ASSOCIATION OF 308TNF AND +252LTA SINGLE NUCLEOTIDE POLYMORPHISMS WITH HEMATOLOGICAL MALIGNANCIES IN CHILDREN FROM THE BASHKORTOSTAN REPUBLIC
Yakupova EV 1,* Krasavtceva TN2, Malyevsky OA2, Viktorova TV1
*Corresponding Author: Dr. Elvira V. Yakupova, Institute of Biochemistry and Genetics, Ufa Research Centre, Russian Academy of Science, Prospect Oktyabrya 69, Ufa 450054, Russia; Tel./Fax: +007-3472-356088; E-mail: ecolab_203@mail.ru
page: 9

MATERIALS AND METHODS

The study comprised 105 children with HM which were treated at the Bashkortostan children republic hospital in 2000 - 2003 years and 141 unrelated healthy individuals that provided samples available for genetic analysis after informed consent. Among patients 86 were with acute lymphoblastic leukemia (ALL), 10 with acute myeloblastic leukemia (AML), 9 with Non-Hodgkins lymphoma (NHL). Pathological confirmation of the diagnosis and clinical history were available in the every case.

Genotyping analysis.  Genomic DNA from peripheral blood cells was extracted by the standard phenol-chloroform method (9).

 The primer pairs F (5' A ATA GGT TTT GAG GGC CAT G 3') and      R (5' TC CTC CCT GCT CCG ATT CCG 3') were used to amplify a 107-bp fragment of the TNF gene, which includes the polymorphic site (G?A transition) at the nucleotide position       -308 (10).  After heating at 95C for 5 minutes, PCR reactions were performed for 30 cycles consisting of heat denaturation (95C for 30 seconds), annealing (53C for 30 seconds), and extension (72C for 45 seconds). Then, PCR-amplified products were directly digested with 5 U of restriction enzyme Bsp19I, at the temperature 37C, during the night. The band cleaved with Bsp19I (87 and 20 bp fragments) represented the TNF*GG allele (wild type), and no cleaved (107 bp) band represented TNF*AA allele with higher spontaneous and inducible expression levels (10-11).

A substitution of A to G at the nucleotide position +252 of LTA gene creates a Bsp19I restriction fragment length polymorphism. PCR-amplified products of 368 bp were obtained with the use of F (5' CTC CTG CAC CTG CTG CCT GGA TC 3') and R (5' GAA GAG ACG TTC AGG TGG TGT CAT 3') primers (12). PCR and restriction digest conditions were similar with described above. The no cleaved (368 bp) band represented LTA*AA genotype (wild type), and bands cleaved with Bsp19I (133 and 235 bp fragments) represented the LTA*GG genotype (with higher DNA expression level) (12).

Statistical analysis was conducted using the Yates corrected χ2 test (13).




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