title: Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability.
abstract: OBJECTIVE
  Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting.
  
  
  METHODS
  Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls.
  
  
  RESULTS
  Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status.
  
  
  CONCLUSIONS
  Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.
authors.affiliation: School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Lovelock
authors.forename: Ee Ming
authors.initials: EM
authors.lastname: Wong
authors.forename: Carl N
authors.initials: CN
authors.lastname: Sprung
authors.forename: Anna
authors.initials: A
authors.lastname: Marsh
authors.forename: Karen
authors.initials: K
authors.lastname: Hobson
authors.forename: Juliet D
authors.initials: JD
authors.lastname: French
authors.forename: Melissa
authors.initials: M
authors.lastname: Southey
authors.forename: Tom
authors.initials: T
authors.lastname: Sculley
authors.forename: Nirmala
authors.initials: N
authors.lastname: Pandeya
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Brown
authors.forename: Georgia
authors.initials: G
authors.lastname: Chenevix-Trench
authors.forename: Amanda B
authors.initials: AB
authors.lastname: Spurdle
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McKay
mesh_terms: D045744:Cell Line, Tumor; D002470:Cell Survival; D004252:DNA Mutational Analysis; D004307:Dose-Response Relationship, Radiation; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008214:Lymphocytes / Q000528:radiation effects*; D015162:Micronucleus Tests; D009154:Mutation; D010641:Phenotype; D011839:Radiation, Ionizing; D016615:Telomere / Q000378:metabolism