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. 2013 Sep 27:3:2789.
doi: 10.1038/srep02789.

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

Lasse Sommer Kristensen  1 , Marianne Bach Treppendahl, Fazila Asmar, Mia Seremet Girkov, Helene Myrtue Nielsen, Tina Ellegaard Kjeldsen, Elisabeth Ralfkiaer, Lise Lotte Hansen, Kirsten Grønbæk
Affiliations

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

Lasse Sommer Kristensen et al. Sci Rep. .

Abstract

The tumor suppressor genes MGMT and DAPK1 become methylated in several cancers including diffuse large B-cell lymphoma (DLBCL). However, allelic methylation patterns have not been investigated in DLBCL. We developed a fast and cost-efficient method for the analysis of allelic methylation based on pyrosequencing of methylation specific PCR (MSP) products including a SNP. Allelic methylation patterns were reliably analyzed in standards of known allelic methylation status even when diluted in unmethylated DNA to below 1% methylation. When studying 148 DLBCL patients MGMT and DAPK1 methylation was observed in 19% and 89%, respectively, and among methylated and heterozygous patients 29% and 55%, respectively, were biallelically methylated. An association between the T-allele of the rs16906252 SNP and MGMT methylation was observed (p-value=0.04), and DAPK1 methylation of the A-allele was associated with shorter overall survival (p-value=0.006). In future cancer research allelic MSP-pyrosequencing may be used to study a wide range of other loci.

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Figures

Figure 1
Figure 1. The MGMT allelic MSP-pyrosequencing assay.
The non-CpG C position analyzed is highlighted in yellow. The antisense strand was analyzed. (A) Allelic MSP-pyrosequencing on a standard having both alleles methylated. (B) Allelic MSP-pyrosequencing on a standard having only the T-allele methylated. (C) Allelic MSP-pyrosequencing on a standard having only the C-allele methylated.
Figure 2
Figure 2. The sensitivity of the allelic MSP-pyrosequencing assays.
The standards having both alleles methylated were serially diluted into unmethylated DNA and tested using the allelic MSP-pyrosequencing assays. The experiments were repeated three times for each of the assays. It can be observed that a signal from both alleles above 25% can be detected down to 0.675% methylation. For MGMT the antisense strand was analyzed. (A) The MGMT assay (B) The DAPK assay.
Figure 3
Figure 3. Allelic MGMT methylation analysis in DLBCL samples.
The non-CpG C position analyzed is highlighted in yellow. The antisense strand was analyzed. (A) Patient ID 23 was methylated at both alleles. (B) Patient ID 32 was methylated at the T-allele. (C) Patient ID 56 was methylated at the C-allele.
Figure 4
Figure 4. Bisulfite sequencing of single clones for patient ID 23, 32, and 56.
ND is no data (A) For patient ID 23, heavily methylated C- and T-alleles could be observed. (B) For patient ID 32, most of the clones successfully sequenced were unmethylated C-alleles, while one heavily methylated T-allele was observed. Part of the sequencing trace for this allele is shown, and the SNP position is underlined. (C) For patient ID 56, most of the sequenced clones were unmethylated T-alleles, while one heavily methylated C-allele was observed.
Figure 5
Figure 5. Overall survival after frontline R-CHOP treatment according to MGMT methylation status and rs16906252 genotypes.
(A) Overall survival according to MGMT methylation status. (B) Overall survival according to rs16906252 genotype.
Figure 6
Figure 6. Overall survival after frontline R-CHOP treatment according to DAPK1 methylation status, rs13300553 genotypes, and allelic DAPK methylation patterns.
(A) Overall survival according to DAPK1 methylation. (B) Overall survival according to rs13300553 genotypes. (C) Overall survival according to allelic DAPK1 methylation patterns in individuals heterozygous for rs13300553.
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References

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