Tumour characterisation and revised multifactorial analysis
Patient recruitment and consent
As described previously [5], pedigrees with UVs in BRCA1 and BRCA2 were ascertained by the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) according to eligibility criteria established by the organisation [15, 16]. With informed consent from participants, breast tumour sections from archival pathology specimens were recalled for research studies. This research study was approved by the human research ethics committees of the Peter MacCallum Cancer Centre, the Queensland Institute of Medical Research, and the University of Queensland.
Pathology review
Criteria for classifying tumours as 'BRCA1-like' or 'not BRCA1-like' have been described previously [5].
Tumour immunohistochemistry
Oestrogen receptor (ER), cytokeratin 5/6 (CK5/6), and cytokeratin 14 (CK14) immunohistochemistry was carried out as described previously [12].
Tumour microsatellite instability
Ten microsatellite markers (BAT25, BAT26, BAT40, BAT34C4, D5S346, D17S250, ACTC, D18S55, D10S197, and MYCL) were analysed for microsatellite instability (MSI) status according to a previously established protocol [17]. Tumour tissue was compared with normal tissue. Tumourclassification was as follows: MSI-high if three or more markers demonstrated instability, MSI-low if one or two markers demonstrated MSI, and MSI-stable if no marker exhibited MSI.
Prior probability of pathogenicity from amino acid conservation, and location of the mutation in specific known functional domains
Missense substitutions and in-frame deletions were classified according to their location within one of two recognised functional domains of the proteins, the C terminus region containing the BRCA1 BRCT repeats, defined loosely as amino acids 1,396 to 1,862, and the BRCA2 DNA-binding domain (amino acids 2,500 to 3,098). Variants were also categorised according to whether the wild-type residue involved in the substitution/deletion was evolutionarily conserved through to the pufferfish Tetraodon, using multiple sequence alignments available on the Web site [18]. Heterogeneity analysis of 1,433 variants in the Myriad Genetic Laboratories, Inc. (Salt Lake City, UT, USA) database was used to estimate the proportion of Myriad deleterious variants in three classifications [19]: (a) invariant position in BRCA1 C terminus domain/BRCA2 DNA-binding domain (BRCT/DBD) domain, proportion = 0.73; (b) variable position in BRCT/DBD domain, proportion = 0.08; and (c) position outside of BRCT/DBD domain, proportion = 0.02. The values were then used as prior probabilities of being deleterious for the classification of the studied variants. All variants in this study fell within the BRCA1 BRCT domain.
Co-occurrence with pathogenic mutations
We queried the Myriad Genetic Laboratories, Inc. database of approximately 100,000 full-sequence tests to determine the number of times a UV was observed, and the number of different deleterious mutations observed to co-occur with each variant, as a measure of the number of times the UV is seen in trans with a deleterious mutation. Phase of the variant and mutation was established for a subset of individuals. Observations for variants were excluded if in cis with a mutation, included if in trans with a mutation, and assumed to be in trans with at least n - 1 observations for n observations with different deleterious mutations of unknown phase.
Histopathology
Available invasive tumour sections were analysed for parameters recognised to be associated with BRCA1 mutation status [12, 20, 21]. Immunohistochemistry scoring was performed as described previously [12]. Scoring was performed by a single pathologist (SRL).
Pedigree causality analysis
Bayes factor analysis of families was performed as described previously and incorporated no information additional to that published previously [3, 5].
Derivation of probabilities and multifactorial likelihood scoring
Probabilities were derived for each of the components included in the study, under the assumption that each factor was independent. For the co-occurrence component, we estimated the likelihood that any given UV was causal, as described previously [4]. The Bayes factor was included directly as a likelihood ratio (LR) score for the pedigree analysis component. Tumour expression of ER, CK5/6, and CK14 was used for calculating histopathological LR scores, based on the previously reported prevalence of the combined immunotypes of these independent predictors of BRCA1 mutation status in breast tumours [12]. The likelihoods for causality were ER-positive (irrespective of cytokeratin score) = 0.14:1; negative for all three markers = 0.87:1; ER-negative, CK14-negative, CK5/6-positive = 5.6:1; ER-negative, CK14-positive, CK5/6-negative = 2.6:1; and ER-negative, CK14-positive, CK5/6-positive = 27.4:1. For the single ER-negative grade 3 tumour with insufficient material available for cytokeratin analysis, the likelihood was calculated based on ER expression and grade (LR 2.95:1), as described previously [5].
The individual LRs were multiplied to calculate an overall multifactorial LR, assuming statistical independence of the sources of information. Bayes rule was then used to calculate a posterior probability that the variant was deleterious from the multifactorial LR and the prior probability as determined by sequence alignment.
Functional analysis
BRCA1cDNA plasmids
For the transcriptional activation assays, the pGal4B vector and pGal4B vector containing a cDNA sequence encoding C-terminal residues 1,528 to 1,863 (571 amino acids) of BRCA1 containing TADs 1 and 2 [22] were kindly donated by Jane Visvader (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia). For generating control templates for single nucleotide primer extension (SNuPE) assays and for the cytoplasmic localisation and centrosome amplification assays, a pZeoSV plasmid containing the full-length BRCA1 cDNA with or without UV [3] was used.
Generation of mutagenised BRCA1cDNA plasmids
Mutations in BRCA1 cDNA plasmids were introduced using a polymerase chain reaction (PCR)-mediated mutagenesis protocol as described previously [3] using Pfu Turbo Taq polymerase (Invitrogen Corporation, Carlsbad, CA, USA) and the following primers incorporating the appropriate sequence change (underlined, bold): BRCA1 1699Q forward 5'-gat gct gag ttt gtg tgt gaa cag aca ctg aaa tat ttt cta gg-3', BRCA1 1699Q reverse 5'-cct aga aaa tat ttc agt gtc tgt tca cac aca aac tca gca tc-3', BRCA1 1708V forward 5'-ctg aaa tat ttt cta gga att gtg gga gga aaa tgg gta gtt ag-3', BRCA1 1708V reverse 5'-cta act acc cat ttt cct ccc aca att cct aga aaa tat ttc ag-3', BRCA1 1738R forward 5'-gag cat gat ttt gaa gtc aga aga gat gtg gtc aat gga aga aac-3', and BRCA1 1738R reverse 5'-gtt tct tcc att gac cac atc tct tct gac ttc aaa atc atg ctc-3'. Mutagenic primers used to generate the BRCA1 1708E variant are described elsewhere [3]. Mutagenised clones were confirmed by sequencing, and large-scale DNA preps were made using commercial preparation kits (Qiagen Inc., Valencia, CA, USA).
Cell culture
Lymphoblastoid cell lines (LCLs) were grown in RPMI with 10% foetal calf serum (FCS) and antibiotic/antimycotic (Gibco-BRL, now part of Invitrogen Corporation). 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS and antibiotic/antimycotic. T47D cells were cultured in RPMI media with 10% FCS, 10 μg/mL insulin, and antibiotic/antimycotic. All cells were incubated at 37°C in 5% CO2.
RNA extraction from cell lines
RNA was extracted from cell lines using Trizol Reagent (Invitrogen Corporation) according to the manufacturer's instructions and DNAse treated using the Ambion DNA-Free kit (Ambion, Inc., Austin, TX, USA). cDNA was made from RNA using the Invitrogen Superscript III Reverse Transcriptase kit according to the manufacturer's instructions and used directly in PCR.
Single nucleotide primer extension assays
Templates for the SNuPE assay were generated by reverse transcription-PCR of RNA extracted from LCLs and PCR of the matching control plasmids (wild-type and mutagenised BRCA1) using either of the primers described previously [3] for the R1699Q, A1708V, and G1738R variants. PCR products from wild-type and mutagenised BRCA1 control templates and LCL cDNAs were then used in the SNuPE assay as described previously [3] using the following primers: SNuPE1699For 5'-gat gct gag ttt gtg tgt gaa c-3', SNuPE1699Rev 5'-cct aga aaa tat ttc agt gtc-3', SNuPE1708F 5'-ctg aaa tat ttt cta gga att g-3', SNuPE1708R 5'-gct aac tac cca ttt tcc tcc c-3', SNuPE 1738F 5'-gag cat gat ttt gaa gtc aga-3', and SNuPE 1738R 5'-ttc ttc cat tga cca cat ctc-3'. Radiolabelled products were resolved on a denaturing polyacrylamide gel and visualised by autoradiography, as described previously [3].
Tryptic digestion profiles
Plasmids carrying the mutagenised BRCA1 cDNA or wild-type BRCA1 cDNA (1,571 base-pair product encompassing exons 12 to 24) were used as templates to generate PCR products with primers described previously [3] for the R1699Q, A1708V, and G1738R variants. The PCR products were transcribed and translated in vitro using the Promega TNT Coupled Reticulocyte Lysate System and sulfur-35 L-methionine (PerkinElmer, Melbourne, Australia) according to the manufacturer's instructions. Protein products were digested in increasing concentrations of trypsin and resolved on a 14% acrylamide gel. Products were visualised using autoradiography or the Typhoon™ Phosphorimaging system (Amersham Biosciences, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Foci formation assays
MCF-7 human breast cancer cells were maintained in DMEM supplemented with 10% FCS and grown at 37°C in a humidified 5% CO2 atmosphere. Cells were seeded onto sterile glass coverslips and transfected at 50% to 60% confluency with 2 to 5 μg of plasmid DNA using Lipofectamine Reagent (Invitrogen Corporation) according to the manufacturer's instructions. At 6 hours after transfection, the transfection mix was removed and replaced with DMEM containing 10% FCS. At 44 hours after transfection, cells were either left untreated or exposed to 15 Gy of radiation from a cesium-137 source (Gammacell 1000 irradiator; Atomic Energy of Canada Limited, Mississauga, ON, Canada) and then allowed to recover at 37°C for 4 hours. Cells were fixed in 3.7% formalin/PBS for 15 minutes, permeabilised in 0.2% Triton-PBS for 10 minutes, and processed for immunostaining. Myc-tagged ectopic BRCA1 was detected by immunofluorescence using the anti-Myc rabbit polyclonal antibody A-14 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Myc antibody was detected with anti-rabbit Alexa Fluor 594 (Invitrogen Corporation). YFP-BARD1 was co-transfected to ensure nuclear localisation of BRCA1 isoforms. Cell nuclei were counterstained with the chromosome dye Hoechst 33285 (Sigma-Aldrich, St. Louis, MO, USA). The intranuclear foci localisation of each ectopic protein was determined by scoring cells using an Olympus BX40 epifluorescence microscope (Olympus, Tokyo, Japan), and the proportion of cells displaying 0, 1 to 10, or greater than 10 nuclear foci per cell was determined as previously described [7]. Digital images were collected using a SPOT camera. P values were determined using two-tailed t tests.
Centrosome amplification assays
293T cells were cultured on glass coverslips and transfected with Myc-BRCA1 wild-type and mutant constructs using Fugene 6 (Roche, Melbourne, Australia) according to the manufacturer's instructions. For indirect immunofluorescence, cells were fixed with cold methanol, permeabilised, and stained with primary anti-centrin-2 (1:800) polyclonal (MC1, kindly provided by Jeffrey Salisbury) and anti-Myc (9E10) (1:200) monoclonal (Santa Cruz Biotechnology, Inc.) antibodies 96 hours after transfection. Alexa 568 goat anti-mouse and 488 goat anti-rabbit secondary antibodies were subsequently added, along with 1 μg/mL Hoechst (Molecular Probes Inc., now part of Invitrogen Corporation). Centriole numbers were counted in a minimum of 100 Myc-expressing cells from each of two independent experiments using a Zeiss LSM510 confocal microscope (Carl Zeiss, Jena, Germany).
BRCA1 transcriptional activation domain reporter assays
Human 293T and T47D cells were transiently transfected with 0.5 μg of the pG5CAT reporter plasmid and 1 μg of the pGal4B plasmids described above in triplicate in six-well plates using Fugene 6 according to the manufacturer's instructions. Cell lysates were assayed for chloramphenicol acetyltransferase (CAT) activity using the Roche CAT enzyme-linked immunosorbent assay kit. CAT activity was normalised to total cell extract protein assayed by the Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). P values were determined using two-tailed t tests.