In this study the molecular genomic polymorphism of age related acute myeloid leukemia was analyzed by twenty one arbitrary primers of decamer oligonucleotides to investigate the genetic polymorphisms. Two categories of RAPD primers were identified, according to their ability to amplify genomic DNA. Thirteen primers were found unable to amplify genomic DNA of acute myeloid leukemia patients. On the other hand, eight primers were able to amplify genomic DNA and were divided into two subgroups, first group showed monomorphic DNA fragments, whereas the second one gave polymorphic bands. One of the polymorphic amplifying primers showed unique patterns of amplified DNA fragments in the genomic DNA of age related acute myeloid leukemia.
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Acute Myeloid Leukemia (AML) is a cancer of the myeloid of blood cells, characterized by the rapid growth of abnormal white blood cells in the bone marrow and interfere with the production of normal blood cells. It is the most common cause of acute leukemia affecting adults, and its incidence increases steadily with age. However, more than 50% of cases being diagnosed in subjects aged 60 years or over (Pinto et al., 1998), while the incidence of Acute Lymphoblastic Leukemia (ALL) is rather uncommon in adults (Bassan et al., 2004).
During the last 20 years, the diagnosis of acute leukemias emerged from cytomorphology alone to a comprehensive bundle of different methods that are necessary not only for the diagnosis and classification but also for individual treatment decisions. Moreover, nowadays it is rather mandatory to further perform Multi-parameter Flow Cytometry (MFC), with metaphase cytogenetic in every case, in which acute leukemia is suspected, the latter has to be accompanied by FISH and PCR analysis or even screening for specific molecular markers (Haferlach et al., 2005).
Molecular genetic studies however suggested that genetic instabilities of genomic DNA are rather implicated in the pathogenesis of cancers. Recent published results showed remarkable progress has been made in the elucidation of the molecular pathogenesis of Acute Myeloid Leukemia (AML) where most AML-typical chromosomal alterations result in fusion of genes which is rather important for myeloid differentiation, and the resulting fusion proteins would interfere with their function (Steffen et al., 2005). Such molecular alteration at the genomic level encouraged researchers to dig widely in molecular approaches for detecting genomic alterations of various types of cancers; one of these approaches is Random Amplified Polymorphic DNA (RAPD) analysis. In this method arbitrary primers of decamer oligonucleotides are used with variable success. This method can simply and rapidly detect genetic alterations in the entire genome without knowledge of specific DNA sequence information (Papadopoulos et al., 2002). The RAPD-PCR analysis was used as a mean for identifying the genetic alterations in human tumors and revealed that genetic alterations are frequently observed in various types of tumors, e.g., leukemia (Odero et al., 2001) and other cancers (Misra et al., 1998; Maeda et al., 1999; Ong et al., 1998; Scarra et al., 2001; Singh and Roy, 2001; Xian et al., 2005; Zhang et al., 2004).
The objective of the study is to investigate the molecular genetic polymorphism of age related Acute Myeloid Leukemia (AML) using RAPD-PCR analysis and to identify RAPD primers demonstrating genomic instability and having potential use for detection of AML.
MATERIALS AND METHODS
Blood Samples Collection and DNA Extraction
Five-millimeters blood samples were collected from AML patients attended Al-Bairuni Hospital in Damascus during the period from 17/5/2007 to 25/11/2007. The blood samples were kept in tubes containing anti-coagulant (K3EDTA) at -20°C till further use (Goossens et al., 1991). The genomic DNA was extracted from whole blood following the instructions of the DNA purification kit obtained from Promega Company. In this study we used DNA pooling procedure for normal male and female individuals.
DNA Amplification and Gel Electrophoresis
Amplification reactions were performed in a total volume of 25 μL consisting of 12.5 μL of PCR master mix (Fermentas, EU), 2.5 μL primer (10 pmol mL-1), 2.0 μL genomic DNA (25 ng μL-1) and 8 μL sterile distilled water. Amplification program was started with 2 min at 94°C for initial denaturation followed by 40 cycles of 94°C for 30 sec (denaturation), 38°C for 1 min (annealing) and 72°C for 2 min (extension). The final extension cycle was 72°C for 10 min (Xian et al., 2005).
Twenty One Primers
(OPA-01, OPA-09, OPA-11, OPA-12, OPA-14, OPB -11, OPB -12, OPB -15, OPB -17, OPB -18, OPC-01, OPE-05, OPF-18, OPI -18, OPJ -01, OPJ-05, OPK-08, OPW-17, OPY-10, OPZ-02, OPZ-19) were used in the amplifications, the primers were obtained from Operon Technologies, Alameda, AL, USA. The amplification products were separated on 1.5% agarose gel and visualized under UV light after staining with ethidium bromide for molecular size determinations in kilo base pair (kbp) of DNA fragments (Sambrook and Russell, 2001).
RAPD-PCR assays were repeated at least three times and only the reproducible bands were scored. Each RAPD-PCR product was assumed to represent a single locus, for considering a marker as polymorphic; the absence of an amplified product in at least one sample was used as a criterion (Seufi et al., 2009).
AML patients were classified into seven categories according to gender and age group (Table 1). The percentages of female and male AML patients were 57.14 and 42.857%, respectively. Considering age related AML incidence, the number of AML patients who were 50 years old and more were 12, representing 57% of AML patients.
In this study 21 different arbitrary primers of decamer oligonucleotides were screened for RAPD analysis using genomic DNA isolated from ten age related AML patients, seven females and three males, the ages of patients were more than 50 years. After the optimization of RAPD-PCR reaction, the obtained results classified the 21 primers into two categories. In the first category included thirteen different primers showed no amplified products, in spite of repeating the experiments. The non amplifying primers were: OPA-01, OPA-11, OPA-12, OPA-14, OPB-11, OPB-12, OPC-01, OPF-18, OPI-18, OPJ-01, OPK-08, OPY-10 and OPZ-19. The second category included eight primers which were able to amplify genomic DNA and may be divided in to two subgroups according to type of DNA bands polymorphisms (Table 2), first group included six primers (OPA-09, OPB-15, OPB-17, OPB-18, OPE-05, OPW-17) which gave polymorphic DNA fragments in AML patients, whereas second group included two primers (OPJ-05 and OPZ-02) which gave mainly monomorphic DNA fragments in both normal individuals and AML patients. Three polymorphic bands produced by primer OPB-17 with moderate or weak intensities appeared in the genomic DNA extracted from elderly female and male patients (Fig. 1). The molecular weights of the three DNA fragments were 1.413, 1.158 and 1.04 kbp (Table 3). These three polymorphic bands were not detected in normal male and female individuals.
AML patients classified according to gender and age groups
Sequences of amplifying deca-oligonucleotide primers used for RAPD-PCR analysis of age related AML patients
Distribution of polymorphic DNA bands produced by primer OPB-17 in AML patients. Bands were classified according to size of DNA fragments (kbp) and their intensities. Male and female patients were represented by numbers which were indicated in Fig. 1
RAPD-PCR polymorphic amplification patterns of genomic DNA of age related AML patients obtained with primer OPB-17. Electrophoresis was performed on (1.5%) agarose gel and run with 3 volt cm-1. The lane 1 represent normal females, lanes from 2 to 9 represent the AML female patients, lane 10 represent normal males; lanes from 11 to 13 represent AML male patients. Lane L indicates the λDNA as a ladder
RAPD-PCR monomorphic amplification patterns of genomic DNA of age related AML patients obtained with primer OPZ-02. Electrophoresis was performed on (1.5%) agarose gel and run with 3 volt cm-1. The lane 1 represent normal females, lanes from 2 to 9 represent the AML female patients, lane 10 represent normal males; lanes from 11 to 13 represent AML male patients. Lane L indicates the λDNA as a ladder
The monomorphic bands amplified by primer OPZ-02 were of three types: good, moderate and weak intensities and found in male and female patients as well as in control group of male and female individuals (Fig. 2).
Age is considered the most important risk factor for most of our life-threatening human malignancies; two-thirds of all cancers are now being diagnosed in people over age 65 years old (Irminger and Benz, 2005). In this study the ages of 57% of AML patients were more than 50 years old. It has been reported that the incidence of Acute Myelogenous Leukemia (AML) increases steadily with age and more cases are diagnosed in subjects aged 60 years or over, however, the accepted incidence of this disease is around or more than 50% in elderly people (Pinto et al., 1998). On the other hand, age has been reported as an adverse prognostic indicator in AML affecting both remission rates and survival (Lowenberg et al., 1999).
The failure of amplification of genomic DNA of normal persons and leukemia patients by thirteen RAPD primers used in this study may be attributed to the absence of suitable priming sites for these primers in the complementary sequences for these primers in their genomes (Papadopoulos et al., 2002). On the other hand, using the other eight primers which were able to amplify the genomic DNA of AML patients, a reasonable degree of DNA polymorphism was detected between normal person and leukemia patients. Moreover in this study it was possible to identify a RAPD primer which gave DNA fragments which might be specific to age related leukemia (AML), these DNA fragment were not detected in genomic DNA of normal individuals, however further work is required to ascertain whether the detected DNA polymorphisms by OPB-17 is specific to age related AML or not. Possible correlations between amplified DNA bands and age related cancers were not reported in previous investigations, we are in process of investigating such possible correlation considering the role of DNA methylation in genomic instabilities in cancer patients (Strathdee and Brown, 2002).
It is expected that the investigation of DNA polymorphism will pave the way for better understanding of molecular mechanisms of age related leukemia and it will help in identification of molecular markers for diagnosis of cancers and follow up of cancer therapy and relapse. It is worth mentioned that DNA polymorphism is an indication of genomic instabilities which is a result of aberration in global CG and CG islands genomic DNA methylation. Genomic instabilities and aberration of DNA methylation are believed to be a driving force in aging and carcinogenesis (Strathdee and Brown, 2002).
Cancer and aging are two coupled developmental processes as reflected by the higher incidence of cancer in the elderly human population group. An understanding of the relationship between aging and cancer is of more than mere academic interest, since biomedical research aims to investigate and develop molecular methods which can prevent both. Genetic polymorphisms are frequent in genomic DNA of Leukemia patients and could be detected by using suitable RAPD primers. Specific bands could be generated as a result of RAPD-PCR analysis which might be used for diagnosis and prognosis of AML. The observation of unique bands in RAPD-PCR profiles of elderly AML patients might suggest the possible correlation between genomic instabilities occurring during process of aging and age related cancers.
The authors would like to thank the Royal Jordanian Scientific Society, Al-Nahrain University, National Syrian Biotechnology Commission and King Hussein Cancer Center for their support of this research.
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