Genetic polymorphisms of XPD Arg156Arg and XPD Lys751Lys DNA repair genes in patients whit Acute Myeloid Leukemia First author: Crauciuc George Second author: Tripon Florin Coordinator: Conf. Dr.Claudia B ã nescu International Congress for students, young physicians and pharmacists - Marisiensis

Introduction Leukemia are clonal diseases which commonly arise as a result of genetic damage deregulating blood cell development or hematopoiesis.(1) Common Uncommon Viral infections: HTLV I HIV Epstein Barr Viral infections: HTLV I HIV Epstein Barr Genetic factors: Chromosomal disorders (Down syndrome, aplasia marrow Fanconi, syndrome Klinefelter) Molecular abdnormarlities FLT3, RAS, RUNX1, NPM1 and XPD 156, 751 Genetic factors: Chromosomal disorders (Down syndrome, aplasia marrow Fanconi, syndrome Klinefelter) Molecular abdnormarlities FLT3, RAS, RUNX1, NPM1 and XPD 156, 751 Physico-chemical factors: Ionizing radiation Chemicals Physico-chemical factors: Ionizing radiation Chemicals

XPDArg156Arg and XPDLys751Lys, is a helicase involved in the nucleotide excision repair pathway, which recognizes and repairs many structurally unrelated lesions(4). It is plausible that small variations in the effiency of repair in the normal population may facilitate cancer development in exposed individuals.(2-3)

Objective The aim of the study was to estabilish some genetic relationship between AML and XPDArg156Arg and XPDLys751Lys genes polymorphisms. Material 98 patients and a control group whit 157 healty persons.

Methods Our study population consisted of blood from 98 patients with AML also 157 healty from which we extracted DNA according to the protocol described by the manufacturer (ZymoResearch DNA kit). Genotypic analysis of the XPD156 C>A and XPD751 A>C polymorphism was determined by the PCR-RFLP using specific primers. Following PCR, 20µl of PCR product was subjected to restriction digestion using10 U of Pst I for XPD751 and Tfil l for XPD 156 restriction enzyme(5) (Thermo Scientific Fast Digest).

The primers used to amplify the XPD gene were as follows: for XPD 156 (exon 6): Forward primer: 5-TGG AGT GCT ATG GCA CGA TCT CT-3 Reverse: 5-CCA TGG GCA TCA AAT TCC TGG GA-3 for XPD 751 (exon 23): Forward primer: 5-ATC CTG TCC CTA CTG GCC ATT C Reverse primer: 5-TGT GGA CGT GAC AGT GAG AAC T-3 (7-8) Amplification was performed with an initial denaturation for 10 min at 95 ⁰ C, followed by 30 cycles of denaturation at 95 ⁰ C for 1 min, annealing at 60 ⁰ C for 1 min, extension at 72 ⁰ C for 1min and a final extension at 72 ⁰ C for 10 min. PCR protocol The digested products were resolved on 3% agarose gels (Sigma Chemical Co), stained with ethidium bromide and analyzed under UV light.(6)

Results The mean age of all persons was years, whit ± 18 years deviation. The success rate of genotyping was 90% in the first stage of work and 100% in the second genotyping for the same DNA.

Results: Patients group In the AML group the gene XPD 156 was found in the following variants: -homozygous normal (CC genotype) at 30 people -heterozygous (CA genotype) at 51 and -homozygous mutant (AA genotype) on 17. In the same group XPD 751 was found as: -homozygous normal (AA genotype) at 33 people -heterozygous (AC genotype) at 52 and -homozygous mutant (CC genotype) on 13.

Results: Control group In the control group the gene XPD 156 was found in the following variants: -homozygous normal (CC genotype) at 66 people -heterozygous (CA genotype) at 59 and -homozygous mutant (AA genotype) on 32. In the same group XPD 751 was found as: -homozygous normal (AA genotype) at 17 people -heterozygous (AC genotype) at 51 and -homozygous mutant (CC genotype) on 30.

The marker used in the agarose gel has 100 pb XPD 751 C 480 pb A 352 pb 250 pb XPD 156 A 900 pb C 761 pb

Statistical Results Wild TypeHeterozygous Homozygous Mutant Heterozygous and Homozygous mutant XPD 156- p= OR: CI: p= OR: CI: p= OR: CI XPD 751- p= OR: CI: p= OR: CI: p= OR: CI:

Conclusion and Discussions Exist a correlation between mutation in the XPD 156 gene and AML, whit statistical and scientific significance. The distributions of the XPD 156 and XPD Lys751Gln genotypes were in accordance with Hardy-Weinberg equilibrium among the controls (p = 0.176; p = 0.083, respectively) and the AML cases. Further studies with a large number of patients are needed as well studies to demonstrate this genetic defects are important factors in determining the desease and the response to chemotherapy and outcome.

Discussions We continued to investigate the importance of XPD mutations dividing individuals in another two groups: +50 years / -50 years. The statistical results remained the same, the age is not related with the mutant genotype. 19 cases are FLT3 positive, No association was found between FLT3 mutation and mutant genotypes studied.

References 1. Benhamou,S. and Sarasin,A. Variability in nucleotide excision repair and cancer risk: a review. Mutat. Res., , 149– Simone Benhamou1,2,3 and Alain Sarasin2,, ERCC2/XPD gene polymorphisms and cancer risk, Mutagenesis vol.17 no.6 pp.463–469, Maurizio Manuguerra1, Federica Saletta1, Margaret R. Karagas, XRCC3 and XPD/ERCC2 Single Nucleotide Polymorphisms and the Risk of Cancer: A HuGE Review, Human Genome Epidemiology 4.Liang G, Xing D, Miao X, et al. Sequence variations in the DNA repair gene XPDand risk of lung cancer in a Chinese population. Int J Cancer 2003;105:669–73. 5.Qiao Y, Spitz MR, Shen H, et al. Modulation of repair of ultraviolet damage in the host-cell reactivation assay by polymorphic XPC and XPD/ERCC2 genotypes.Carcinogenesis 2002;23:295–9. 6.Matullo G, Peluso M, Polidoro S, et al. Combination of DNA repair gene single nucleotide polymorphisms and increased levels of DNA adducts in a population-based study. Cancer Epidemiol Biomarkers Prev 2003;12:674–7. 7.Tang D, Cho S, Rundle A, et al. Polymorphisms in the DNA repair enzyme XPD are associated with increased levels of PAH–DNA adducts in a case-control study of breast cancer. Breast Cancer Res Treat 2002;75:159– Rita Misra et all. Polymorphisms in the DNA repair genes XPD, XRCC1, XRCC3, and APE/ref-1, and the risk of lung cancer amongmale smokers in Finland. Cancer Letters Volume 191, Issue 2, Pages , 10 March 2003

THANK YOU !