POLYMORPHISM OF BIOTRANSFORMATION GENES AND RISK OF RELAPSE IN CHILDHOOD ACUTE LEUKEMIA
Gra OA1,2, Kozhekbaeva ZhM1,2,3, Makarova OV4,Samochatova EV4, Nasedkina TV1,*
*Corresponding Author: Tatyana V. Nasedkina, Ph.D., Department of Biological Microarray, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; Tel.: +7-499-135-62-59; Fax: +7-499-135-14-05; E-mail: nased@biochip.ru, nased@eimb.ru
page: 21

MATERIALS AND METHODS

Patients. We examined 332 children, including 258 diagnosed with primary ALL and 74 with ALL relapse. Their mean age was 6 years (from 3 months to 16 years; 57.8% males). We also examined 71 children with AML, including 41 diagnosed with primary AML and 30 with AML relapse. Their mean age was 8 years (from 1 to 17 years; 47.9% males). All patients were residents of the European part of Russia and originated from the East Slavonic population. The parents of all patients gave their informed consent according to the Declaration of Helsinki; the study was approved by the authorities of the Engelhardt Institute of Molecular Biology, Moscow, Russia. Diagnosis was made according to the 1997 World Health Organization (WHO) classification [23]. Blood/bone marrow specimens were obtained for diagnosis of primary disease, relapse, or remission in the oncohematology divisions of the Russian Children’s Clinical Hospital (Moscow), Morozov Children’s Clinical Hospital (Moscow), and the Moscow Regional Oncological Dispensary (Balashikha) from 2000 to 2004. The ALL and AML patients received scheduled therapy according to the protocol ALL-MB-02 or AML-2000 (Moscow-Minsk), respectively. The drugs administrated are summarized in Table 1.

We examined 332 children, including 258 diagnosed with primary ALL and 74 with ALL relapse. Their mean age was 6 years (from 3 months to 16 years; 57.8% males). We also examined 71 children with AML, including 41 diagnosed with primary AML and 30 with AML relapse. Their mean age was 8 years (from 1 to 17 years; 47.9% males). All patients were residents of the European part of Russia and originated from the East Slavonic population. The parents of all patients gave their informed consent according to the Declaration of Helsinki; the study was approved by the authorities of the Engelhardt Institute of Molecular Biology, Moscow, Russia. Diagnosis was made according to the 1997 World Health Organization (WHO) classification [23]. Blood/bone marrow specimens were obtained for diagnosis of primary disease, relapse, or remission in the oncohematology divisions of the Russian Children’s Clinical Hospital (Moscow), Morozov Children’s Clinical Hospital (Moscow), and the Moscow Regional Oncological Dispensary (Balashikha) from 2000 to 2004. The ALL and AML patients received scheduled therapy according to the protocol ALL-MB-02 or AML-2000 (Moscow-Minsk), respectively. The drugs administrated are summarized in Table 1.

Specimens. DNA samples were isolated from whole blood or bone marrow leukocytes, using a Wizard genomic DNA purification system (Promega, Madison, WI, USA).

Oligonucleotide Synthesis and Biochip Preparation. The oligonucleotides to be immobilized in a biochip were synthesized on a 394 automated DNA/RNA synthesizer (Applied Biosystems, Carlsbad, CA, USA) according to the standard phosphoramidite procedure [24]. The 3' end of the oligonucleotides was linked to a spacer with a free amino group, which was introduced during synthesis with the use of a 3'-Amino-Modifier C7 CPG 500 (Glen Research, Sterling, VA, USA). The nucleotide sequences of the immobilized nucleotides [7,21] are available on request. Biochips were obtained by photoinducible copolymerization of the oligonucleotides with the components of polyacrylamide gel [25].

Multiplex Polymerase Chain Reaction (PCR). DNA fragments of all genes were amplified in two rounds of nested multiplex PCR except for the GSTT1 andGSTM1 genes, which were amplified in one round of PCR. The primers were designed using the Oligo 6 program (Molecular Biology Insights, Cascade, CO, USA). The target mutations and the primer sequences [7,21] are available on request. Five parallel multiplex reactions were run. The primer combinations and the PCR conditions were as described [7, 21].

The fluorescently-labeled products of the second rounds of multiplex PCR were hybridized to the biochip. The hybridization mixture (30 mL) contained 25% formamide (Serva, Heidelberg, Germany), 5´ SSPE (Promega), and the PCR product (3 mL from each multiplex reaction), and denatured at 95°Ń for 5 min, quickly chilled in ice for 1 min, and applied onto the biochip. The biochip was incubated at 37°Ń overnight, washed with 1´ SSPE at room temperature for 10 min and then dried.

Image Analysis. The fluorescent signal from cells of the microchip was detected using a portable biochip analyzer supplied with a CCD camera and Imageware software (Biochip-IMB, Moscow, Russia) [21-22].

Sequencing. Specimens with different polymorphic variants of CYP1A1 were sequenced using a 3100 ABI PRISM™ genetic analyzer (Applied Biosystems).

Statistical Analysis. This was performed using the GraphPad InStat program (San Diego, CA, USA). Genotype frequency distributions were tested for correspondence to the Hardy-Weinberg equilibrium by the c2 test and proved to obey it for all biotransformation genes. For each genotype we estimated odds ratios (OR) and 95% confidence intervals (95% CI). The OR value is widely adopted in epidemiological studies to estimate the probability to have the disease for the carrier of a particular genotype (see also [7]). To calculate the OR in case-control study the 2´2 contingency table is used.
The OR = a/b,               
               c/d
 where a = n1, b = N1n1, c = n2, d = N2n2;N1 andN2
 
are the sample sizes; and n1and n2 are the numbers of individuals with the given character in the two samples. The polymorphic variants of investigated genes were estimated for non-random association with disease recurrence by Fisher’s exact test (double-sided). The null hypothesis is that each two values in row and columns are random. The null hypothesis is rejected at critical level of confidence 5%, if the calculated probability
p = (a+ b)!(a + c)!(b + d )!(c + d )!
                    N!a!b!c!d!
 
 is less than 0.05.

The OR values below are given with a 95% confidence interval; 0.33-0.92,p = 0.05 was used as a threshold to estimate the statistical significance of difference in genotype frequencies.

 



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