Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing.
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.
Department of Hematology, Rigshospitalet, Copenhagen, Denmark.
Lasse Sommer
LS
Kristensen
Marianne Bach
MB
Treppendahl
Fazila
F
Asmar
Mia Seremet
MS
Girkov
Helene Myrtue
HM
Nielsen
Tina Ellegaard
TE
Kjeldsen
Elisabeth
E
Ralfkiaer
Lise Lotte
LL
Hansen
Kirsten
K
Grønbæk
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D058846:Antibodies, Monoclonal, Murine-Derived; D000971:Antineoplastic Combined Chemotherapy Protocols; D002460:Cell Line; D003520:Cyclophosphamide; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D064550:Death-Associated Protein Kinases / Q000235:genetics*; D004317:Doxorubicin; D005260:Female; D005838:Genotype; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000188:drug therapy / Q000235:genetics* / Q000401:mortality / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D020641:Polymorphism, Single Nucleotide; D011241:Prednisone; D000069283:Rituximab; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D016896:Treatment Outcome; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D014750:Vincristine; D055815:Young Adult
D058846:Antibodies, Monoclonal, Murine-Derived; C571759:R-CHOP protocol; D025521:Tumor Suppressor Proteins; D000069283:Rituximab; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; D015254:DNA Modification Methylases; C515491:MGMT protein, human; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D045643:DNA Repair Enzymes; D011241:Prednisone