Qin Chen

Recurrent DNMT3A R882 Mutations in Chinese Patients with Acute Myeloid Leukemia and Myelodysplastic Syndrome

Somatic mutations of DNMT3A gene have recently been reported in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We examined the entire coding sequences of DNMT3A gene by high-resolution melting analysis and sequencing in Chinese patients with myeloid malignancies. R882 mutations were found in 12/182 AML and in 4/51 MDS, but not in either 79 chronic myeloid leukemia (CML), or 57 myeloproliferative neoplasms (MPNs), or 4 chronic monomyelocytic leukemia. No other DNMT3A mutations were detected in all patients. R882 mutations were associated with old age and more frequently present in monoblastic leukemia (M4 and M5, 7/52) compared to other subtypes (5/130). Furthermore, 14/16 (86.6%) R882 mutations were observed in patients with normal karyotypes. The overall survival of mutated MDS patients was shorter than those without mutation (median 9 and 25 months, respectively). We conclude that DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS, and may be an adverse prognostic event in MDS.

IDH1 and IDH2 mutation analysis in Chinese patients with acute myeloid leukemia and myelodysplastic syndrome

The somatic mutations of isocitrate dehydrogenase genes (IDH1 and IDH2) have been identified in a proportion of hematologic malignancies. We examined IDH1 R132 and IDH2 R140/R172 mutations by high resolution melting analysis and direct sequencing in Chinese patients with different myeloid malignancies including 198 acute myeloid leukemia (AML), 82 myelodysplastic syndrome (MDS), 85 chronic myeloid leukemia, and 57 myeloproliferative neoplasms. IDH1 and IDH2 mutations were found in four (2.0%) and ten (5.0%) AML and in two (2.4%) and three (3.6%) MDS cases, but not in other patients. IDH1 and IDH2 mutations were heterozygous and mutually exclusive. IDH1/2 mutations were significantly more frequently observed in cytogenetically normal AML or MDS compared to those without mutations. There was no difference in overall survival of both AML and MDS patients with or without IDH1/2 mutations (P = 0.177 and 0.407, respectively). In conclusion, IDH1/2 mutations are recurrent but rare molecular aberrations in Chinese AML and MDS.

Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform

BACKGROUND Detection of the JAK2 mutation has recently been included under the essential diagnostic criteria for myeloproliferative neoplasm (MPN). High-resolution melt (HRM) curve analysis, a nongel-based, automated system, is introduced as a means of mutation scanning without the requirement of any post-PCR handling. METHODS We studied the sensitivity and reproducibility of LightScanner™ platform in the detection of JAK2 V617F mutation and the availability for diagnostic use in MPN. RESULTS The reproducible sensitivity of HRM analysis with LightScanner™ platform was 5% for the detection of JAK2 V617F mutation. In the test of blind screening of 105 samples (48 Ph- MPN and 57 Ph+ chronic myeloid leukemia), the identical judgement was interpreted by two blinded investigators. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing. CONCLUSIONS The HRM method developed here is an extremely sensitive, accurate and reliable technique and allows high-throughput, fast pre-screening to select for sequencing only those specimens that most likely contain mutant JAK2 V617F allele(s).

Abnormal methylation of GRAF promoter Chinese patients with acute myeloid leukemia

The epigenetic disturbances are recognized as an alternative mechanism contributing to the pathogenesis of acute myeloid leukemia (AML). GTPase regulator associated with focal adhesion kinase (GRAF), a putative tumor suppressor gene, was revealed with mutations and promoter methylation in AML and myelodysplastic syndrome. In this study, we investigated the methylation status of GRAF promoter in Chinese AML patients. Aberrant methylation of GRAF promoter was detected in 66.7% (88/132) of the cases analyzed. The methylation of GRAF gene could be detected in all FAB subtypes and in all cytogenetic risk groups. There were no significant differences in clinical features, FAB subtypes and cytogenetic risk groups between patients with and without GRAF methylation. GRAF transcript was significantly lower in AML group compared to controls (3.30 vs 56.06, P<0.001). Both patients with methylated GRAF gene and those without methylated GRAF gene had significantly lower GRAF transcript than controls (P<0.001). Furthermore, GRAF transcript was significantly lower in patients with methylated GRAF than those without methylated GRAF (1.64 vs 6.42, P=0.005). These findings suggest that the hypermethylation of GRAF promoter might be one of early events in the development of AML.

Rapid and reliable detection of IDH1 R132 mutations in acute myeloid leukemia using high-resolution melting curve analysis

OBJECTIVE The mutations of isocitrate dehydrogenase 1 (IDH1) gene have been identified in a proportion of hematologic malignancies including acute myeloid leukemia (AML). The aim of the present study was to explore the reliability of the high-resolution melting analysis (HRMA) for the identification of IDH1 R132 mutations in AML. DESIGNS AND METHODS We evaluated the sensitivity of HRMA in the detection of IDH1 R132 mutation and screened IDH1 mutations in 110 AML patients using HRMA. The results of HRMA were validated by direct DNA sequencing. RESULTS The reproducible sensitivity of HRMA was 5% for the detection of IDH1 R132 mutation, higher than 10% of direct DNA sequencing. Heterozygous IDH1 mutations were identified in 4 (3.6%) AML cases, which were R132H in 3 cases and R132S in 1 case confirmed by DNA sequencing. CONCLUSION The HRMA is a rapid, accurate, reliable, high-throughput method to screen IDH1 gene mutations.

Aberrant hypomethylation of SALL4 gene in patients with myelodysplastic syndrome

The abnormalities of SALL4 gene, which encodes a zinc-finger transcription factor and is essential for developmental events, have been found to be involved in tumorigenesis. In this study, we investigated the methylation status of the CpG island of SALL4 promoter region in myelodysplastic syndrome (MDS) using methylation-specific PCR (MSP). Aberrant hypomethylation of SALL4 gene was found in 21.7% (18/83) of the cases analyzed. A significantly positive correlation was identified between the level of SALL4 transcript and the status of SALL4 hypomethylation (R=0.641, P<0.001). No correlation was found between SALL4 hypomethylation and clinical parameters. However, the frequency of SALL4 hypomethylation significantly increased in higher risk MDS (14% in Low/Int-1 versus 39% in Int-2/High, P=0.031). The association between SALL4 hypomethylation and the mutations in three methylation modifiers (IDH1, IDH2 and DNMT3A) was not observed. Although the estimated 50% survival time of the SALL4-hypomethylated group was shorter than that of SALL4-methylated group (11.0 months vs. 20.0 months), the difference was not statistically significant (P=0.430). These findings suggest that hypomethylation of SALL4 promoter is a common event in MDS.

Aberrant hypomethylation of SALL4 gene is associated with intermediate and poor karyotypes in acute myeloid leukemia

OBJECTIVE SALL4 gene has been identified to stimulate the expansion of hematopoietic stem cell (HSCs) and enhance the self-renewal of HSCs. Overexpression of SALL4 has been found in several cancers. The present study was aimed to investigate the methylation status of SALL4 promoter region in acute myeloid leukemia (AML). DESIGNS AND METHODS The methylation status of SALL4 promoter was analyzed in 84 patients with AML using methylation-specific PCR (MSP) and its clinical significance was evaluated. RESULTS Aberrant hypomethylation of SALL4 gene, which was correlated with SALL4 expression, was found in 17.8% (15/84) cases. The patients with SALL4 hypomethylation had significantly older age and higher WBCs than those without SALL4 hypomethylation. The incidence of SALL4 hypomethylation was higher in M1 subtype than in M2 and other subtypes (50%, 26% and 6%, respectively, P=0.001). SALL4 hypomethylation was associated with cytogenetically intermediate and poor groups. Although survival time of the SALL4-hypomethylated AML was shorter than that of SALL4-methylated group (4 months vs 9 months), the difference was not statistically significant (P=0.356). CONCLUSIONS Hypomethylation of SALL4 promoter is a common event and is associated with the intermediate and poor karyotypes in AML.

Development of a high-resolution melting analysis for the detection of the SF3B1 mutations.

SF3B1, located on chromosome 2q33.1, encodes a core component of RNA-splicing machinery, and its mutation has been described in myelodysplastic syndromes (MDS) characterized with ring sideroblasts (RS). To explore the reliability and sensitivity of the high-resolution melting analysis (HRMA) technique for the identification of the SF3B1 mutations, mutations in 92 patients with MDS were detected in this study. The sensitivity could reach 5%, obviously higher than the 25% of direct DNA sequencing. A low frequency (5.4%) of SF3B1 mutations were identified in patients with MDS, including three cases of K700E, one case of H662Q, and one case of K666M. Further, SF3B1 mutations were more frequently recurrent in the 33% of patients with MDS characterized with RS, whereas in other subtypes of MDS, only 2.3% of patients were detected with SF3B1 mutations (p=0.006). In conclusion, a rapid, reproducible, sensitive, and high-throughput HRMA assay has been established for the scanning of SF3B1 mutations.

The Methylation Status of the DDX43 Promoter in Chinese Patients with Chronic Myeloid Leukemia

Aberrant DNA methylation is a common epigenetic alteration and an important feature in human cancers. The DEAD box polypeptide 43 (DDX43) has been found to be overexpressed in various solid tumors and some hematologic malignancies. In the present study, we investigated the methylation status of the DDX43 promoter in 87 Chinese patients with chronic myeloid leukemia (CML) using real-time quantitative methylation-specific polymerase chain reaction and examined the DDX43 transcript in 35 patients using real-time quantitative polymerase chain reaction. DDX43 promoter hypomethylation was observed in 22 (25.3%) CML patients. No significant correlation was found between the hypomethylation of the DDX43 promoter with the age, sex, white blood cell counts, hemoglobin concentration, platelet counts, and chromosomal abnormalities of CML patients (p>0.05). The frequency of DDX43 hypomethylation in patients in the chronic phase, in the accelerated phase, and in blast crisis was 23.4% (15/64), 25.0% (2/8), and 33.3% (5/15), respectively (p>0.05). There was a significant correlation between DDX43 hypomethylation and DDX43 transcript (r=0.469, p=0.004). Our data suggest that hypomethylation of the DDX43 promoter may be an early and frequent molecular event in the development of CML in Chinese patients.

Aberrant hypomethylation of DDX43 promoter in myelodysplastic syndrome

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 I. 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 lg) 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) (RomanGomez 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 pMD19-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 lmol/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 ll reaction volume with 0·2 mmol/l of dNTP, 4 mmol/l of MgCl2, 0·4 lmol/l of primers (Roman-Gomez et al, 2007), 1·2 ll 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 I). 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 I). Aberrant hypomethylation of DDX43 promoter was observed in each subtype of Correspondence