Clinical Relevance of DNA Methyltransferase and Histone Deacetylase Gene Single Nucleotide Polymorphism in Myelodysplastic Syndrome

This study will include the patients who signed the subject informed consent form among the patients with MDS who were chosen to be treated with Decitabine (Part I), plus additional 140 MDS patients as a historical control (Part II). Approximately, 68 patients will be included who satisfy the following inclusion and exclusion criteria in the Part I study.

Prospective collection of DNA from peripheral blood will be performed in the patients with MDS before commencement of decitabine therapy. We will evaluate the efficacy decitabine therapy according to the DNMT or HDAC gene SNPs in terms of following parameters: 1) hematolotic response (HR) or improvement (HI), or requirement of decitabine dose to achieve HR or HI, 2) complete (CR) or partial response (PR), or requirement of decitabine dose to achieve CR or PR, and 3) time to relapse or progression of MDS.

Genotyping will be undertaken using the Sequenom® iPLEX platform™, according to the manufacturer's instructions (www.sequenom.com; Sequenom Inc, San Diego, CA, USA). Whole blood samples will be obtained according to the declaration of Helsinki. DNA will be extracted using the Puregene DNA purification Kit (Gentra Systems Inc, Minneapolis, MN, USA). The detection of SNPs will be performed by the analysis of primer extension products generated from previously amplified genomic DNA using a Sequenom chip-based matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry platform. Multiplex SNP assays will be designed using SpectroDesigner software (Sequenom). Ninety-six well plates containing 2.5 ng DNA in each well will be amplified by PCR following the specifications of Sequenom. Unincorporated nucleotides in the PCR product will be deactivated using shrimp alkaline phosphatase. Allele discrimination reactions will be conducted by adding the extension primer(s), DNA polymerase, and a cocktail mixture of deoxynucleotide triphosphates and di-deoxynucleotide triphosphates to each well. MassExtend clean resin (Sequenom) will be added to the mixture to remove extraneous salts that could interfere with MALDI-TOF analysis. The primer extension products will be then cleaned and spotted onto a SpectroChip. Genotypes will be determined by spotting an aliquot of each sample onto a 384 SpectroChip (Sequenom), which is subsequently read by the MALDI-TOF mass spectrometer.

All statistical tests will be two-sided with the significance level set as 0.05 unless otherwise stated. The statistical data will be obtained using an SAS version 9.1 (SAS Institute, Cary NC, USA). Followings are the endpoints for the study.

1. Primary endpoint evaluation data