Peripheral EDTA-blood samples were derived from 32 leukemic patients (20 females and 12 males) and 52 healthy individuals (34 females and 18 males). Median ages were 47.1 ± 28.6 years (range 2 to 78 years) in the leukemic patient group and 32.6 ± 16.3 years (range 1 to 82 years) in the healthy group. In addition, bone marrow aspirates were derived from 38 MDS patients (18 females and 20 males) with different IPSS classifications (12 patients with low-risk, 13 with intermediate-1-risk, five with intermediate-2-risk, and eight with high-risk classifications). Median ages were 68.8 ± 9.0 years (range 47 to 78 years) in the MDS patient group.
Furthermore, two EDTA-blood samples of 18 patients with high-risk MDS or AML, which were included in the clinical RELAZA trial  and who were treated with azacitidine (Vidaza; Celgene Corporation, Summit, NJ, USA; 75 mg/m2/day subcutaneously on days 1–7 of one course) were received for measurements of PLA2R1 gene methylation at the beginning and end of the treatment. Finally, EDTA-blood samples of one patient (58 years old female) with AML, FAB M2, were received for serial measurements of PLA2R1 gene methylation. After allogeneic hema-topoietic stem cell transplantation (HSCT) and with minimal residual disease (MRD), the patient underwent 3 courses of azacitidine treatment (75 mg/m2/day subcutaneously on days 1–7 per one course).
All patients provided written informed consent for the following studies and the use of the patient blood samples was approved by the Ethical Board of the University Hospital of Dresden. Patient demographics and disease characteristics are summarized in Additional file 1: Table S1, Additional file 2: Table S2 and Additional file 3: Table S3.
Culture cell lines and incubation
Jurkat (human T lymphocyte acute leukemia), U937 (human hystiocytic lymphoma), Bv173 (human B-cell precursor leukemia), K-562 (human chronic myeloic leukemia in blast crisis), OC1-AML3 (human acute myeloid leukemia), KG-1A (human acute myeloid leukemia) and RPMI 8226 (human myeloma) cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Cells were cultured in standard cell medium RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. Exponentially growing cells were used in all experiments. In addition, genomic DNA from Raji (human B-cell leukemia), MCF7 (human mammary adenocarcinoma), and A431 (human melanoma) cell lines was purchased from BioCat GmbH (Heidelberg, Germany).
Jurkat and U937 leukemic cells were treated with 5-aza-2´-deoxycytidine (5-aza-dC), added to a final concentration of 1 μM. Cells were prepared at the end of the 3 day treatment period for analysis of PLA2R1- and TATA box binding protein (TBP)-specific mRNA.
Extraction of genomic DNA and RNA
Genomic DNA was isolated from Jurkat, U937, Bv173, K-562, OC1-AML3, KG-1A and RPMI 8226 cell lines, peripheral blood samples, and bone marrow aspirates using a Blood & Cell Culture DNA Mini Kit from Qiagen GmbH (Hilden, Germany) and following the manufacturer`s instructions. RNA was isolated after lysis of Jurkat and U937 cells in TRI Reagent (Sigma-Aldrich, Deisenhofen, Germany) according to the manufacturer`s instructions.
Quantitative RT-PCR analyses
Isolated RNA was converted to cDNA using the GeneAmp RNA-PCR Kit (PerkinElmer LAS GmbH, Jügesheim, Germany). For quantitative RT-PCR, portions of the reverse transcribed reaction products were then amplified for identification of PLA2R1 expression in comparison to those of TBP as a reference gene. Real-time RT-PCR was performed using Rotor-Gene Q (Qiagen GmbH) and Rotor Gene SYBR Green PCR kit according to manufacturer`s instructions. The applied primer pairs were 5`-CAG AAG AAA GGC AGT TCT GGA TTG-3` and 5`-AAA GCC ACA TCT CTG GCT CTG ATT-3´ for PLA2R1, giving PCR products with a length of 496 bp, and 5`-GAA TAT AAT CCC AAG CGG TTT G-3` and 5`-ACT TCA CAT CAC AGC TCC CC-3` for TBP amplifying products with 226 bp length. The primers were applied in a final concentration of 0.8 μM. The conditions for amplification were as follows: 40 courses at 95°C for 5 sec and 58°C for 10 sec. Amplified products were than analyzed by electrophoresis on agarose gels.
Analysis of the promoter and down-stream regions of the PLA2R1 gene
MethPrimer software (http://www.urogene.org/methprimer) was used to analyze the proximal promoter and down-stream regions -700 to +1340 bp relative to exon 1 of PLA2R1, and thereby assess the presence of 5`-CpG islands in the promoter region.
Bisulfite genomic sequencing
DNA methylation was analyzed using bisulfite-modified genomic DNA and by subsequent bidirectional sequencing. Aliquots of 500 ng of isolated genomic DNA were bisulfite modified [17–19] using the EpiTect Bisulfite Kit (Qiagen GmbH) according to manufacturer`s instructions. Three fragments expanding from −662 bp to +1275 bp relative to exon 1 were amplified by nested or semi-nested PCR. The applied primer pairs were 5`-TTT GTT GGT TAT TTG AAG GAG GA-3` and 5`-ACC ATC TAC CCA TCC CAA AA-3` as extrinsic and 5`-TAT ATT TTA GTT AGG GTT GTT TTA T-3` and 5`-TTC CTA CCT TTA AAA TAA AAA CAA A-3` as intrinsic primers for fragment 1 of PLA2R1 (−662 bp to −107 bp from exon 1), giving PCR products with a length of 556 bp; for fragment 2 (−105 bp to +783 bp from exon 1) 5`-TTT GTT TTT ATT TTA AAG GTA GGA A-3` and 5`-ACC CTA TCT CAA AAA ACA AAC AA-3` as extrinsic and 5`-TTT TTG GGA TGG GTA GAT GG-3`and 5`-ACC TAA CTT AAA AAT CAC TCC TA-3` as intrinsic primers, giving PCR products with a length of 888 bp; and for fragment 3 (+761 bp to +1275 bp from exon 1) 5`-TAG GAG TGA TTT TTA AGT TAG GT-3` and 5`-CTC TCC TCC CTC TCT TTA CA-3` as extrinsic and 5`-TAG GAG TGA TTT TTA AGT TAG GT-3` and 5`-CAA CCT TCT AAA TCT CAT ATA TAA-3` as intrinsic primers in a semi-nested PCR, amplifying products with 515 bp length.
The primers were applied to a final concentration of 0.8 μM. The conditions for amplification were as follows: 15 courses with extrinsic primers at 94°C for 30 sec, 60-50°C as touch-down for 30 sec and 72°C for 1 min followed by 30 courses with intrinsic primers at 94°C for 30 sec, 62-52°C for 30 sec and 72°C for 1 min. Buffers and reagents were from GeneAmp Kit (PerkinElmer LAS GmbH). After amplification, PCR products were subjected to agarose gel electrophoresis to establish the purity of amplificates. Five ng of PCR products were then prepared for sequencing using ABI PRISM BigDye Terminator v3.1. Samples were purified using Agencourt CleanSEQ system (Beckman/Coulter Company; MS, USA) and sequenced using 3730 XL ABI/Hitachi. Sequences of bisulfite-treated genomic DNA were compared with those of untreated genomic DNA to verify the efficiency of bisulfite treatment. Ratios of cytosine:thymine and guanine:adenine residues in the forward or reverse sequencing were respectively calculated to assess the extent of methylation at different 5`-CpG sites.
Methylation specific-high resolution melting (MS-HRM) analyses
MS-HRM analyses were carried out to quantify the extent of methylation in the distinct region −437 bp to −270 bp from exon 1 of the PLA2R1 gene. These analyses were carried using Rotor-Gene Q (Qiagen GmbH) and the EpiTect MS-HRM PCR Kit according to manufacturer`s instructions. Bisulfite modified unmethylated and methylated standard DNA (Qiagen GmbH) were mixed giving samples with 0%, 10%, 20%, 30%, 50%, and 100% methylation ratios for calibration. A standard curve with known methylation ratios was included in each run. PCR was performed in 12.5 volumes. The applied primer pairs were 5`-GGG GTA AGG AAG GTG GAG AT-3` and 5`-ACA AAC CAC CTA AAT TCT AAT AAA CAC-3` giving PCR products with a length of 168 bp. The primers were applied at a final concentration of 0.8 μM. The conditions of amplification were as follows: 40 courses at 95°C for 10 seconds, 58°C for 30 seconds and 72°C for 15 sec. Immediately after PCR, products were analyzed by high resolution melting analysis with fluorescence measured during the linear temperature transition from 50-95°C at 0.01°C/second.
Data were analyzed by two-tailed and unpaired Student`s t-test. Differences were considered significant at p < 0.05. Pearson Product Moment Correlation was applied to analyze the HRM values in relation to IPSS classification of MDS patients.