DOI: 10.32471/exp-oncology.2312-8852.vol-43-no-3.16474

Chronic lymphocytic leukemia (CLL) is characterized by a heterogeneous course varying from indolent to highly aggressive [1]. A large number of CLL prognosis markers have been proposed, among them the mutational status of genes coding the variable region of the heavy chain of immunoglobulin (IGHV) remains the most reliable and universal to date [2, 3]. CLL patients with mutated (M) IGHV genes have a better prognosis than patients with unmutated (UM) IGHV genes.

The gene of lipoprotein lipase (LPL) was identified as a gene differentially overexpressed in patients with UM IGHV genes [4, 5] and is now considered to be a reliable RNA-based marker for CLL prognosis [6]. It is believed that the expression of LPL in patients with UM IGHV cases is a consequence of the demethylation of the CpG island involving the whole exon 1 and the first nucleotides of intron 1, whereas in cases with M IGHV genes, the promoter remains methylated [7]. As it was shown in the most studies, the expression level of LPL in CLL patients with UM IGHV genes cases varies significantly [8–13]. The reasons for this are not fully understandable.

One of the possible functions of the LPL in CLL cells is enzymatic, associated with lipolysis and subsequent fatty acid-mediated survival advantage [14–16]. McCaw et al. [17] found that different lipids, including free fatty acids, produced by the LPL activity, amplify cytokine-signaling in CLL cells that were not seen in normal lymphocytes. Numerous data indicate that the TP53 gene is involved in the regulation of cellular metabolism, in particular, lipid metabolism. Some differences in metabolic processes depending on certain polymorphisms of the TP53 gene have been established [18–21]. Therefore, the aim of this study was to determinate the association of the LPL expression as one of the key enzymes of lipid metabolism with the genetic variants of the TP53 gene.

MATERIALS AND METHODS

The studied group consisted of 45 male patients with previously untreated CLL referred to the State Institution “National Research Centre for Radiation Medicine of the National Academy of Medical Sciences of Ukraine”. The study was approved by the local Ethics Review Committee, all patients provided informed consent prior to participation. The diagnosis of CLL was based on clinical history, lymphocyte morphology and immunophenotypic criteria according to iwCLL guidelines [22]. Mutational status of IGHV genes was assessed as described previously [23]. Only patients with UM IGHV genes (≥ 98% of homology to the germ line) were included in the study.

Genomic DNA and total RNA were extracted from peripheral blood mononuclear cells with the QIAamp Blood Mini Kit (Qiagen, United Kingdom) and TRI Reagent (Molecular Research Centre Inc., USA), respectively. The concentration and purity of RNA and DNA were assessed with NanoDrop Spectrophotometer (NanoDrop Technology, USA).

Expression of LPL was evaluated using real-time quantitative reverse transcription polymerase chain reaction (PCR) and CT (2^−ΔΔCt) method [24]. LPL expression in lymphocytes obtained from healthy donors was used as a calibrator for relative quantification.

TP53 genotyping was performed by PCR amplification followed by direct sequencing with the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) as described earlyer [25]. Single nucleotide polymorphism (SNP) variants of the TP53 gene with the minor allele frequency ≥ 5% in the total European population were analyzed, namely: rs1042522, rs1642785, rs17883323, rs2909430, rs145153611, rs113530090, rs12947788, rs12951053, and rs17878362.

Hardy — Weinberg equilibrium (HWE) was evaluated by the chi–square (χ²) test. SNPstats tool (bioinfo.iconcologia.net/snpstats/start.htm) was used for comparison of genotype and haplotype frequencies in groups of CLL patients with low and high LPL expression. Linkage disequilibrium (LD) between SNPs was estimated by calculating the D’ and r² measures using CubeX online tool (www.oege.org/software/cubex) and/or SNPstats tool. All tests were two-sided and considered statistically significant when p ≤ 0.05. Statistical calculations were performed with SPSS for Windows (version 19.0; SPSS Inc., USA).

RESULTS

Relative LPL expression levels in observed CLL patients with UM IGHV genes ranged from 0.3 to 1024. As it was established earlier, the cut-off value of 17.0 showed the best degree of discrimination between the CLL cases with M and UM IGHV genes [9]. In 11 cases, LPL expression level did not exceed or was slightly above the cut-off value (mean 11.17 ± 2.66, median 13.7, range 0.1–24.3). These patients were included in the group with low LPL expression (I group). The remaining 34 patients with LPL expression levels ranging from 42.2 to 1024 (mean 275.48 ± 39.37, median 84.5) composed the group with high LPL expression (II group). Differences between groups were significant (p = 0.016).

The genotype frequencies of the most studied TP53 gene polymorphisms corresponded to HWE. Only two SNPs (rs12947788 and rs12951053) exhibited the deviation from HWE (p < 0.05). Exonic rs1042522 was in LD with intronic rs1642785 (D’ = 0.9459; r² = 0.8884; p < 0.0001); rs17883323 (D’ = 0.9972; r² = 0.2686; p = 0.0108); rs17878362 and rs2909430 (D’ = 0.9993; r² = 0.6221; p < 0.0001). Intronic rs1642785 was associated with rs17883323 (D’ = 0.9973; r² = 0.2686; p = 0.0073), rs2909430 and rs17878362 (D’ = 0.9994; r² = 0.6223; p < 0.0001); rs12951053 and rs12947788 (D’ = 0.7458; r² = 0.4517; p < 0.001). Strong association was found between rs12951053 and rs12947788 (D’ = 0.9995; r² = 0.9995; p < 0.001) (Figure).

Data on the distribution of TP53 gene genotypes in both groups of patients are presented in the Table. Significantly reduced frequency of C/C homozygotes (Arg72Arg) of rs1042522 (p = 0.0036), and, conversely, increased number of heterozygous C/G variants (p = 0.0058) and carriers of G allele (p = 0.0036) were found in patients of the group I.

Table. Associations between TP53 SNPs and relative LPL expression level

Similar data were obtained regarding the distribution of rs1642785 polymorphic variants: a significant decrease in the number of homozygotes of dominant allele (G/G) and an increase in the number of heterozygous genotype (G/C) in the group of patients with low LPL expression. In this group, an increased number of heterozygotes of rs2909430 and rs17878362 was revealed as well.

No associations of LPL expression level with rs17883323, rs12947788, and rs12951053 were found. SNP rs145153611 and rs113530090 were monomorphic and presented only by homozygotes of dominant allele in both groups of patients.

According to data of SNPstats tool analysis, haplotype G–С–G–A2 (odds ratio (OR) = 9.69; 95% confidence interval (CI) 1.74–54.0; p = 0.013) was associated with significantly increased risk of low LPL expression when compared with the reference haplotype С–G–A–А1 (rs1042522/rs1642785/rs2909430/rs17878362, respectively).

DISCUSSION

The TP53 gene encodes p53 protein, which plays a central role in the arrest of cell cycle and apoptosis following DNA damage [26, 27]. At present, over 200 SNPs in TP53 have been identified (www-p53.iarc.fr). The association of TP53 SNPs with the susceptibility to different types of cancer has been most widely studied [28–31]. Recently, however, much attention was paid to the participation of p53 in the development of metabolic disorders and diseases, associated with atherosclerosis [18–21].

There is evidence that the frequencies of G allele of rs1042522 increased in disorders that are often accompanied by abnormalities of the serum lipid spectrum and LPL activity [32]. Elevated frequencies of carriers of G allele of rs1042522 were found among obese people [33], coronary heart disease [34, 35], type II diabetes [36, 37]. Therefore, our results on reduced LPL expression in patients with CLL in the presence of the G allele of rs1042522 are in the context of previously obtained data and may reflect the general mechanisms of the effect of TP53 on lipid metabolism in different cell types.

Two intron SNPs (rs17878362 in intron 3 and rs1642785 in intron 2) are in close LD (according to our data D’ = 0.9994; r² = 0.6223; p < 0.0001). A region of intron 3, containing rs17878362, forming G-quadruplex structures in p53 pre-mRNA is involved in the alternative splicing of TP53 intron 2. Differential splicing of intron 2 leads to the generation of mRNAs that encode canonical suppressor p53 protein and the Δ40p53 isoform, which lacks the transactivation domain and acts as a negative regulator of p53 activity [38]. In experiments with lymphoblastoid cell lines established from the peripheral blood of breast cancer patients, it was found that transcripts containing the C allele of rs1642785 had lower stability than that containing the G allele. The highest basal and radiation-induced p53 and Δ40p53 levels were associated with combined rs1642785-GG/rs17878362-A1A1 alleles, whereas the presence of rs1642785-C with either rs17878362 allele was associated with lower p53 pre-mRNA [39]. Morten et al. [40] have shown that Δ40p53: р53 ratio was significantly lower in samples of human breast cancer that were homozygous for the polymorphic allele of rs17878362 (A2/A2 genotype) compared with heterozytotes (A1/A2) and especially low compared with homozygous for the dominant allele (A1/A1). The authors suggest that a low Δ40p53:р53 ratio is associated with better disease-free survival of patients carriers of the rs17878362 A2 allele. Δ40p53 lacks the first 39 amino acids contained by full-length p53 that encode the first transcriptional activation domain and can alter p53 target gene expression [41, 42].

Thus, in CLL patients with UM IGHV genes and low LPL expression, we found an increased frequency of rs1642785 C and rs17878362-A2 alleles, which are associated with lower stability of TP53 mRNA gene and, possibly, with an altered Δ40p53:p53 ratio. These preliminary data suggested that in the leukemic B cells of CLL patients there was some association between LPL expression level and the functional state of the TP53 gene, the mechanisms of which have not been elucidated to date. One of such mechanisms may be a changed content of miR29, which is under the strict transcriptional control from p53 [43] and, in turn, influence on the transcription of the LPL gene [44].

In conclusion, some TP53 SNP may be genetic modifiers for LPL expression level in CLL leukemic B-cells. Further research is required in a larger cohort of patients to confirm our findings.

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