Dissertation committee:
Παρασκευή Ρούσσου, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Καλλιόπη Μανωλά, Ερευνήτρια Α΄ βαθμίδας, ΙΠΡΕΤΕΑ, Ε.Κ.Ε.Φ.Ε. «Δημόκριτος»
Ευανθία Διαμάντη Κανδαράκη, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Βασιλική Παππά, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Παναγιώτης Τσιριγώτης, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ευστάθιος Παπαγεωργίου, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μαρία Αγγελοπούλου, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Chronic Lymphocytic Leukemia (CLL), one of the most common forms of hematologic malignancies, is characterized by in vivo accumulation of monoclonal B-lymphocytes in peripheral blood and bone marrow, which infiltrate lymphatic tissues and spleen, often causing inflation of the lymph nodes and dysfunction of the immune system. The disease presents great clinical variation which is intertwined with the diversity observed at genetic and molecular level. Some patients survive for many years without treatment while others suffer from an aggressive form of the disease. The molecular and genetic basis of CLL has not been fully elucidated yet however, genetic and epigenetic mechanisms appear to be present in the pathogenetic pathway of the disease and the large clinical variability that it presents.
Cytogenetic findings contribute to the diagnosis, prognosis and selection of the appropriate treatment protocol for CLL patients. Nowadays, in the bone marrow (BM) cell culture of CLL patients we use a combination of B-cell stimulators, such as DSP-30 and interleukin-2 (IL-2) combination, which significantly increase the percentage detection of clonal chromosomal abnormalities, personalizing the prognosis of patients.
Molecular analysis has shown alterations in genes whose products regulate important cell functions such as growth, division and proliferation. These changes involve both gene mutations and epigenetic changes. Methylation of cytosines in CpG islands of gene promoters is one of the most studied epigenetic DNA modifications in humans and affects the structure of chromatin as well as the transcriptional activity. Although the investigation of molecular mechanisms in leukemia is currently at the center of research interest, the study of these mechanisms in CLL is still at an early stage.
Very recent studies indicate that methylation of DNA is a significant epigenetic modification that is associated with CLL. In this thesis we study methylation of RAD21 gene which is located in the long arm of chromosome 8, chromosomal region 8q24.11. The produced RAD21 protein is one of the basic subunits of the cohesin complex which is involved in the mechanism that controls the correct separation of sister chromatids during cell division and participates in DNA repair mechanisms. Methylation of RAD21 gene promoter leads to transcriptional inactivation and thus gene silencing. Studying methylation of RAD21 gene and associating findings with CLL and its specific cytogenetic alterations may contribute to a better understanding of the pathogenetic course of the disease and also to the design of new therapeutic methods using demethylation agents that are already used in the treatment of other diseases.
In addition, studies of CLL patients have shown the involvement of genetic predisposing factors in the onset of the disease. Paroxonases (PONs) are enzymes that protect cells from genotoxic factors such as organophosphates and free oxygen radicals, agents that cause metabolic lesions and DNA damage by contributing to carcinogenicity. PON1 gene (paroxonase 1, a subunit of the paroxonases complex) located on the chromosome region 7q21.3, presents two SNP polymorphisms in its coding region, at positions 55 and 192, which result in the replacement of an amino acid and affect the concentration, stability and activity of the enzyme. In particular, the L55M polymorphism (Leu55Met [rs854560]) involves replacement of leucine (L) by methionine (M) and polymorphism Q192R (Gln192Arg [rs662]) replacing glutamine (Q) by arginine (R). In this dissertation we studied the two polymorphisms of PON1 gene in order to investigate their possible involvement in the genetic predisposition of CLL and the formation of characteristic chromosomal lesions.
The purpose of this doctoral thesis is the cytogenetic, molecular and epigenetic study of CLL patients. In particular, the present thesis focuses on investigating the methylation pattern of RAD21 gene promoter and the study of L55M and Q192R polymorphisms of PON1 gene which, in combination with cytogenetic findings, may contribute to the elucidation of the pathogenesis and the mechanisms of the disease.
For the purposes of this thesis, 300 bone marrow samples from CLL patients, received at the NCSR Lab "Demokritos" for their cytogenetic analysis were recruited. After BM cell culturing, harvesting and fixation, chromosome banding was performed using GTG banding method. Microscopic and karyotypic analysis was performed using Ikaros software (Metasystems). Karyotypes were described according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016. In patients deemed necessary, we applied fluorescent in situ hybridization (FISH) method to mesophasic and metaphase cells (FISH interphase and metaphase) using appropriate probes.
Studying RAD21 methylation we included 105 CLL patients, selected based on their cytogenetic findings, and 17 healthy donors. Total genomic DNA was isolated from all of CLL patients’ BM samples and from 106 healthy donors’ peripheral blood samples for molecular analysis. Using Real-Time PCR and the EpiTect Methyl II PCR Assay we studied the methylation status of RAD21 gene promoter and then the results were correlated with patients’ cytogenetic findings to investigate possible involvement in the formation of specific CLL abnormalities.
In the study of the two polymorphisms of PON1 gene, 300 CLL patients and 106 healthy donors were participated. Genotyping methods using Polymerase Chain Reaction (PCR) and digestion with restriction endonucleases (PCR-RFLPs method) were standardized. For the determination of normal and mutant genotypes electrophoresis in 2% agarose gel was performed. After the distribution of the genotypes and alleles of the polymorphisms the results were associated with patients’ cytogenetic findings. The correlations of all results were examined and compared with the Chi-Square test, χ2. Statistical analysis was performed using SPSS software (Statistical Package for Social Sciences) version 20. The study was based on a 95% confidence interval and a significance level of 5%. Fisher's Exact Test was used where necessary.
From the cytogenetic analysis of the 300 CLL patients, 39.3% (118/300) of patients had normal karyotype (confirmed with FISH) and 60.7% (182/300) pathological karyotype. Among the 182 patients with pathological karyotype 33.5% (61/182) had del(13q), 30.8% (56/182) trisomy 12, 21.9% (40/182) del(11q), 13.2% (24/182) del(17p), 11.5% (21/182) of patients had abnormalities in 14q32 region [abn(14q32)], 6.6% (12/182) chromosome 8 alterations [abn(8)], 4.9% (9/182) of patients del(6q) and 12.1% (22/182) of patients had other chromosomal alterations.
Concerning RAD21 methylation, this study was successful in 96% of patients and 100% of healthy donors. A statistically higher frequency of methylated samples was observed in patients (25.75%), compared to controls (0%) (p= 0.022). Correlation between RAD21 gene methylation and karyotypic results showed comparable methylation frequency between the different cytogenetic subgroups. Specifically, methylation was observed in 28.57% of patients with normal karyotype and in 25% of patients with pathological karyotype suggesting that methylation of this gene was not associated with chromosomal alterations. This thesis is the first study that examines the methylation of RAD21 gene in patients with CLL.
Examining PON1 gene polymorphisms, the Q192R polymorphism study was 100% successful in CLL patients and 98.1% in healthy donors. Genotypic and allele distribution ofQ192R polymorphism showed a statistically significant higher frequency of the mutant genotypes (QR heterozygotes and RR homozygotes) (p=0.028) and the mutant R allele in patients compared to controls (p=0.012). Comparing each karyotypic subgroup of patients with the results of the healthy donors a statistically higher incidence was showed at mutated genotypes and alleles in the pathological karyotype group, specifically in those carrying abn14q32 and del(6q) (p=0.030 and p=0.021 respectively). A statistically increased frequency for the mutant allele was also revealed in patients with del(11q) (p=0.035).
The study of L55M polymorphism was 100% successful in CLL patients and 99.1% in healthy donors. But unlike Q192R polymorphism, L55M polymorphism showed similar distributions between patients and controls indicating that L55M polymorphism, in contrast to Q192R polymorphism, does not appear to predispose to the appearance of CLL.
In conclusion, this thesis was the first study to investigate the methylation status of RAD21 gene in patients with CLL and showed an increased presence of methylation in CLL patients compared to healthy donors, suggesting a possible pathogenetic role of RAD21 promoter methylation in the development of CLL probably via self-renewal of CLL cells and not via the formation of chromosomal abnormalities.
Our results from the study of the two polymorphisms of PON1 gene, indicates a possible role of Q192R polymorphism in CLL predisposition and the formation of specific chromosome aberrations in contrast with L55M polymorphism.
Keywords:
Chronic Lymphocytic Leukemia, Cytogenetics, Epigenetics, Polymorphisms, Methylation, PON1 gene, RAD21 gene
