Methylation analysis of the von Hippel-Lindau gene in acute myeloid leukaemia and myelodysplastic syndromes

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DNA methylation in genomic regions called CpG islands represents an important epigenetic modification in cancer. Methylation is associated with transcriptional silencing of genes that function in cell differentiation, apoptosis, growth regulation, cell signalling and tumour invasiveness, conferring selective growth advantage to the malignant cells.1 Acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) are diseases in which aberrant methylation has been described in a number of genes.2 The von Hippel-Lindau gene (VHL) is expressed in fetal and adult tissues. Expression is not restricted to the organs affected in the VHL disease, and the VHL gene is a candidate tumour suppressor. The purpose of our study was to determine whether aberrant methylation occurs in the VHL CpG island in AML and MDS.

The study population comprised 58 previously untreated patients (32 AML and 26 MDS) classified according to the WHO classification, and 10 control individuals with no haematological malignancy or solid tumour infiltration. Patients’ characteristics are shown in Table 1. The study was approved by the Institutional Review Board and by the University of Ioannina Research Committee. The presence of adequate representation of malignant cells in bone marrow samples was verified by histopathological analysis. Genomic DNA was extracted from bone marrow samples using the QIAmp DNA Minikit (Qiagen, Hilden, Germany). Bisulphite modification of genomic DNA was as described by Manning et al.3 The methylation status of the promoter region was determined using methylation-specific polymerase chain reaction (MSP) and bisulphite sequencing. MSP was performed using the primers and conditions described by Kuroki et al.4 Amplified products were resolved on 2.5% agarose gels and visualized under ultraviolet light after staining with ethidium bromide. MSP reactions were run in duplicates with positive controls (universal methylated DNA; Intergen Company, Purchase, NY, USA), negative controls from donors with no haematological malignancies and water samples with no DNA template (blanks). Primers for bisulphite sequencing were as follows:

Table 1 Characteristics of patients participating in the study

Bis F1 5′-IndexTermGAGTTTTTTTAGGTTATTTTTTGTAAT-3,′

Bis R1 5′-IndexTermTCACCCTAAATATATCCTACCTCAAAA-3′;

Bis F2 5′-IndexTermTTGAGTTAGGGAGGTTAAGGTTGTA-3,′

Bis R2 5′-IndexTermCAAAAAAATCCTCCAACACC-3′;

Bis F3 5′- IndexTermTTTTGAGGTAGGATATTAGGTGA-3,′

Bis R3 5′-IndexTermTACAACCTTAACCTCCCTAACTCAA-3.′

Purified PCR products were ligated into a TA cloning vector (Invitrogen Ltd, Paisley, UK), transformed into Top 10 Escherichia coli competent cells and the sequence of multiple clones determined by dideoxy cycle sequencing. For each case, a minimum of 10 clones were sequenced to determine the overall level of methylation within CpG islands.

We initially sought evidence of aberrant methylation within the VHL CpG island using MSP with previously recognized primers. Under these conditions, there was no evidence of methylation in any of the AML or MDS cases, regardless of the disease WHO subtype, the IPSS score or the karyotype, only unmethylated alleles being amplified from the genomic DNA of each patient (Figure 1). The sensitivity and specificity of the MSP reaction were verified by amplification of the unmethylated and methylated control DNA reactions (Figure 1). Because MSP analysis allows only a small number of individual CpG dinucleotides to be sampled, a negative result does not exclude methylation elsewhere in the CpG island. We therefore performed rigorous bisulphite sequencing analysis of two regions of the VHL CpG island in ten of the cases deemed negative by MSP. We used three sets of primer pairs that together allow analysis of the majority of the CpG island. No methylation was detected with primer pairs 1 and 3. However, with primer pair 2, which amplifies dinucleotides 1–26, there was methylation at nucleotides 7, 8, 9 and 10 of the CpG island in four of the five analysed cases (Figure 1). Methylation was not detected in genomic DNA isolated from white blood cells of healthy controls (data not shown).

Figure 1
figure1

(a) Diagrammatic map of the VHL CpG island. CpG dinucleotides are shown as vertical lines under the numbered horizontal line. Positions of primers used to amplify fragments for bisulphate sequencing are indicated. The enlarged region is that which is amplified by primer pair 2. Within this, methylated CpG sites are shown as black blocks. Five levels of methylation are indicated: 0, no black blocks; 1–25%, 1 black block; 25–50%, 2 black blocks; 50–75%, 3 black blocks; 75–100%, 4 black blocks. (b) Bisulphite sequencing trace of the region of the VHL CpG island amplified by primer pair 2. The position of the methylated CpG sites is indicated by arrows. The top trace shows unmethylated CpGs, the bottom trace shows methylated CpGs. (c) MSP analysis. MSP was performed using previously published primer pairs as described in Methods. M, methylated reaction; U, unmethylated reaction; Blk, reagent blank; Cntr, positive control DNA for the methylated VHL promoter region. 1, 2 and 3: DNA extracted from bone marrow from three patients; 4: DNA extracted from negative control. Only the band for the un-methylated promoter region was detected for samples 1,2, 3 and 4. L, molecular weight marker (100 bp ladder; Invitrogen) was included for size determination. The PCR product for the M reaction was 158 bp long and that for the unmethylated reaction was 165 bp long. MSP, methylation-specific polymerase chain reaction; VHL, von Hippel-Lindau gene.

Von Hippel-Lindau gene regulates various hypoxia-inducible genes, has hypoxia-inducible factor-dependent and hypoxia-inducible factor-independent functions, binds directly to fibronectin interacting with the extracellular matrix and regulates cell cycle. Loss of VHL expression by inactivating mutations or methylation results in upregulation of hypoxia-inducible factor with consequent overexpression of hypoxia-inducible factor's target genes involved in mitogenesis, angiogenesis, invasion, metastasis and apoptosis.5, 6 These observations imply that VHL may possess tumour suppressor properties and methylation of the VHL gene CpG island has been studied in various types of cancer. A lack of methylation has been reported in colorectal cancer, cervical cancer, testicular germ cell tumours, testicular lymphomas, multiple myeloma and acute promyelocytic leukaemia. In contrast, methylation has been described in 13% of oesophageal squamous cell carcinomas, and up to 19% of renal cancer cases.5, 4, 7, 8, 9, 10, 11, 12 Here, we have examined a well-characterized series of AML and MDS samples for evidence of VHL methylation. Although initial MSP analysis was negative, more detailed bisulphite sequencing clearly identified methylation in a small region of the CpG island. Methylation was specific for neoplasia as this area was uniformly unmethylated in normal white blood cells. As such, these changes are likely to be acquired during neoplastic transformation. From the available evidence, the effect of these epigenetic changes on basal and/or inducible expression of VHL, and on clinico-pathological parameters, cannot be determined. Studies to address these questions and to assess epigenetic change in VHL in large numbers of AML and MDS cases, using bisulphite sequencing, are in progress.

Our data illustrate the complexity of epigenetic changes in haematological malignancy. Moreover, they show that studies using only MSP to analyse methylation will inevitably fail to detect methylation restricted to specific regions of CpG islands and as such imply that methylation cannot be excluded without bisulphite sequence analysis of the entire CpG island.

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Benetatos, L., Dasoula, A., Syed, N. et al. Methylation analysis of the von Hippel-Lindau gene in acute myeloid leukaemia and myelodysplastic syndromes. Leukemia 22, 1293–1295 (2008) doi:10.1038/sj.leu.2405053

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