title: The Icelandic founder mutation BRCA2 999del5: analysis of expression.
abstract: BACKGROUND
  A founder mutation in the BRCA2 gene (BRCA2 999del5) accounts for 7-8% of female breast cancers and for 40% of male breast cancers in Iceland. If expressed, the mutant gene would encode a protein consisting of the first 256 amino acids of the BRCA2 protein. The purpose of this study was to determine whether this mutant protein is produced in heterozygous individuals and, if so, what might be the functional consequences of mutant protein production.
  
  
  METHODS
  The presence of BRCA2 999del5 transcripts in fibroblasts from heterozygous individuals was assayed by cDNA synthesis and sequencing. The potential protein-coding portion of BRCA2 999del5 was cloned into the pIND(SP1)/V5-His vector and expressed in COS7 cells. The presence of the mutant protein in cell lysates from heterozygous fibroblasts and from COS7 cells was tested by a number of methods including immunoprecipitation, affinity purification with nickel-coated agarose beads, Western blotting and ELISA, using antibodies to the N-terminal end of BRCA2, antiserum specific for the 16 nonrelevant amino acids at the carboxyl end and antibodies to fusion partners of recombinant proteins.
  
  
  RESULTS
  The frequency of the BRCA2 999del5 transcript in heterozygous fibroblasts was about one-fifth of the wild-type transcript; however, no mutant protein could be detected. Overexpression of BRCA2 999del5 mRNA in COS7 cells failed to produce a mutant protein unless degradation by proteasomes was blocked.
  
  
  CONCLUSIONS
  Our results show that the protein product of BRCA2 999del5 is extremely unstable. Therefore, an increase in breast cancer risk in BRCA2 999del5 carriers is due to haploinsufficiency at the BRCA2 locus.
authors.affiliation: Molecular and Cell Biology Laboratory, The Icelandic Cancer Society, Reykjavik, Iceland. sjenia@yahoo.com
authors.forename: Evgenia K
authors.initials: EK
authors.lastname: Mikaelsdottir
authors.forename: Sigridur
authors.initials: S
authors.lastname: Valgeirsdottir
authors.forename: Jorunn E
authors.initials: JE
authors.lastname: Eyfjord
authors.forename: Thorunn
authors.initials: T
authors.lastname: Rafnar
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D024682:BRCA2 Protein / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D018703:Founder Effect; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D024522:Genes, BRCA2; D006579:Heterozygote; D006801:Humans; D007055:Iceland; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009154:Mutation / Q000235:genetics*; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000096:biosynthesis; D017384:Sequence Deletion / Q000235:genetics*; D014157:Transcription Factors
chemical_list: D024682:BRCA2 Protein; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014157:Transcription Factors; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine