Differential Allelic Expression in Leukoblast from Patients with Acute Myeloid Leukemia Suggests Genetic Regulation of CDA, DCK, NT5C2, NT5C3, and TP53

Abstract

ABSTRACT:mRNA expression levels of certain genes have shown predictive value for the outcome of cytarabine-treated AML-patients. We hypothesized that genetic variants play a role in the regulation of the transcription of these genes. We studied leukoblasts from 82 patients with acute myeloid leukemia and observed various extent and frequency of differential allelic expression in the CDA, DCK, NT5C2, NT5C3, and TP53 genes. Our attempts to identify the causative regulatory single nucleotide polymorphisms by a bioinformatics approach did not succeed. However, our results indicate that genetic variations are at least in part responsible for the differences in overall expression levels of these genes.The deoxynucleoside analog cytarabine (1-␤-D-arabinofuranosylcytosine) is a major component of the chemotherapeutic treatment of patients with acute myeloid leukemia (AML). The most important limitation for the use of deoxynucleoside analogs in the clinic is the presence of primary or acquired resistance. Several studies have identified clinically relevant mechanisms of resistance in patients with leukemia or other malignant diseases (Jordheim and Dumontet, 2007). In particular, the mRNA expression level of several genes has been correlated to the outcome of the treatment with cytarabine or gemcitabine (2Ј-2Ј-difluorodeoxycytidine), another analog of deoxycytidine. In all of these studies, large variations in the expression levels of genes involved in cytarabine metabolism have been observed between patients, suggesting the presence of important regulatory mechanisms. In addition to differences in levels and activities of transcription factors and stability of mRNA, variations in the genomic sequence of the gene and its regulatory elements can influence the mRNA level. In fact, at least 25 to 35% of interindividual differences in gene expression are supposed to be caused by cis-acting variations (Pastinen and Hudson, 2004).When a heterozygous genetic variation induces a difference in mRNA expression level, the two corresponding alleles are expressed at different levels. This is called differential allelic expression (DAE) or allelic expression imbalance (Pastinen and Hudson, 2004). Currently, DAE is studied in samples heterozygous for an exonic variation (exonic single nucleotide polymorphisms; cSNP) used as a marker to determine the relative amount of transcripts from the two alleles. This method allows the distinction between cis-and transacting effects because the cellular environment and mRNA extraction are exactly the same (Stamatoyannopoulos, 2004). The cSNP used for the assessment of DAE is not necessarily responsible for the allelic expression imbalance, and additional investigations are needed to identify the functional regulatory variant (regulatory SNP; rSNP) or the underlying epigenetic modification (Milani et al., 2007).Specific mRNA expression levels can be used to predict the outcome of cancer patients treated with chemotherapy. Because genetic variants are partially responsible for variations in gene expr...

Allelic imbalance (AI) identifies novel tissue-specificcis-regulatory variation for humanUGT2B15

“…Multiple approaches have been used to detect AI, including SNaPShot [Bray, et al, 2003a; Bray, et al, 2003b; Fukuda, et al, 2006; He, et al, 2005; Johnson, et al, 2008; Liu, et al, 2005; Valle, et al, 2008; Wilkins, et al, 2007; Zhang, et al, 2005], sequencing [Fukuda, et al, 2006; Tuupanen, et al, 2008], pyro-sequencing [Loeuillet, et al, 2007; Mahr, et al, 2006; Wang and Elbein, 2007], iPLEX [Ding, et al, 2004], RNA difference plot [Murakami, et al, 2004], band intensity in gel [Heighway, et al, 2005; Hirota, et al, 2004] and other technologies [Jordheim, et al, 2008; Ware, et al, 2006]. However, since all these methods are end-point readings of PCR, they may not be accurate in quantifying the ratio between the alleles.…”

Detecting differential allelic expression using high-resolution melting curve analysis: application to the breast cancer susceptibility gene CHEK2

“…This methodology has major advantages over more conventional methods for investigating DAE based on the comparison of gene expression between individuals as discussed elsewhere [8,10,20]. Since they come from the same tissue sample and have therefore been subjected to the same environmental influences (such as genetic trans -acting factors and experimental exposures, including mRNA degradation) both alleles should be equally expressed in the absence of cis -acting sequence variation or epigenetic effects affecting the expression of the target mRNA.…”

“…Statistical significance for the allelic imbalance was calculated using Student's t-test. Criteria for DAE were the following: i) the point estimate of the difference between genomic DNA and cDNA ratios should be greater than 20%; ii) the Student's t-test p-value should be ≤ 0.05, and iii) the 95% confidence interval of the point estimate should not include 0 [14,19,20,24]. …”

“…Data acquisition on HRM instruments consists of monitoring changes in the fluorescence intensity of the probe, as it dissociates from the two allelic templates, while the probe-target duplexes are continuously heated. We have already reported the use of this methodology to compare the relative abundance of allelic transcripts in a study investigating mRNA degradation due to NMD in BRCA2 [19], and in a group of selected genes involved in the cellular response to the cytotoxic agent cytarabine [20]. In these studies, DAE analysis was limited by the single-capillary throughput of the HRM device used, the HR-1™ instrument, and allelic imbalance was quantified manually.…”

NT5C3 polymorphisms and outcome of first induction chemotherapy in acute myeloid leukemia

“…Large variations have been observed in response to AraC, and one possible explanation is that genetic variation in genes involved in the activation and inactivation of these drugs might contribute toward differences in drug response. An intronic single nucleotide polymorphism (SNP), rs4316067, was also associated with the mRNA expression level of NT5C3 in leukoblast from AML patients [6]. Gene expression level of NT5C3 was significantly associated with cytotoxicity for AraC in human lymphoblastoid cell lines and knockdown of NT5C3 altered cancer cell sensitivity to this drug.…”