression of GRα m RNA after prednisolone exposure was related to GC sensitivity.