title: Hypermethylation of the FANCC and FANCL promoter regions in sporadic acute leukaemia.
abstract: OBJECTIVE
  Inactivation of the FA-BRCA pathway results in chromosomal instability. Fanconi anaemia (FA) patients have an inherited defect in this pathway and are strongly predisposed to the development of acute myeloid leukaemia (AML). Studies in sporadic cancers have shown promoter methylation of the FANCF gene in a significant proportion of various solid tumours. However, only a single leukaemic case with methylation of one of the FA-BRCA genes has been described to date, i.e. methylation of FANCF in cell line CHRF-288. We investigated the presence of aberrant methylation in 11 FA-BRCA genes in sporadic cases of leukaemia.
  
  
  METHODS
  We analyzed promoter methylation in 143 AML bone marrow samples and 97 acute lymphoblastic leukaemia (ALL) samples using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). Samples with aberrant methylation were further analyzed by bisulphite sequencing and tested for mitomycin C sensitivity using Colony Forming Units assays.
  
  
  RESULTS
  MS-MLPA showed promoter methylation of FANCC in one AML and three ALL samples, while FANCL was found methylated in one ALL sample. Bisulphite sequencing of promoter regions confirmed hypermethylation in all cases. In addition, samples with hypermethylation of either FANCC or FANCL appeared more sensitive towards mitomycin C in Colony Forming Units assays, compared to controls.
  
  
  CONCLUSIONS
  Hypermethylation of promoter regions from FA-BRCA genes does occur in sporadic leukaemia, albeit infrequently. Hypermethylation was found to result in hypersensitivity towards DNA cross-linking agents, a hallmark of the FA cellular phenotype, suggesting that these samples displayed chromosomal instability. This instability may have contributed to the occurrence of the leukaemia. In addition, this is the first report to describe hypermethylation of FANCC and FANCL. This warrants the investigation of multiple FA-BRCA genes in other malignancies.
authors.affiliation: Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands.
authors.forename: C J
authors.initials: CJ
authors.lastname: Hess
authors.forename: N
authors.initials: N
authors.lastname: Ameziane
authors.forename: G J
authors.initials: GJ
authors.lastname: Schuurhuis
authors.forename: A
authors.initials: A
authors.lastname: Errami
authors.forename: F
authors.initials: F
authors.lastname: Denkers
authors.forename: G J L
authors.initials: GJ
authors.lastname: Kaspers
authors.forename: J
authors.initials: J
authors.lastname: Cloos
authors.forename: H
authors.initials: H
authors.lastname: Joenje
authors.forename: D
authors.initials: D
authors.lastname: Reinhardt
authors.forename: G J
authors.initials: GJ
authors.lastname: Ossenkoppele
authors.forename: C M
authors.initials: CM
authors.lastname: Zwaan
authors.forename: Q
authors.initials: Q
authors.lastname: Waisfisz
mesh_terms: D000328:Adult; D001854:Bone Marrow Cells / Q000201:enzymology* / Q000473:pathology; D002648:Child; D002675:Child, Preschool; D003432:Cross-Linking Reagents / Q000627:therapeutic use; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism; D052218:Fanconi Anemia Complementation Group C Protein / Q000235:genetics*; D052240:Fanconi Anemia Complementation Group L Protein / Q000235:genetics*; D051856:Fanconi Anemia Complementation Group Proteins / Q000235:genetics; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D016685:Mitomycin / Q000627:therapeutic use; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014410:Tumor Stem Cell Assay
chemical_list: D003432:Cross-Linking Reagents; C497651:FANCC protein, human; D052218:Fanconi Anemia Complementation Group C Protein; D051856:Fanconi Anemia Complementation Group Proteins; D016685:Mitomycin; D015254:DNA Modification Methylases; C499908:FANCL protein, human; D052240:Fanconi Anemia Complementation Group L Protein