Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability.
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.
Lovelock
Paul K
PK
School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia.
Wong
Ee Ming
EM
Sprung
Carl N
CN
Marsh
Anna
A
Hobson
Karen
K
French
Juliet D
JD
Southey
Melissa
M
Sculley
Tom
T
Pandeya
Nirmala
N
Brown
Melissa A
MA
Chenevix-Trench
Georgia
G
Spurdle
Amanda B
AB
McKay
Michael J
MJ
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