title: Frequent but borderline methylation of p16 (INK4a) and TIMP3 in medulloblastoma and sPNET revealed by quantitative analyses.
abstract: Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 (INK4A), p14 (ARF), TIMP3, CDH1, p15 (INK4B )and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 (INK4A) (17/43), p14 (ARF) (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 (INK4A )and TIMP3 were methylated consistently in medulloblastomas (p16 (INK4A ) 14%, TIMP3 11%) and p16 (INK4A) also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1-5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital Muenster, 48129 Muenster, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Mühlisch
authors.forename: T
authors.initials: T
authors.lastname: Bajanowski
authors.forename: C H
authors.initials: CH
authors.lastname: Rickert
authors.forename: W
authors.initials: W
authors.lastname: Roggendorf
authors.forename: G
authors.initials: G
authors.lastname: Würthwein
authors.forename: H
authors.initials: H
authors.lastname: Jürgens
authors.forename: M C
authors.initials: MC
authors.lastname: Frühwald
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D002528:Cerebellar Neoplasms / Q000235:genetics*; D002648:Child; D002675:Child, Preschool; D019175:DNA Methylation; D064550:Death-Associated Protein Kinases; D005260:Female; D020868:Gene Silencing; D019942:Genes, p16; D006801:Humans; D007223:Infant; D008297:Male; D008527:Medulloblastoma / Q000235:genetics*; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics; D018242:Neuroectodermal Tumors, Primitive / Q000235:genetics*; D011971:Receptors, Immunologic / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics*; D000097905:Roundabout Proteins
chemical_list: D051017:Apoptosis Regulatory Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D064550:Death-Associated Protein Kinases; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D019717:Tissue Inhibitor of Metalloproteinase-3; D000097905:Roundabout Proteins; C088390:TIMP3 protein, human; C579096:DAPK1 protein, human