Prepared and Application of New Microarray-Based Method for Detecting SNP in the Risk of Hematological Malignancy.

Objective To investigate a new microarray-based method for single nucleotide polymorphisms (SNP) genotyping, and to study the role of 5,10-methylenetetrahydrofolate reductase(MTHFR) SNP in hematological malignancy pathogenesis in the Chinese population.

Methods Prepared a aimed gene microarray based on cDNA microarray theory, dual-color fluorescenece hybridization was used for detecting SNP loci, sequencing was performed to confirm the results. In order to prepare the function SNP detection microarray, three different functionalized glass slides were selected, which were modified with 3-aminopropyltriethoxysilane (APTES), poly-l-lysine and glutaraldehyde, respectively. In order to obtain satisfied signal intensity, the ultraviolet crosslinking energy and the immobilized DNA concentration were optimized. By using the above SNP microarray, MTHFR C677T and A1298C SNP loci of 157 controls and 127 patients with hematological malignancy[30 multiple myeloma(MM),28 non-Hodgkin’s lymphoma(NHL), 22 acute lymphoblastic leukemia(ALL),40 acute myeloid leukemia(AML),7 chronic myeloid leukemia (CML)] from Jiangsu province were detected.

Results Based on the immobilization efficiency, APTES modified glass slides were selected to fabricate DNA microarrays. The optimal ultraviolet crosslinking energy was 400 mJ and the optimal concentration of immobilized DNA was 300 mg/ml. Homozygous wild type, heterozygote type and homozygous mutant type yielded green, yellow and red fluorescence, respectively. Sequencing validated the results.The allele frequencies of MTHFR677C, 677T were 58.7%, 41.3% in patients and 66.9%, 33.1% in controls, respectively; the frequencies of 677CC, 677CT, 677TT genotype were 37.8%, 41.7%, 20.5% in patiens and 45.9%, 42.0%, 12.1% in controls, respectively. The allele frequencies of MTHFR1298A, 1298C were 83.1%, 16.9% in patients and 84.1%, 15.9% in controls, respectively; the frequencies of 1298AA, 1298AC, 1298CC genotype were 70.9%, 24.4%, 4.7% in patiens and 69.4%, 29.3%, 1.3% in controls, respectively. In multivariate analysis, significant difference was observed in the allele frequencies of C677T(χ²=4.08, P=.043) and in the genotype frequency of 677TT(OR 2.09, 95%CI 1.04–4.20, P=.039). The frequencies of 677TT (33.3%) and 1298CC(6.7%) in MM group were found significantly higher than in control group(OR 8.92, 95%CI 1.06–75.24, P=.006; OR=4.80, 95%CI 1.56–14.73, P=.044), resectively. No associations were found between any polymorphisms and susceptibilities to ALL, AML and NHL.

Conclusions This microarray-based method is accurate, high-throughput and cheap, suitable for SNP genotyping in a large of individuals. MTHFRC677T polymorphisms influence the risk of hematological malignancy in Chinese population. Both MTHFR677TT and MTHFR1298CC genotype are susceptive to MM.