Aberrant hypomethylation of <i><scp>DDX</scp>43</i> promoter in myelodysplastic syndrome (2012)

The gene DDX43 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 43; also known as HAGE) was first identified as a cancer/testis antigen (CTA) gene in a human sarcoma cell line (Martelange et al, 2000) and has been found to be overexpressed in various solid cancers and haematological malignancies (Adams et al, 2002; Condomines et al, 2007; Roman-Gomez et al, 2007; Liggins et al, 2010; Mathieu et al, 2010). Recently, abnormal hypomethylation of DDX43 promoter was shown in chronic myeloid leukaemia (CML) and was associated with mRNA overexpression (Roman-Gomez et al, 2007). The present study aimed to analyse the methylation status of DDX43 promoter and its clinical and prognostic impacts in myelodysplastic syndrome (MDS) patients. Genomic DNA was isolated from bone marrow mononuclear cells (BMNCs) of 90 patients with primary MDS at diagnosis using the DNA Purification Kit (Gentra, Minneapolis, MN, USA) according to the manufacturer's instructions. Total RNA from BMNCs was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's recommendations. Samples were collected after obtaining informed consent. The diagnosis and classification of MDS were made according to the French-American-British (FAB) classification and the 2008 revision of the World Health Organization classification (Vardiman et al, 2009). Clinical parameters of all patients are provided in Table 1. Follow-up data were obtained for 78 patients. Genomic DNA and total RNA from 24 cases with iron deficiency anaemia (IDA) were used as controls. Genomic DNA (1 μg) was modified according to the manufacturer's protocol using the CpGenome™ DNA Modification kit (Chemicon, Ternecula, CA, USA). Amplification The DDX43 unmethylated sequence was amplified by methylation-specific real-time quantitative polymerase chain reaction (RQ-MSP) using the primers reported previously (Roman-Gomez et al, 2007). The relative quantification of unmethylated DDX43 sequence was calculated in relation to the reference Alu sequence (Qian et al, 2011). To further determine the comprehensive methylation status of DDX43 promoter and exon 1, bisulfite-modified DNA sequencing PCR was performed using the specific primers 5′-TTTTTTTTTGGAATAATGTTTTATTA-3′(forward) and 5′-TAACCCCACCTAT CCTACCCTAC-3′(reverse) (Roman-Gomez et al, 2007). PCR conditions were 95°C for 4 min, 40 cycles for 45 s at 94°C, 30 s at 57·2°C, 30 s at 72°C, followed by a melting program at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s, and 60°C for 15 s. The 307-bp PCR products, which covered 21 CpG sites (+12 bp~+250 bp) within a CpG-rich region located in DDX43 promoter and exon 1, were cloned into pMD®.19-T Vector (TaKaRa, Dalian, China). Ten independent clones of each sample were sequenced (Sangon, Shanghai, China). Two micro-gram of total RNA was reverse-transcribed into cDNA using 10 μmol/l of random primers, 200 units of MMLV reverse transcriptase, 0·5 mmol/l of dNTPs, 10 mmol/l of dithiothreitol, and 25 units of RNase inhibitor. Real-time quantitative PCR (RQ-PCR) was performed to detect the expression of DDX43 transcript on a 7300 Thermo cycler (Applied Biosystems, Foster City, CA, USA), using 100 ng of cDNA in a 25 μl reaction volume with 0·2 mmol/l of dNTP, 4 mmol/l of MgCl2, 0·4 μmol/l of primers (Roman-Gomez et al, 2007), 1·2 μl of EvaGreen, and 1·0 U of Taq DNA Polymerase (MBI Fermentas, Amherst, NY, USA). The mRNA abundance of DDX43 gene was calculated relative to the expression of the housekeeping gene, ABL1. 23 IDA bone marrow specimens displayed slight hypomethylation of DDX43, which was confirmed by the presence of specific unmethylated products in the melting curves. Nunmethylation-DDX43 ratio of all controls was 0–100% (31·78 ± 27·00%). Thus, a Nunmethylation-DDX43 ratio equal to or above 112·78% (determined as the mean + 3 standard deviations) was chosen to define the DDX43 hypomethylation in MDS samples. Hypomethylation of DDX43 promoter was observed in 27 of 90 (30%) primary MDS patients (Table 1). The RQ-MSP results were confirmed by evaluating the methylation density of DDX43 promoter in five samples from one control, two DDX43-hypermethylated and two DDX43-hypomethylated MDS according to RQ-MSP. The results revealed the high relevance between RQ-MSP and bisulfite DNA sequencing (Fig S1A and S1B). We also examined DDX43 expression in 27 MDS patients with available mRNA. A significantly positive correlation was observed between the level of DDX43 transcript and the level of DDX43 hypomethylation (R = 0·520, P = 0·005). Patients with DDX43 hypomethylation (n = 2) had significantly higher DDX43 expression than those with DDX43 methylation and than controls (P = 0·001 and 0·025, respectively). Haemoglobin level was the only clinical characteristic that showed a significant difference between patients with and without DDX43 methylation (Table 1). Aberrant hypomethylation of DDX43 promoter was observed in each subtype of the FAB, International Prognosis Scoring System (IPSS) and WHO classifications. There was no significant difference in hypomethylation incidence among FAB, IPSS or WHO subtypes (P > 0·05) (Table 1). The estimated 50% overall survival (OS) of all patients was 21 months. Although the patients with DDX43 hypomethylation had a better OS (median 24 months, 95% confidence interval [CI] 5·9–42·1 months) than those with DDX43 methylation (median 14 months, 95% CI 5·9–22·1 months), the difference was not statistically significant (χ2 = 2·664, P = 0·103, Fig 1A). Among the subset of patients with normal karyotypes, however, DDX43-hypomethylated patients showed a significantly longer OS (median not obtained) than those with DDX43-methylation (median 16 months, CI 4·9–27·1 months) (χ2 = 3·906, P = 0·048, Fig 1B). Roman-Gomez et al (2007) reported that DDX43 hypomethylation was associated with disease progression and poor outcome in CML. However, our results showed that the aberrant hypomethylation of DDX43 promoter was frequently observed in every subtype of MDS and was a favourable prognostic factor, which suggests that DDX43 may play a different role in different diseases. Somatic mutations of three genes regulating cytosine methylation, including isocitrate dehydrogenase 1 (IDH1, R132), IDH2 (R140 and R172), and DNA methyltransferase 3 alpha (DNMT3A), were recently analysed in MDS (Lin et al, 2011, 2012). 4 DNMT3A mutations (3 R882H and 1 R882C) and 5 IDH1/IDH2 mutations (1 IDH1 R132H and 4 IDH2 R140Q) were identified. The association between the methylation status of DDX43 promoter and the mutations in three methylation modifiers was analysed. However, no significant difference was observed in DDX43 hypomethylation between patients with and without IDH1/IDH2 or DNMT3A mutations (Table 1). Further analysis also did not find an association of DDX43 hypomethylation with DH1/IDH2 or DNMT3A mutations in patients with normal karyotypes. In conclusion, our study shows for the first time that the DDX43 promoter is frequently hypomethylated in MDS and DDX43 hypomethylation is associated with favourable outcome in cytogenetically normal MDS. This study was supported by Natural Science foundation of Jiangsu Province (BK2009206), 333 Project of Jiangsu Province (BRA2011085), and Social Development Foundation of Zhenjiang (SH2010015).