Pascaline Gaildrat

Sophie Krieger

Jean-Christophe Thery

Audrey Killian

Antoine Rousselin

Pascaline Berthet

Thierry Frébourg

Agnès Hardouin

Alexandra Martins

Mario Tosi
email: mario.tosi@univ-rouen.fr

THE BRCA1 c.5434C>G (p.PRO1812ALA) VARIANT INDUCES A DELETERIOUS EXON 23 SKIPPING BY AFFECTING EXONIC SPLICING REGULATORY ELEMENTS

published or not. The documents may come

INTRODUCTION

Genetic counseling is often limited by lack of knowledge about the pathogenic role of a large fraction of germline sequence variations. These are usually referred to as variants of unknown significance or unclassified variants (UVs). These types of variants are especially problematic for the breast and ovarian cancer susceptibility gene, BRCA1, which exhibits a large spectrum of sequence variation. Among these variants, most of the missense changes identified in the BRCA1 gene, as well as many intronic sequence changes, are currently listed as UVs in the database of the Breast Cancer Information Core (BIC, http://research.nhgri.nih.gov/projects/bic).

Over the past ten years, it became clear that a large fraction of missense UVs could be deleterious by impacting pre-mRNA splicing. [START_REF] Cartegni | Listening to silence and understanding nonsense: exonic mutations that affect splicing[END_REF][START_REF] Baralle | Splicing in action: assessing disease causing sequence changes[END_REF] Exonic mutations alter either splice site sequences or splicing regulatory elements such as ESE (exonic splicing enhancer) or ESS (exonic splicing silencer). Most ESE elements promote exon inclusion by interacting with activating splicing factors, usually members of the serine/argine-rich protein family. In contrast, ESS elements play a role in splicing repression usually through the binding of proteins, mostly belonging to the heterogenous nuclear ribonucleoprotein (hnRNP) family.

Mutations in these cis-acting elements may induce abnormalities in pre-mRNA splicing, that could result either in the degradation of the mature aberrant mRNA by the nonsense mediated decay (NMD) pathway or in the production of an altered protein. [START_REF] Pagani | Genomic variants in exons and introns: identifying the splicing spoilers[END_REF] Here, we describe the characterization of the c.5434C>G (p.Pro1812Ala) variant found in the BRCA1 gene in a French ovarian cancer patient. This variant was previously identified in several families with breast and/or ovarian cancers and was reported as an UV but was not listed in the BIC database of BRCA mutations. [START_REF] Martínez-Ferrandis | Mutational analysis of BRCA1 and BRCA2 in Mediterranean Spanish women with early-onset breast cancer: identification of three novel pathogenic mutations[END_REF][START_REF] Díez | Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects[END_REF][START_REF] Kaufman | The P1812A and P25T BRCA1 and the 5164del4 BRCA2 mutations: occurrence in high-risk non-Ashkenazi Jews[END_REF] Because it co-segregates with the high risk phenotype at least in one family, [START_REF] Kaufman | The P1812A and P25T BRCA1 and the 5164del4 BRCA2 mutations: occurrence in high-risk non-Ashkenazi Jews[END_REF] this variant was suspected to be deleterious.

It was initially thought that it could interfere with the activity of the BRCA1 protein, considering the high degree of conservation across species of the proline at position 1812 and its location in the second BRCT (BRCA1 carboxyl terminal) domain. However, a previous study showed that the BRCA1 p.Pro1812Ala variant induces a modest decrease in the activity of the BRCA1 protein, based on a transcriptional transactivation assay. [START_REF] Kaufman | The P1812A and P25T BRCA1 and the 5164del4 BRCA2 mutations: occurrence in high-risk non-Ashkenazi Jews[END_REF] More recently, the effects of this variant were also tested on structural integrity, protein stability and interaction with various phosphopeptides but these data were not sufficient to characterize this mutation as pathogenic. [START_REF] Drikos | Characterization of cancer-linked BRCA1-BRCT missense variants and their interaction with phosphoprotein targets[END_REF] Thus, the deleterious nature of this missense mutation was unclear at the protein level and this uncertainty prompted us to investigate its impact on pre-mRNA splicing. Here, we show that the BRCA1 c.5434C>G variant induces a major splicing defect, resulting in skipping of exon 23.

METHODS

Patient

The BRCA1 c.5434C>G variant tested in this study was detected in an index case selected from families undergoing genetic counseling in the Western French breast cancer genetics network. The criteria for diagnostic mutation screening of BRCA1/2 genes in patients were according to the current French recommendations. The patient gave signed informed consent.

Nomenclature

The DNA sequence numbering is based on the cDNA sequence for BRCA1 (NCBI RefSeq NM_007294), following the recommendations of the Human Genome Variation Society (HGVS, first position of the translation initiation codon ATG=1).

Genetic analysis

Patient peripheral blood samples were collected in EDTA tubes. Genomic DNA was isolated using an automated procedure (EZ1 DNA blood kit on biorobot EZ1 workstation, Qiagen, Hilden, Germany). Screening for BRCA1 and BRCA2 mutations was performed using denaturing High Performance Liquid Chromatography (dHPLC) or High Resolution Melting (HRM), followed by direct sequencing. Screening for BRCA1 and BRCA2 large rearrangements was performed using a combination of the multiplex ligation-dependent probe amplification (MLPA) and quantitative multiplex PCR of short fluorescent fragments (QMPSF) methods.

RT-PCR analysis of patient blood RNA

Whole blood samples were collected from patients and voluntary healthy donors in PAXgene Blood RNA tubes (Qiagen). RNA extraction was performed according to the manufacturer's protocols, including DNase treatment. RT-PCR was performed from 250 ng of total RNA, a forward primer complementary to BRCA1 exon 20 (5'-GAAGAAACCACCAAGGTCCAA-

Splicing minigene reporter assay

Generation of minigene constructs. The splicing minigene assay has been previously

described. [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF][START_REF] Bonnet | Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene[END_REF][START_REF] Gaildrat | Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants[END_REF] Wild-type and mutant c.5434C>G BRCA1 exon 23, together with 236 bp of the 5' flanking intron and 202 bp of the 3' flanking intron, were amplified by PCR using forward primer (5'-GACCAGATCTATTAGCCAGGGGTGGTGGTA-3') and reverse primer

(5'-GACCACGCGTCACCCCATGGAAACAGTTCA-3'), carrying 5' tails with BglII and MluI restriction sites (underlined), respectively. After digestion with BglII and MluI, the PCR products were inserted into the intron of the two-exons pCAS1 splicing reporter minigene vector (Fig. 1B). [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF][START_REF] Bonnet | Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene[END_REF][START_REF] Gaildrat | Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants[END_REF] Transfection and RT-PCR analysis. After identification by plasmid sequencing, the wild-type and mutant minigene constructs were transiently transfected into HeLa cells using FuGENE 6 transfection reagent, according to manufacturer's instructions (Roche Applied Science). Cells were then collected 24 h post-transfection. Total RNA was extracted using the TriPure Isolation Reagent (Roche Applied Science) and analyzed by RT-PCR. The reverse transcription (RT) step was performed using the Superscript II reverse transcriptase (Invitrogen) primed with oligod(T) 18 (New England Biolabs). The resulting cDNA was amplified by PCR (25, 30, 35 cycles) using the forward primer F (5'-CTCCGCAGGTCCGCT-3') and the reverse primer R (5'-ATTGGTTGTTGAGTTGGTTGTC-3') (Fig. 1B). RT-PCR products were separated by electrophoresis on 2.5% agarose gel containing ethidium bromide and visualized by exposure to ultraviolet light. Semi-quantitative analysis of the RT-PCR products was carried out using a Gel doc TM XR 170-8170 instrument (Biorad) and the software Quantity One (Biorad). Each DNA band was gel-extracted using Nucleospin Extract II kit (Macherey-Nagel) and sequenced using Big Dye Terminator cycle sequencing kit (Applied Biosystems) on an automated sequencer (ABI 3130, Applied Biosystems).

ESE-dependent splicing assay

Generation of pcDNA-Dup constructs. BRCA1 exon 23 wild-type fragment c.5420-5449 was tested for the presence of ESE splicing regulatory sequences by using an ESE-dependent splicing assay. [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF] Briefly, the exonic fragment was obtained by annealing complementary 5'-phosphorylated oligonucleotides designed to encompass 30 nucleotides around the mutation position and to carry 5'-EcoRI and 3'-BamHI compatibles ends (Fig. 3A). This duplex was inserted into the EcoRI and BamHI sites of the pcDNA-Dup plasmid. This expression vector contains a splicing cassette consisting of a β-globin-derived three-exon minigene (Dup) under the control of the cytomegalovirus (CMV) promoter (Fig. 3A). [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF] The

RESULTS

Identification of the BRCA1 c.5434C>G variant in an ovarian cancer patient

The patient was referred for genetic counseling, based on the diagnosis of ovarian cancer at 53 years and her family history. Her mother and maternal aunt were diagnosed with ovarian cancer at ages 53 and 65 years, respectively. Her paternal aunt was diagnosed with breast cancer at age 65 years. Using the Manchester scoring system, [START_REF] Evans | A new scoring system for the chances of identifying a BRCA1/2 mutation, outperforms existing models including BRCAPRO[END_REF] prior probabilities of a pathogenic mutation were 51% in the BRCA1 gene and 32% in the BRCA2 gene. Based on this information, the genomic DNA of this patient was screened for germ-line mutations and large genomic rearrangements in both BRCA1 and BRCA2 genes. No genetic alteration was found in BRCA2. However, a C to G transvertion was identified at position c.5434 of BRCA1.

At the time of genetic counseling, this BRCA1 c.5434C>G variant was not listed in the BRCA1 mutation BIC database nor in the French database of BRCA1 mutation (Dr. Rosette Lidereau, personal communication). It was therefore considered as an unclassified variant. In order to assess the pathogenicity of this variant, partial segregation analysis was performed.

As the mother of the proband was deceased at the time of the analysis, her affected maternal aunt was tested and found to be a carrier of BRCA1 c.5434C>G. This observation suggested a germline transmission of the variant in the maternal branch, indicating that it could represent the disease causing mutation or a genetic marker linked to it.

The BRCA1 c.5434C>G variant is associated with abnormal splicing of BRCA1 mRNA in patient blood cells

In order to evaluate the consequence of the BRCA1 c.5434C>G sequence variant on BRCA1 pre-mRNA splicing, RT-PCR analysis was performed using total RNA extracted from peripheral lymphocytes of the proband and a control subject (Fig. 1A). A single product with the expected size (456 bp) was detected in the control subject and sequencing confirmed that it corresponds to the region covering exon 20 to the 3'UTR of the normal BRCA1 transcript, including exon 23 (Fig. 1A). In contrast, the analysis of RNA from the patient carrying the BRCA1 c.5434C>G variant revealed the presence of an additional RT-PCR product with a smaller size (395 bp), expressed at a slightly lower level, that was identified by sequencing as an aberrant transcript lacking BRCA1 exon 23 (Fig. 1A). Semi-quantitative analysis indicated that this RT-PCR product accounts for 35% of the total transcripts (data not shown).

Therefore, skipping of exon 23 in the transcript corresponding to the mutant allele could be incomplete, or the aberrant mRNA could be degraded in part by the NMD pathway.

Sequencing of the top band corresponding to the normal BRCA1 transcript showed a barely detectable signal from the mutant sequence, strongly suggesting that very low levels of normal mRNA were expressed from the mutant allele. This result demonstrates that the presence of the BRCA1 c.5434C>G variant is associated with a major splicing defect, resulting in the skipping of exon 23.

The BRCA1 c.5434C>G variant induces BRCA1 exon 23 skipping in a minigene splicing assay

In order to test the hypothesis that this aberrant skipping of BRCA1 exon 23 was directly caused by the substitution C to G at position c.5434, the effect of the variant on splicing was assessed using a minigene splicing assay (Fig. 1B). pCAS minigene constructs carrying either the wild-type or the mutant BRCA1 c.5434C>G exon 23 were transiently expressed in HeLa cells and the minigene transcripts were analyzed by RT-PCR (Fig. 1C). Because the patient is heterozygous for the frequent SNP c.5407-193A>G (rs8176310, minor allele frequency: 0.456) located in intron 22, the wild-type and mutant c.5434C>G minigene constructs carried the G and A polymorphic nucleotides, respectively. All the processed RNA expressed from the wild-type BRCA1 exon 23 minigene retained BRCA1 exon 23, indicating that the less frequent G nucleotide at the intronic polymorphism rs8176310 has no negative effect on splicing. In contrast, the major spliced transcript expressed from the mutant BRCA1 c.5434C>G exon 23 minigene exhibits skipping of exon 23 (Fig. 1C). Semi-quantitative analysis indicated that, in this monoallelic assay, 75% of the total transcripts have skipped exon 23 (data not shown). These data demonstrate that the variant BRCA1 c.5434C>G is directly responsible for a major splicing defect.

The BRCA1 region c.5420-5449 in the centre of exon 23 contains sequences with global enhancer properties, which are affected by the c.5434C>G mutation

The c.5434C>G variant is located in the centre (position +28) of the short exon 23 (61 bp) of the BRCA1 gene, in a region highly conserved at nucleotide level across species (Fig. 2).

Nucleotide sequence conservation is particularly striking in a stretch of 6 amino acids with highly degenerate codons (IVVVQP). Such an exonic mutation, associated with exon skipping and located at distance from the splice sites, is likely to affect splicing regulatory elements. In order to determine if the BRCA1 exonic region affected by the c.5434C>G mutation contains a splicing enhancer element (ESE), we took advantage of an ESEdependent splicing assay (Fig. 3A). [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF] A ~30 bp fragment was inserted into the middle exon of a three-exon minigene under the control of a CMV promoter. The different minigenes were transiently expressed in HeLa cells and the splicing patterns of the minigene transcripts were analyzed by RT-PCR. Due to a weak 3' splice site, this middle exon is not recognized by the splicing machinery and therefore is excluded from the processed transcript, unless enhancer elements are inserted within its sequence. [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF] As expected, the middle exon was included into the mature transcripts expressed from a positive control minigene (SF2/ASF-3x) carrying triplet of enhancer elements (ESE), known to bind to the splicing regulatory SR protein SF2/ASF (Fig. 3B), whereas the middle exon without enhancer element was excluded from the mature transcripts expressed from minigenes without an inserted sequence (empty), or containing a randomly chosen intronic sequence derived from the BRCA2 intron 11 (BR2Int11) (Fig. 3B). The wild-type c.5420-5449 segment of BRCA1 exon 23 was able to induce inclusion of the middle exon (Fig. 3B), strongly suggesting that this specific region contains a sequence with enhancer properties. In contrast, the middle exon was excluded from the mature transcripts expressed from the minigene carrying the BRCA1 exon 23 c.5420-5449 region with the c.5434C>G mutation. This result indicates that the c.5434C>G variant induces skipping of BRCA1 exon 23 either by disrupting an enhancer element (ESE) or by creating a silencer element (ESS).

We also analyzed the impact on splicing of a reported SNP at the same position (BRCA1 c.5434C>T; rs1800751) by using the same ESE-dependent assay (Fig. 3B). In contrast to the c.5434C>G mutation, the c.5434C>T variant had modest negative effect on the inclusion of the middle exon. This result suggests that the effect on splicing at position c. b , as determined using hnRNPA1 experimental motif from HSF [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF] c , the nucleotide underlined indicates the position of the mutation *, scores that differ significantly considering the threshold

DISCUSSION

We have shown, using patient RNA analyses and minigene splicing assays, that the BRCA1 variant c.5434C>G (p.Pro1812Ala) induces a major splicing defect, resulting in the skipping of exon 23. The amount of exon skipping was estimated at 75% in the monoallelic splicing minigene assay, a value that is consistent with the analysis of RT-PCR products obtained from patient blood cell RNA. Translation of the aberrant transcript is predicted to result in a protein with a premature stop codon, p.Gly1803GlnfsX11, which disrupts the second BRCT domain.

This functional region is essential for the tumor suppressor activity of the BRCA1 protein, as it is involved in DNA repair and transcriptional control. [START_REF] Monteiro | Evidence for a transcriptional activation function of BRCA1 C-terminal region[END_REF][START_REF] Glover | Insights into the molecular basis of human hereditary breast cancer from studies of the BRCA1 BRCT domain[END_REF] Altogether, these results strongly suggest that the mutation BRCA1 c.5434C>G induces the production of a nonfunctional protein, as a consequence of exon 23 skipping, and argue for its classification as a pathogenic mutation. This study adds to the growing list of examples indicating that missense changes, even those affecting extremely conserved amino-acid residues located in functional domains, should not be evaluated exclusively at protein level. [START_REF] Baralle | Missed threads. The impact of pre-mRNA splicing defects on clinical practice[END_REF] Over the past ten years, exonic mutations identified in genetic screening of several genes have been shown to induce exon skipping by altering splicing regulatory elements. [START_REF] Pagani | Genomic variants in exons and introns: identifying the splicing spoilers[END_REF] But, so far, in the BRCA1 gene, only two mutations, both located in exon 18, have been shown experimentally to affect splicing regulatory elements: the nonsense variant c.5080G>T (p.Glu1694X) [START_REF] Mazoyer | A BRCA1 nonsense mutation causes exon skipping[END_REF][START_REF] Liu | A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes[END_REF][START_REF] Goina | Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant[END_REF] and the missense variant c.5123C>T (p.Ala1708Glu). [START_REF] Millevoi | The c.5242C>A BRCA1 missense variant induces exon skipping by increasing splicing repressors binding[END_REF] We investigated the mechanism underlying the effect of the mutation BRCA1 c.5434C>G on splicing. Using an ESE-dependent splicing assay, we show that the central 30 nucleotide-long region of exon 23 encloses a sequence with global enhancer properties, which are strongly affected by the c.5434C>G mutation. In contrast, the c.5434C>T variant, which corresponds to a SNP without allele frequencies listed in the dbSNP database of NCBI, has little effect in the ESE-dependent splicing assay and its functional consequences are debated. [START_REF] Rajasekaran | Identification and in silico analysis of functional SNPs of the BRCA1 gene[END_REF] The bioinformatics predictions shown in Table I indicate, for both variants, a strong reduction of scores for two ESE elements and predict, only for the c.5434C>G variant, the generation of a new hnRNPA1 splicing silencer motif. Our data and these bioinformatics predictions suggest that the exonic variant BRCA1 c.5434C>G creates a sequence with silencer properties, predicted to bind the splicing repressor hnRNPA1. A similar model has been proposed to explain the impact on splicing of the nonsense BRCA1 c.5080G>T mutation, which causes exon 18 skipping [START_REF] Mazoyer | A BRCA1 nonsense mutation causes exon skipping[END_REF] and was initially predicted to disrupt a splicing enhancer element for the SF2/ASF protein. [START_REF] Liu | A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes[END_REF] It has recently been shown that this variant creates a silencer element, which interacts with the inhibitory splicing factor hnRNPA1/A2 and DAZAP1. [START_REF] Goina | Binding of DAZAP1 and hnRNPA1/A2 to an exonic splicing silencer in a natural BRCA1 exon 18 mutant[END_REF] The other variant in exon 18 of BRCA1 reported to induce exon skipping is the missense variant c.5123C>A, which is thought to increase the RNA binding of the splicing repressors hnRNPA1 and hnRNPH/F. [START_REF] Millevoi | The c.5242C>A BRCA1 missense variant induces exon skipping by increasing splicing repressors binding[END_REF] Similar models could explain the effect on exon 23 splicing of the BRCA1 c.5434C>G mutation. However, at this point, we cannot rule out other models, based on the inactivation of an ESE or on a combination of ESE inactivation and generation of an ESS. In the BRCA2 gene, exon skipping has been associated with the missense mutations c.8165C>G (p.Thr2722Arg) in exon 18 [START_REF] Fackenthal | BRCA2 T2722R is a deleterious allele that causes exon skipping[END_REF] and c.6853A>G (p.Ile2285Val) in exon 12 [START_REF] Li | Functional redundancy of exon 12 of BRCA2 revealed by a comprehensive analysis of the c.6853A>G (p.I2285V) variant[END_REF], but the molecular bases of these splicing defects have not been described.

Retrospectively, one may ask if bioinformatics predictions could have pointed to the c.5434C>G variant as a splicing mutation. It should be noted that we initially decided to examine the effect of this variant on splicing not because of bioinformatics predictions, but because the available information on the functional consequences of this change at the protein level were inconclusive and family data suggested that it was directly or indirectly associated with the predisposition to breast/ovarian cancer.[6, and the present study] Most published evaluations of bioinformatics predictions of splicing defects deal with UVs that affect splice site changes, either by affecting 5' or 3' splice sites, or by creating or activating cryptic splice sites. These predictions are usually considered sensitive and rather specific. [START_REF] Vreeswijk | Intronic variants in BRCA1 and BRCA2 that affect RNA splicing can be reliably selected by splice-site prediction programs[END_REF] On the other hand, bioinformatics predictions of the effects of UVs on exonic regulatory elements are much less reliable and lack specificity. [START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF][START_REF] Houdayer | Evaluation of in silico splice tools for decision-making in molecular diagnosis[END_REF][START_REF] Spurdle | Prediction and assessment of splicing alterations: implications for clinical testing[END_REF] Therefore, bioinformatics analyses of exonic splicing regulatory elements need the addition of other criteria to improve specificity.

Strong sequence conservation at the nucleotide level could be among such additional criteria, as already suggested. [START_REF] Pettigrew | Evolutionary conservation analysis increases the colocalization of predicted exonic splicing enhancers in the BRCA1 gene with missense sequence changes and in-frame deletions, but not polymorphisms[END_REF][START_REF] Pettigrew | Colocalisation of predicted exonic splicing enhancers in BRCA2 with reported sequence variants[END_REF] Moreover, sequence variations in small size exons could be more prone to affect exonic splicing regulatory elements than those located in large size exons, [START_REF] Anczuków | Unclassified variants identified in BRCA1 exon 11: Consequences on splicing[END_REF] where compensatory effects may exist.

In summary, our results provide in vivo and ex vivo evidences that the BRCA1 c.5434C>G variant is pathogenic through a major skipping of exon 23. As is often the case with splicing mutations, the resulting defect is not complete, but was estimated to be present on about 75% of the transcripts from the mutant allele. This finding and the predicted deleterious consequences on the corresponding BRCA1 protein, together with segregation data from this family and from another family [START_REF] Kaufman | The P1812A and P25T BRCA1 and the 5164del4 BRCA2 mutations: occurrence in high-risk non-Ashkenazi Jews[END_REF] places the c.5434C>G variant of BRCA1 into class 4, i.e. "Likely pathogenic" of the classification system that has been recently

proposed by Plon et al. [START_REF] Plon | Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results[END_REF] In addition, analyses of the effect of this variant on exonic splicing regulatory elements contribute to a better understanding of the mechanisms underlying splicing regulation.

KEY POINTS:

• The variant BRCA1 c.5434C>G (p.Pro1812Ala) was found to induce a major splicing defect, resulting in exon 23 skipping in hybrid minigene assays and in blood cells from an ovarian cancer patient, arguing for its classification as a likely pathogenic splicing mutation.

• The central segment c.5420-5449 of BRCA1 exon 23 exhibits splicing enhancer properties, which are abolished by the c.5434C>G mutation.

• Bioinformatics analyses predict that the mutation BRCA1 c.5434C>G creates an hnRNPA1-dependent splicing silencer motif.

LEGENDS OF THE FIGURES

  effects of the mutations BRCA1 c.5434C>G and c.5434C>T were tested in parallel in the ESE-dependent splicing assay. Control experiments were performed, as previously described,[START_REF] Tournier | A large fraction of unclassified variants of the mismatch repair genes MLH1 and MSH2 is associated with splicing defects[END_REF] using pcDNA-Dup derived minigenes, including i) the empty pcDNA-Dup plasmid, ii) the pcDNA-Dup-BR2Int11 construct which contains a fragment without known regulatory elements (5'-TGTTGTCCAGGTCACATTCAATAG-3') derived from BRCA2 intron 11 and iii) the pcDNA-Dup-SF2/ASF-3x construct containing a triplet of binding sites for the SR protein SF2/ASF (5'-AGAAGAACAAGAAGAACAAGAAGAACG-3').Transient transfection and RT-PCR analysis. The different pcDNA-Dup constructs have been transiently transfected into HeLa cells, as described above. Cells were harvested 24 h post-transfection and total RNA was extracted using the TriPure Isolation Reagent (Roche Applied Science). The RNA preparations were treated by Amplification Grade DNase I (AMP-D1, Sigma-Aldrich), according to the manufacturer's instructions and subjected to RT-PCR analysis. The one-step RT-PCR reaction was performed from 0.5 µg DNase-treated RNA in a 25 µl reaction volume using the OneStep RT-PCR kit (Qiagen), according to the manufacturer's instructions. Reverse transcription was done using the reverse primer Dup-2R(5'-GGACTCAAAGAACCTCTGGG-3') that binds to the last exon of the minigene and PCR amplification was performed with 25 cycles using the forward primer T7-Pro (5'-TAATACGACTCACTATAGGG-3'), which binds immediately upstream of the Dup minigene, and Dup-2R. RT-PCR products were separated by electrophoresis on 2.5% agarose gel containing ethidium bromide and visualized by exposure to ultraviolet light. Each DNA band was extracted from the gel by using Nucleospin Extract II kit (Macherey-Nagel) and sequenced using Big dye Terminator cycle sequencing kit (Applied Biosystems) on an automated sequencer (ABI 3130, Applied Biosystems).

Figure 1 .

 1 Figure 1. The BRCA1 c.5434C>G variant induces aberrant splicing of exon 23

Figure 2 .

 2 Figure 2. The central region of BRCA1 exon 23 is highly conserved at the nucleotide

Figure 3 .

 3 Figure 3. The c.5420-5449 region of BRCA1 exon 23 contains sequences with global

Figure 3

 3 Figure 3

Table 1 . Bioinformatics predictions of splicing regulatory elements

 1

	5434 is

ACKNOWLEDGEMENTS

We thank the patients and their families who have contributed to this project. This work was supported by the Fondation de France (N°Engt:2004012859) and the French North West Canceropole. P. Gaildrat has received a postdoctoral fellowship from the Fondation de France and is supported by the French North West Canceropole. J.-C. Théry was supported by a fellowship from the Association pour la Recherche sur le Cancer (ARC).

Competing interests: none declared

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in Journal of Medical Genetics and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence


Claudia Spits
email: claudia.spits@uzbrussel.be

Sara Seneca

Pierre Hilven

Inge Liebaers

Karen Sermon

METHYLATION OF THE CPG SITES IN THE MYOTONIC DYSTROPHY LOCUS DOES NOT CORRELATE WITH CTG EXPANSION SIZE OR WITH THE CONGENITAL FORM OF THE DISEASE

We have studied the methylation status of the sequence 152 nucleotides upstream of the CTG repeat of the DM1 locus in patients' peripheral blood. We used the methylationsensitive endonucleases SacII, HpaII and HhaI, followed by PCR. This allowed to correlate the methylation status of each CTG allele with its size. Contrary to previous findings (Steinbach P et al., 1988, Am.J.Hum.Genet. 62:278-285), only the SacII site is often but not always differentially methylated amongst expanded CTG alleles.

Importantly, this methylation was not restricted to congenital DM1, nor to large expansions, as it was also present in DM1 patients with a classical phenotype and various expansion sizes. On the other hand, we did not find any methylated alleles on the HhaI

and HpaII sites, as was reported by Steinbach et al, which is in line with the results of Shaw and collaborators (1993, J.Med.Genet. 30:189-192). The size range of the repeat expansions with methylation was from as small as 300 to as large as 2800 repeats.

REPORT

Myotonic dystrophy type 1 (DM1, [MIM 160900]) is a dominantly inherited multisystemic disorder, with an incidence of approximately 1 in 8000 individuals. DM1 has a complex phenotype, with symptoms including progressive skeletal muscle wasting, impaired muscle relaxation (myotonia), cardiac conduction defects resulting in arrhythmias, early-onset iridescent cataracts, insulin resistance and hyperinsulinemia [START_REF] Harper | Myotonic Dystrophy. 3 rd edn[END_REF]. These symptoms are caused by several mechanisms, which include the aberrant processing of the DMPK mRNA, leading to decreased levels of DMPK [START_REF] Krahe | Effect of myotonic dystrophy trinucleotide repeat expansion on DMPK transcription and processing[END_REF], toxic gain of function of the mRNA [START_REF] Mankodi | Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat[END_REF], decreased expression of the neighbouring SIX5 gene [START_REF] Klesert | Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP[END_REF][START_REF] Thornton | Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene[END_REF] and disrupted alternative splicing of numerous other genes [START_REF] Ranum | RNA-Mediated Neuromuscular Disorders[END_REF].

DM1 is caused by a CTG expansion in the 3' untranslated region of the DMPK gene located on chromosome 19q13.3. Expanded CTG tracts show both somatic and germinal instability; unaffected individuals have from 5 to 40 CTG repeats, whereas individuals with the disease carry more than 100 repeats. Disease severity and age of onset correlate with repeat size, and expansions of >1500 repeats often result in a severe congenital form of DM1.

CpG methylation has been suggested to play a role in the stability of repetitive sequences in general (Gourdon et al., 1997, Nichol and[START_REF] Nichol | CpG methylation modifies the genetic stability of cloned repeat sequences[END_REF], and more particularly in the behaviour of the CTG repeat in the DM1 locus [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF][START_REF] Brock | Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands[END_REF][START_REF] Gorbunova | Genome-wide demethylation destabilizes CTG.CAG trinucleotide repeats in mammalian cells[END_REF] as well as in the pathogenesis of the disease [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF][START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF][START_REF] Filippova | CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus[END_REF].

The relationship between the semiology of the disease and the methylation of the CpG sites in the DM1 locus was first investigated by Shaw and collaborators (1993), who could not find any clear correlation between the methylation and the age of onset or the severity of the disease. On the other hand, Steinbach and coworkers found that several CpG sites close to the CTG repeat were fully methylated in blood samples containing expansions larger than 1000 repeats. They also reported that, since these expansions appeared as single well-defined signals on Southern blot, the methylation of these CpG sites would correlate with triplet repeat stability. This idea was later supported by in vitro and in silico work [START_REF] Brock | Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands[END_REF][START_REF] Gorbunova | Genome-wide demethylation destabilizes CTG.CAG trinucleotide repeats in mammalian cells[END_REF][START_REF] Gourdon | Intriguing association between disease associated unstable trinucleotide repeat and CpG island[END_REF][START_REF] Nichol | CpG methylation modifies the genetic stability of cloned repeat sequences[END_REF]. The work of Filippova built further on the idea that methylation would be related to the pathogenesis of congenital DM1 (CMD). The authors discovered that there are two binding sites for the zinc-finger protein CTCF flanking the CTG repeat, and that the hypermethylation of these sequences, which coincide with the region described by Steinbach and collaborators (1998), would be incompatible with the binding of the CTCF and its proper function mediating the inhibition of promoterenhancer interactions by insulator elements [START_REF] Bell | The protein CTCF is required for the enhancer blocking activity of vertebrate insulators[END_REF]. Conversely, Libby and collaborators (2008) found that CpG methylation of CTCF binding sites may rather lead to repeat instability, instead of stability as previously suggested.

In this work we studied 8 of the 18 CpG sites in 152 nucleotides upstream of the CTG repeat of the DM1 locus (see figure 1). These sites are part of the CpG sites investigated by Steinbach and collaborators and did not include the sites affecting the CTCF binding [START_REF] Filippova | CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus[END_REF]. We did not investigate the distal SacII site, located over 1 Kb upstream of the CTG repeat, which Steinbach and collaborators reported as constitutively methylated, nor the HpaII and HhaI sites located in this same region. We analysed our samples in duplicate by methylation sensitive endonuclease digestion, followed by PCR.

The PCRs were designed so that they included both the restriction site and the CTG repeat and were performed on small quantities of DNA. Consequently, smears could be resolved into individual bands, and sizing was much more accurate than by using Southern blot on total genomic DNA. Furthermore, it enabled us to correlate the methylation status with the exact length of each allele, which would not have been possible with Southern blot on total genomic DNA or bisulphite sequencing. It is important to notice that in the case of the HpaII restriction enzyme, the studied region contained 6 sites. This means that we could not distinguish the methylation state of each independent site, but could only asses whether at least one of the sites was unmethylated.

The aim of this study was to establish the threshold for the expansion size necessary for the hypermethylation of the CpG sites close to the CTG repeat and this within one DNA sample. We also aimed to investigate the possibility that hypermethylation is patient-or allele specific.

DNA samples from peripheral blood from 22 DM1 patients were digested with the methylation-sensitive restriction enzymes SacII, HhaI and HpaII (Biolabs, Westburg, Leusden, The Netherlands). Both digested and undigested samples were analysed by PCR. In the case of the SacII digested samples, we used the primer set indicated in figure 1 as SacII primer and reverse primer. The DNA input was of 200 pg. For the HhaI and HpaII samples, we used the HhaI-HpaII and the reverse primers. Initially, the DNA input was of 200 pg, but after the first results, DNA input was increased to 2 ng to ensure the detection of possible uncleaved alleles present at a very low quantity. The PCR protocol was further performed as previously described [START_REF] De Temmerman | CTG repeat instability in a human embryonic stem cell line carrying the myotonic dystrophy type 1 mutation[END_REF]. The expansions were visualized by denaturing Southern blot using a GAC probe, and the size calculated by comparison to two molecular weight markers (markers VI and VII, Roche diagnostics, Vilvoorde, Belgium).

Table 1 shows the results for the 22 DM1 patients included in this study. The table outlines the results of the PCR before and after endonuclease cleavage by SacII, along with the disease status and inheritance of the mutation in all those patients for which these data were known. The cohort of patients includes cases of congenital DM1, infantile and classic DM1, both paternally and maternally inherited. The age at sampling ranges from birth to 43 years, and expansions were between 180 to 2800 repeats. Figure 2 shows an example of the Southern blot results after SacII restriction enzyme cleavage and PCR. The results for HhaII and HpaII cleavage, and the complete data for the SacII digestion can be found in the supplementary figures 1, 2 and 3.

After HhaI endonuclease cleavage, none of the DNA samples showed amplification, whereas the undigested samples worked as a positive control for the PCR. This meant that all alleles were unmethylated, both the wild type alleles as well as all of the expansions. We verified that the lack of amplification in the digested samples was not due to DNA degradation by amplifying the material with a PCR for 4 short tandem repeats located on chromosome 19q13. 41, 19q13.33, 19p13.3 and 19p13.2 (tandem repeats located using the database www.microsatellites.org). The primer sequences and PCR conditions are available upon request. The results showed that the DNA was intact in all samples, and proved that the restriction by HhaI had been specific for its target.

These results suggest that, contrary to the findings of Steinbach and collaborators, not only is this CpG site constitutively unmethylated, but also that it is not necessarily methylated in expansions larger than 1000 repeats or in congenital DM1 patients.

Identical results were obtained after cleavage with HpaII. Both the wild type and the expanded alleles were unmethylated (for at least one of the six HpaII sites in the sequence) in all the samples studied. These results are in concordance with the findings of Shaw and collaborators (1993) and contradict those of Steinbach and co-workers (1998).

From our results it seems that of the CpG sites we studied the SacII site immediately upstream of the repeat is the only one in the region that can be differentially methylated between wild type alleles, which are never methylated, and expanded alleles, which may be methylated. Our data are difficult to harmonize with the previously published study [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF]. We did not find a correlation between the methylation status of this site on one hand and the allele size or on the other hand with the presence of a smear on the Southern blot -indicative of tripet repeat instability-, as was suggested by Steinbach et al. Alleles as small as 400 repeats could be methylated (patient 20), and alleles of 1600 repeats unmethylated (patient 8), indicating that there is no correlation between the allele size and the methylation status. We found several patients showing intermediate methylation patterns (some alleles were methylated while others were not), and even patients without any methylation at this site. A patient could present a smear in which all alleles appeared to be methylated (i.e. patient 7), indicating that the methylation status apparently does not correlate with the stability of the repeat. However, it is clear that the presence of CTG length mosaicism either with or without methylation does not necessarily make one the cause or effect of the other. The time during which one occurred may or may not have coincided with the other. Furthermore, the link between methylation of this site and congenital DM1 does not seem to be as strict as previously assumed. It is true that both congenital DM1 cases analysed here showed methylation of the SacII site in all the expanded CTG alleles (patients 4 and 6), but non-congenital DM1 patients also presented it (patients 5 and 7). Furthermore, we did not find the correlation previously described [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF] between the methylation of the SacII site and of the HhaI and HpaII sites, since we never found methylation in the latter two, not even in the congenital DM1 patients. The fact that we did not find a methylation pattern specific to the CMD patients contradicts the hypothesis that differential methylation may be responsible for the distinct features of CMD by modulating the binding of CTCF in the DM1 locus (Flippova et al., 2001). It is, however, important to bear in mind that we did not study the methylation of all the CpG sites present in the CTCF binding sites, and that methylation may still play an important role in the pathogenesis of DM1. These discordances do not seem attributable to incomplete restriction or technical differences. In the works of [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF] and [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF], as in ours, incomplete restriction would have been detected by the incomplete cleavage of the wild type allele. To ensure that the results for HpaII and HhaI were not due to the degradation of the samples after restriction, we confirmed the presence of intact DNA by PCR for microsatellite markers. The fact that in our work we analysed PCR products by Southern blot, whereas [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF] and [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF] performed direct Southern blotting of the restricted DNA only adds to the resolution of the expanded alleles. Furthermore, it is possible that our method is more sensitive in picking up a few unrestricted alleles. Nevertheless, for the HhaI and HpaII digests, we never found any uncleaved allele.

In the work of [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF] it is suggested that the patient's age might play a role on the methylation levels in CMD. The authors suggest that, since their CMD patients were rather of mature age, the methylation could have been lost. It is important to bear in mind that these authors were investigating a possible relationship between imprinting of the DM1 locus and CMD, and hypothesized that this imprinting would only be of importance in utero, and would be subsequently lost after birth. In Steinbach's paper, the oldest CMD patient is 24 years, and shows full methylation of the expanded allele for all the SacII, HhaI and HpaII sites. [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF] did not mention the exact age of the patients, but if they were older than 24 years, their hypothesis would harmonize the difference in results between Shaw and Steinbach's papers, but not with ours, where the two CMD patients were very young, and showed no methylation for the HhaI and HpaII

sites. An age effect on the classical DM1 patients can be ruled out, as both Steinbach's work as ours has patients of all ages (Steinbach's range was 19-62, ours 4-45 years). Furthermore, there does not seem to be a parent of origin effect.

In conclusion, we have studied the methylation status of the proximal sequence upstream of the CTG repeat of the DM1 locus. From our results, we establish that, contrary to previous findings, only one of the studied sites is differentially methylated in wild type and expanded CTG alleles, and that this methylation does not strictly correlate with the length of the allele, nor with the disease status of the patient. Further research on this topic is necessary to elucidate the reasons for the discrepancy with some of the previously published results.

AKNOWLEDGEMENTS

The authors wish to thank their colleagues at the Centre for Medical Genetics for the fruitful collaboration, and Prof. C.E. Pearson for his constructive comments on the manuscript. This work has been supported by grants from the Fund for Scientific Research Flanders (Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen). CS is a postdoctoral fellow at the FWO Vlaanderen.

FIGURE LEGENDS

Figure 1. Schematic overview of the sequence analyzed in this study and its relationship to the genomic regions studied by [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF] and [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF]. The figures are modified versions of the original figures found in the respective publications. A: Genomic region studied by [START_REF] Shaw | A study of DNA methylation in myotonic dystrophy[END_REF]. Three fragments were created using EcoRI and EcoRV, and then restricted by HpaII. The results showed that at least some of the HpaII sites contained unmethylated cytosines, independently of the form of the disease or expansion size. B: Genomic region studied by [START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF]. The samples were restricted using combinations of SacI and HindIII, and SacII, HpaII or HhaI. The results showed that the upstream SacII site was constitutively methylated, and the downstream SacII site only methylated on very largely expanded alleles of patients with congenital DM1. The HpaII and HhaI sites were also all

TABLETable 1 .

 1 Details of the studied patients and results after SacII cleavage : disease onset is not congenital, CMD: disease onset is congenital. Full methylation indicates that all alleles appeared to carry a methylated SacII site.

	Patient Disease status and origin of mutation 1 DM, paternal 2 Unknown 3 DM, paternal 4 CMD, probably maternal 5 DM, paternal 6 CMD, maternal Newborn 700-1300 Age at sampling Expansion size (repeats) 33 900-2800 30 400-1200 33 200-400 3 months 1400 41 300-900 7 Unknown 4 700-1300 8 DM, maternal 20 700-1600 9 Unknown Unknown 200-500 10 DM, paternal 33 700-1600 11 DM, maternal 30 600-1200 12 DM, paternal 24 200-480 13 DM, probably 33 200-480 maternal 14 DM, maternal 27 680-900 15 DM, paternal 45 500-1300 16 DM, paternal 38 500-1300 17 Unknown 43 500-1650 18 Unknown 30 220-370 19 DM, maternal 29 250-1400 20 DM Unknown 180-2200	Description of the expansion Numerous discrete bands Smear Two groups of very close bands One band Two groups of very close bands Smear with predominant band at 1300 Smear with predominant band at 1300 Smear with predominant allele at 1600 Smear Smear with predominant allele at 1600 Smear Smear Smear Smear Smear Smear Smear Smear Smear Smear	Alleles after SacII digestion 900-2800 Fully cleaved Fully cleaved 1400 300-900 700-1300 700-1300 860 Fully cleaved 700-1160 680-1170 Fully cleaved Fully cleaved 680-860 840-990 Fully cleaved 1300 Fully cleaved 850-1000 400-1700	Description of the alleles after SacII digestion Numerous discrete bands: full methylation Fully unmethylated Fully unmethylated One band: full methylation Two groups of very close bands: full methylation Smear with predominant band at 1300: full methylation Smear with predominant band at 1300: full methylation One band: partial methylation Fully unmethylated Bands of 700, 990, 840 and 1160: partial methylation Bands of 680 and 1170: partial methylation Fully unmethylated Fully unmethylated Bands of 680 and 860: partial methylation Bands of 840 and 990: partial methylation Fully unmethylated One band: partial methylation Fully unmethylated Bands of 850 and 1000: partial methylation discrete bands of 400, 900 and 1700: partial
	21	Unknown	Unknown 300-2600	Smear	600-2600	methylation discrete bands of 600, 950, 1200, 1500, 1600
							and 2600: partial
	22	Infantile form, maternal inheritance	14	1000-2600	Smear	1300	methylation One band: partial methylation

DM

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Competing interests

None declared methylated in these patients. C: Sequence studied in this paper. Genomic DNA was restricted using SacII, HpaII or HhaI and amplified by PCR using the primers indicated on the sequence as arrows. The highlighted sequence corresponds to the CTCF binding site 1 (Fillippova et al., 2001). The results showed no correlation between the methylation of the SacII site and the disease status of the patient or the expansion size. No methylation of the HhaI site was detected, and the results for HpaII suggested that there was at least always one unmethylated site present in the sequence. Some samples have been analyzed in duplicate on the same blot, and in some cases a replicate can be found on another blot. In the case of the HpaII and HhaI cleavage, the blots show smears in the control lanes because the PCRs were performed with a higher input to ensure the detection of possible uncleaved alleles present in a low quantity.

Nevertheless, none or the digested samples showed any amplification, revealing that they all contained unmethylated sites. The signal at the lower end of the blot corresponds 1 to the wild type allele. Its full cleavage serves as an internal control for the digestion, as it is assumed to be always unmethylated. In some lanes, heteroduplexes can be seen as a signal between the wild type and the expanded allele.


Evans Dgr

Professor DGR Evans

Risk of breast cancer in male BRCA2 carriers

The risk of breast cancer for unaffected men who test positive for a BRCA2 mutation is based on very few retrospective studies. We have used both retrospective and prospective analysis in 321 families with pathogenic BRCA2 mutations. Three breast cancers occurred in male first degree relatives after family ascertainment in 4140 years of follow up suggesting a risk of breast cancer to 80 years of 8.9%. A second analysis excluding index cases identified 16 breast cancers in 905 first degree male relatives on which Kaplan Meier analysis was performed after assigning carrier status. This analysis confirmed that breast cancer risk in males was 7.1% (standard error 5.2-8.6%) by age 70 years and 8.4% (standard error 6.2-10.6%) by age 80 years.

The risk of breast cancer in male BRCA2 carriers is still in question. Only three previous studies have assessed lifetime risk in males [START_REF] Easton | Cancer risks in two large breast cancer families linked to BRCA2 on chromosome 13q12-13[END_REF][START_REF] Breast | Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium[END_REF][START_REF] Tai | Breast Cancer Risk Among Male BRCA1 and BRCA2 Mutation Carriers[END_REF]. The second of these emphasised the need for further studies [START_REF] Breast | Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium[END_REF]. We have assessed the risk of breast cancer amongst males in families with proven BRCA2 mutations from the adjoining health regions centralised around Manchester and Birmingham in Central North West England. Families were ascertained in the two genetic centres covering a population of around 10 million people. Testing generally starts with an affected family member in a family with more than one affected individual and was cascaded out from the index case as requested by the family. Uptake of testing in males was considerably lower than in females [START_REF] Tai | Breast Cancer Risk Among Male BRCA1 and BRCA2 Mutation Carriers[END_REF]. We have developed a dedicated database that enters all male first degree relatives (FDRs) of mutation carriers aged >20 years [START_REF] Evans | Comparison of proactive and usual approaches to presymptomatic testing for BRCA1 and BRCA2 mutations[END_REF][START_REF] Evans | Penetrance estimates for BRCA1 and BRCA2 based on genetic testing in a Clinical Cancer Genetics service setting[END_REF].

There were 321 families with proven pathogenic BRCA2 mutations. In these families there were 20 male index cases of whom 15 had developed breast cancer (age range 29-79 years median 65.3) One had bilateral breast cancer. There were 905 male FDRs of proven BRCA2 mutation carriers. 18/905 (2%) had developed breast cancer (age range 46.8-74 years median 61.7). Eight further male breast cancers occurred amongst second degree relatives including one with bilateral disease, two were confirmed as mutation positive. In 4140 years of follow up from date of family ascertainment in the genetics departments breast cancers had occurred in 3/667 male FDRs (0.72 per 1000). 238 male FDRs had died before the family ascertainment date. As all male FDRs >20 years were on the dataset this would mean that each male had a potential 60 years of risk. This would amount to a lifetime risk of 4.32%. However, only half of the FDRs would be mutation carriers as such the estimated risk in males would be 8.6%. Given the small number of male cancers in follow up we validated this risk by assessing male breast cancer amongst proven or assumed BRCA2 mutation carriers excluding the index case. In this mostly retrospective analysis in addition to all males testing positive, FDR males were assigned mutation status on a 50% basis omitting every other male on the basis of successive age at last follow up. FDR males with breast cancer were assumed to be mutation carriers as the relative risk of breast cancer in male BRCA2 carriers is at least 100 fold [START_REF] Risch | Population BRCA1 and BRCA2 mutation frequencies and cancer penetrances: a kin-cohort study in Ontario, Canada[END_REF] and male breast cancer phenocopies have not been described to our knowledge [START_REF] Smith | The trouble with phenocopies: are those testing negative for a family BRCA1/2 mutation really at population risk?[END_REF]. Male FDRs with pancreatic, and prostate cancer were assigned mutation status on a 4:1 basis omitting the fifth case in successive age order of cancer. Stomach cancers were assigned on a 2:1 basis. This decision was based on the described 2.5-5.9 fold relative risk in BRCA2 carriers for pancreas (3.51-5.9) and prostate cancer (2.5-4.65) and 1.2-2.59 relative risk of stomach cancer [START_REF] Breast | Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium[END_REF][START_REF] Van Asperen | Cancer risks in BRCA2 families: estimates for sites other than breast and ovary[END_REF]. After exclusion of the index case 122 males were identified as proven carriers from family testing six of these had breast cancer. Forty one male FDRs had tested negative in predictive testing. Ten male FDRs of unknown mutation status were assumed to be carriers as they had suffered from breast cancer. Of 29 prostate cancers of unknown mutation status in FDRs 24 were assigned mutation positive status as were 5/6 with pancreatic cancer and 10/14 stomach cancers. Of the remaining 683 male FDRs of unknown status 333 were assigned as positive (table 1). Thus 508 male BRCA2 carriers or assumed carriers were identified. Cumulative risk of breast cancer was assessed by Kaplan Meier analysis (Figure 1). This showed a risk of 7.1% (standard error 5.2-8.6%) by age 70 years and 8.4% (standard error 6.2-10.6%) by age 80.

In the first report to assess male breast cancer risks two large BRCA2-linked families that contained four male breast carcinoma cases were evaluated [START_REF] Easton | Cancer risks in two large breast cancer families linked to BRCA2 on chromosome 13q12-13[END_REF]. The estimated cumulative risk for male breast carcinoma by age 70 years was 6.3%. In a separate study of 164 families with BRCA2 mutations, the estimated cumulative risk of breast cancer for male BRCA2 mutations carriers was 2.8% by 70 years of age and 6.9% by 80 years [START_REF] Breast | Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium[END_REF]. More recently, the estimated cumulative risk of breast carcinoma for male BRCA2 mutation carriers was 6.8% (95% CI = 3.2-12%) by age 70 years and 8.3% to age 80 years [START_REF] Tai | Breast Cancer Risk Among Male BRCA1 and BRCA2 Mutation Carriers[END_REF]. The latter study was based on 23 families ascertained through a male breast cancer. This study based risk estimates on the relative risks of male breast cancer in BRCA2 versus population risks assuming certain BRCA2 allele frequencies. A further study found that the relative risk of breast cancer for male BRCA2 carriers was estimated to be 102 fold [START_REF] Smith | The trouble with phenocopies: are those testing negative for a family BRCA1/2 mutation really at population risk?[END_REF]. Our study is the first to have assessed male breast cancer risk prospectively from family ascertainment. Although the numbers are small and confidence intervals will clearly be wide the rates of breast cancer prospectively are in keeping with a lifetime risk to 80 years of close to 9%. To corroborate this we carried out an analysis of cumulative risk in male FDRs taking advantage of clinical testing but excluding the bias of the index case. There are flaws in such a study in that excluding the index case alone may not be sufficient, but a contrary argument would be that there would be a tendency to preferentially test a male breast cancer case first in families with multiple affected individuals. In any case the risks to 70 and 80 years are similar to those found in the most recent study using a different approach to analysis [START_REF] Tai | Breast Cancer Risk Among Male BRCA1 and BRCA2 Mutation Carriers[END_REF]. Ours is also now the largest study of BRCA2 affected families. Based on the now four studies to assess male breast cancer risks using different study designs it would appear appropriate to quote lifetime risks (to age 70-80 years) of breast cancer to males in the Western world of between 6-9%.

These risk are sufficient to increase awareness of breast cancer amongst males in BRCA2 families and to stress the importance of early presentation with breast symptoms.

Table 1 :

 1 Assignment of male FDRs based on cancer diagnosis Kaplan Meier cumulative onset of breast cancer in 508 male BRCA2 carriers and presumed carriers. Risk to age 70 years: 7.1% standard error 5.2-8.6% Risk to age 80 years: 8.4% standard error 6.2-10.6%

	Cancer	Number	Number	Number	Number	Total
		testing	testing	at risk	assigned	number
		positive	negative	with	positive	in
		or		unknown	status	analysis
		(obligate		mutation
		carriers)		status
	Breast cancer	6 (1)	0	10	10	16
	Pancreatic cancer 4 (2)	0	6	5	9
	Stomach cancer	5 (4)	0	14	10	15
	Prostate cancer	17 (7)	0	29	24	41
	melanoma	1	1	4	2	3
	Lung cancer	0	0	22	11	11
	Bladder cancer	3 (1)	1	3	2	5
	Cholangiocarinoma 1	0	2	2	3
	Ampullary cancer	0	0	1	1	1
	Other cancer	13 (11)	0	33	16	29
	Unaffected	72 (19)	39	606	303	375
	Total	122 (45)	41	752	386	508

Acknowledgements:

This work was supported by the Biomedical Research Centre at Central Manchester Foundation Trust and a grant from the Genesis Appeal.

Conflict of interest: None

"the Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in JMG and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jmg.bmj.com/ifora/licence.pdf)."


Evans D Gareth
email: gareth.evans@cmft.nhs.uk

Munaza Ahmed

Stuart Bayliss

Emma Howard

Lalloo Fiona

Andrew Wallace

Prof. D Gareth Evans

Genetic Medicine

BRCA1, BRCA2 and CHEK2 c.1100 delC mutations in patients with double primaries of the breasts and/or ovaries

Background: Previous publications and utilisation of risk models for BRCA1 and BRCA2 mutation identification suggests that multiple primary disease in an individual is a strong predictor of a BRCA1/2 mutation and that this is more predictive than the same cancers occurring in close relatives.

Methods: This study assessed the pathological mutation detection rates for BRCA1, BRCA2 and the CHEK2 c.1100 delC mutation in 2022 women with breast cancer including 100 with breast/ovary double primary and 255 with bilateral breast cancer. Results and discussion: Although detection rates for mutations in BRCA1/2 are high at 49% for breast/ovarian double primary and 34% for bilateral breast cancer, the differential effect of multiple primaries in an individual appears to have been overestimated, particularly in those families with only a few malignancies. Nonetheless, bilateral breast cancer does differentially enhance detection rates in strong familial aggregations. CHEK2 1100 DelC mutation rates were lower in bilateral than for unilateral cases at 0.8% compared to 2%. The detected mutation rates for isolated double primary breast and ovarian cancer was 14% (3/22) compared to 17% (17/99) for the same two primaries in two close relatives in families with no other cases of breast/ovarian cancer. Risk models may need to be adjusted if further studies corroborate these findings.

Background

Since the identification of the BRCA1 [START_REF] Miki | A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1[END_REF] and BRCA2 [START_REF] Wooster | Identification of the breast cancer susceptibility gene BRCA2[END_REF] genes, there has been widespread testing of families and isolated cases with breast or ovarian cancer or a combination of the two. Whilst the chances of identifying a BRCA1/2 mutation increases with the increasing number and decreasing age of cancers in the family, there has been a suggestion that families containing individuals with breast and ovarian double primaries in particular have very high rates of mutation [START_REF] Schorge | Germline BRCA1-2 mutations in non-Ashkenazi families with double primary breast and ovarian cancer[END_REF][START_REF] Fishman | Patients with double primary tumors in the breast and ovary-clinical characteristics and BRCA1-2 mutations status[END_REF][START_REF] Cvelbar | Risk factors and prognostic factors in patients with double primary cancer: epithelial ovarian cancer and breast cancer[END_REF]. In contrast double primary/bilateral breast cancer has not been thought to be such a strong predictor [START_REF] Steinmann | Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer[END_REF] and may be over scored in current risk models [START_REF] Ready | Accuracy of the BRCAPRO model among women with bilateral breast cancer[END_REF]. More recently a third susceptibility gene, CHEK2 was associated with increased risk of breast cancer in families without BRCA mutations [START_REF] Schorge | Germline BRCA1-2 mutations in non-Ashkenazi families with double primary breast and ovarian cancer[END_REF]. In contrast to BRCA1/2, the CHEK2 c.1100 delC mutation has been reported to be strongly associated with bilateral breast cancer [START_REF] Fletcher | Family history, genetic testing, and clinical risk prediction: pooled analysis of CHEK2 c.1100delC in 1,828 bilateral breast cancers and 7,030 controls[END_REF][START_REF] Johnson | Interaction between CHEK2*c.1100delC and other low-penetrance breast-cancer susceptibility genes: a familial study[END_REF][START_REF] Broeks | Excess risk for contralateral breast cancer in CHEK2*c.1100delC germline mutation carriers[END_REF].

In our population we did not feel that the predictive value of double primary disease was as great as the literature suggested or that the existing models predicted. We therefore aimed to assess the predictive value of double primary disease in identifying mutations in BRCA1, BRCA2 and CHEK2. This study reviewed the screening for BRCA1/2 mutations in North West England amongst non Jewish kindreds and assessed the impact of the presence of multiple primary disease on the identification of BRCA1/2 mutations and the presence of CHEK2 c.1100delC.

Methods

Index cases and relatives

Breast and ovarian cancer families from North-West England have been screened for BRCA1/2 mutations since 1996. The greater Manchester area is below average for socio-economic status and life expectancy is shortened and cancer incidence generally higher although breast cancer rates are similar to UK averages. Both confirmation of cancer diagnoses and mutation testing is undertaken with informed consent. The study was carried out with Local Ethical committee approval. Women who attend the specialist genetic clinics in this region with a family history of breast/ovarian cancer have a detailed family tree elicited with all first, second and if possible third degree relatives recorded. All cases of breast or abdominal cancers are confirmed by means of hospital/pathology records, from the Regional Cancer Registries (data available from 1960) or from death certification with particular attention to confirming a diagnosis of ovarian cancer and contralateral breast cancer to accurately identify cases of double primaries.

Cases were selected for local NHS mutation testing if they were assessed as having at least a 20% chance of a mutation being identified using the Manchester Score., This included all individuals with double primary breast/ovary. Cases below the 20% threshold, but in whom there were at least two breast cancers in the family were selected for research testing. Only individuals affected with breast or ovarian cancer were selected for testing as per UK guidelines. Index cases were tested with a whole gene approach which since 2000 has consisted of sequencing of all BRCA1 and BRCA2 coding exons including the flanking splice donor and acceptor sites and Multiple Ligation dependant Probe Amplification (MLPA) (MRC-Holland P002 and P045 probe sets). As part of the MRC-Holland P045 MLPA kit a test for the CHEK2 c.1100delC mutation is incorporated. 1140 samples not meeting NICE guidelines for NHS testing have been tested on a research basis at the Institute of Cancer Research in Sutton employing a similar whole gene approach. The full coding sequence and intron-exon boundaries of both BRCA1 and BRCA2 were screened in 86 fragments by Conformation Sensitive Gel Electrophoresis (CSGE) as previously described [START_REF] Evans | A new scoring system for the chances of identifying a BRCA1/2 mutation, outperforms existing models including BRCAPRO[END_REF] as well as MLPA and CHEK2 c.1100 delC testing. All families were subject to the same degree of cancer confirmation. All pathogenic mutations for BRCA1 and BRCA2 are noted and placed onto a dedicated Filemaker Pro 7 database. Women with self reported Jewish ancestry were initially multiplex tested for the three common mutations and all such women were excluded as were families identified through testing in other genetics centres.

All 2022 females (882 NHS + 1140 research) affected with any case of breast cancer and tested for BRCA1/2 mutations were included in this study along with 36 cases of ovarian malignancy with a family history of a single breast cancer (for comparison with an isolated double primary). Data were collected on the family history, age of cancer diagnosis, the presence of further primary cancer and whether or not a male breast cancer or ovarian cancer was confirmed in a relative. The Manchester scoring system was used to assess the significance of the breast/ovarian cancer history [START_REF] Evans | A new scoring system for the chances of identifying a BRCA1/2 mutation, outperforms existing models including BRCAPRO[END_REF]. This system assesses the likelihood of a BRCA1/2 mutation and scores breast and ovarian cancers individually in the family, giving a higher score the younger the age at diagnosis [START_REF] Evans | A new scoring system for the chances of identifying a BRCA1/2 mutation, outperforms existing models including BRCAPRO[END_REF]. A combined score of 20 reflects a 20% likelihood of identifying a BRCA1/2 mutation, with a score of 15 representing a 10% threshold. All details including the Manchester score were entered onto the Filemaker Pro 7 database. Proportions of each subset of double primary identified with mutations were compared where appropriate with chi squared testing. Carrier probabilities with BRCAPRO [START_REF] Parmigiani | Determining carrier probabilities for breast cancer susceptibility genes BRCA1 and BRCA2[END_REF] and Myriad tables [START_REF] Frank | Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: analysis of 10,000 individuals[END_REF] were calculated with the CancerGene software package from the University of Texas Southwestern

Medical Center (CaGene version 4.0). Additional information about

CancerGene is available at http://www.utsouthwestern.edu.

Results

In total, 2022 females with breast cancer and 36 with ovarian cancer only were screened for mutations in BRCA1 and BRCA2 with a whole gene approach. A pathogenic BRCA1 or BRCA2 mutation was identified in 322 (16%) women.

Table 1 shows the proportion of mutations identified in families containing either a case with breast-ovary double primary or a pair of first degree relatives where one had breast cancer and the other ovarian cancer. All cases of breast/ovary double primary were confirmed mostly from pathology and cancer registry data.

Although the overall mutation rate in families containing a case with breastovary double primary was high at 49/100 (49%),there was no difference in mutation detection between an isolated (sporadic) breast-ovary double primary 3/22 (14%) and a breast and ovarian First degree relative (FDR) pair without any other family history 17/99 (17%) (p = 0.93). Only the FDR pairs with breast and ovarian cancer diagnosed with a Manchester score of 20 or higher breached the 20% UK threshold for testing [START_REF] Mcintosh | Clinical Guidelines and Evidence Review for The Classification and Care of Women at Risk of Familial Breast Cancer[END_REF].

Comparison of the mutation detection rates in women with bilateral breast cancer are shown in table 2. Attempts were made to confirm all double primaries and >90% were confirmed as separate primaries mostly from pathology and cancer registry data. In 21 cases there was clear evidence of a previous contralateral breast cancer treatment but histology was not obtainable and these were still included. There was a range of 0-36 years between primaries with a mean of 8.2 years. In eight cases the first primary was DCIS and in three the contralateral was in situ disease. The detection rate was higher in those with bilateral disease 34% (87/255; 44 BRCA1; 43 BRCA2) versus unilateral disease14.5% (255/1767) (table 3). Rates increased to 61% (27% BRCA1 and 34% BRCA2) and 34% (25% BRCA1 and 9% BRCA2) in families with an ovarian cancer case and 82% (9% BRCA1 and 73% BRCA2) and 28% (3% BRCA1 and 25% BRCA2) respectively in families with a male breast cancer (MBC) case. The rates decreased to 23% in bilateral cases and 9.5% in unilateral breast cancer cases without a family history of either ovarian or male breast cancer. Selection of women with their first primary aged <45 years also increases the likelihood of detecting a BRCA1/2 mutation but this effect in bilateral cases is largely accounted for by the age and number of breast cancer cases in the family as there is not any differential increase in detection rates in families with similar Manchester scores. The distribution of unilateral and bilateral cases also showed a clear dependence (data not shown). CHEK2 c.1100delC mutations were found slightly less frequently amongst the bilateral breast cancer group with 2/240 (0.8%) compared to 33/1660 (2%) in the unilateral group (p=0.3). This difference narrowed when the individuals with BRCA1 or BRCA2 mutations were excluded (table 3). BRCAPRO appears to overestimate the likelihood of a BRCA1 mutation in particular in individuals with multiple primaries (p=0.0002 for bilateral breast cancer; p=0.08 for breast/ovary). For a breast/ovary double primary this is by a factor of 33% (49 expected 36 found; table 1) and for bilateral breast cancer by close to 100% (table 2). Myriad tables are particularly poor at prediction in bilateral cases significantly underestimating the number of mutations (p=0.0001). Table 4 shows the predictions for various likelihood levels for Manchester scoring, BRCAPRO and Myriad. The poor discrimination of the Myriad tables is demonstrated by the fact that 23/125 (18%) of samples with a Myriad score of <10% have mutations. The sensitivity of the models and scoring system are shown in table 5 at the 10 and 20% threshold.

There was a consistently higher detection rate for bilateral cases than unilateral cases for Manchester scores above the 10% 16 point threshold (table 6). At the 16-19 points and 20-24 point levels this was borderline significant (p=0.052; and 0.048 respectively) at the 30-39 points this was highly significant statistical significance (p<0.0001). Detection rates at <16 points were virtually identical with only 1/62 bilateral cases having a mutation. By adjusting the bilateral scores upwards by adding the combined BRCA2 scores for the bilateral breast cancers a second time (equivalent to a 50% upward adjustment-table 6), but only for combined scores above the 15 point threshold, this equalised the detection rates between unilateral and bilateral cases at similar Manchester scores.

Discussion

This report has documented the involvement of BRCA1, BRCA2 and CHEK2 c.1100 delC mutations in breast cancer patients with double primaries of the breasts and/or ovaries. It has previously been reported that the mutation rates in BRCA1 and BRCA2 are higher with double primaries than in series of unilateral breast cancer. However, the literature is unclear as to whether identification of an individual with two separate primaries is more predictive of a BRCA1 or BRCA2 mutation than the same two primaries in a first degree pair. The results of our analysis do not suggest that any mutations are more likely with a breast and ovarian double primary than with the same two primaries in two close relatives. Although the overall rate of mutations is high at 49% (49/100), the rate in a case with breast-ovary double primary without a family history, is below 20%. This is in contrast to the high rates suggested from previous reports [START_REF] Schorge | Germline BRCA1-2 mutations in non-Ashkenazi families with double primary breast and ovarian cancer[END_REF][START_REF] Fishman | Patients with double primary tumors in the breast and ovary-clinical characteristics and BRCA1-2 mutations status[END_REF]. However, this rate is similar to that for isolated FDR pairs one with breast cancer and one with ovarian cancer. The Manchester scoring system [START_REF] Evans | A new scoring system for the chances of identifying a BRCA1/2 mutation, outperforms existing models including BRCAPRO[END_REF] does not alter if an individual has two primaries as each tumour receives a score dependant on age at diagnosis. The data from this study validates this approach for small aggregations particularly under the 10% threshold. It is not clear from the publications whether other risk models predict an increase in the likelihood of a mutation based on the presence of a double primary of breast and ovary compared to the same cancers in a FDR pair [START_REF] Parmigiani | Determining carrier probabilities for breast cancer susceptibility genes BRCA1 and BRCA2[END_REF][START_REF] Antoniou | The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions[END_REF]. However, it is possible to assess this using a nuclear family of an unaffected mother aged 75 and two daughters aged 55 years. An assessment of the extra weight of the individual with two primaries as opposed to a sibling pair can be made. If a breast and ovarian cancer occurred only in one sister with no cancer in the other, the combined likelihood of a BRCA1/2 mutation was 32.3% with BRCAPRO and 31.8% with BOADICEA. This dropped to 16% and 15.1% respectively if the two cancers occurred separately in the two sisters. This apparent doubling of likelihood is contradicted by the almost identical rates of BRCA1/2 mutation in the similar nuclear families tested 3/22 (14%) for double primary and (17%) 17/99 for FDR pair. The only group in which the BRCA1/2 mutation rate increased was in the group of patients with both bilateral breast cancer and ovarian cancer without any other significant family history (ie triple primary). However, this is based on small numbers of cases. BRCAPRO did overpredict the number of mutations amongst breast/ovary double primaries but this was not significant (p=0.08) and the numbers are probably not powered to detect this.

In contrast to cases with double primary breast and ovary diagnoses, the detection rates in bilateral breast cancer appeared to be higher than expected based on the two tumours in FDR pairs. Based on the same range of Manchester score the detection rates were consistently higher for bilateral than unilateral cases. In the range of 20-24 points and 30-39 points this reached statistical significance (p=0.048; and >0.0001 respectively). However, this difference was not apparent for lower Manchester scores. If a similar scenario to the nuclear example for breast and ovary is worked for bilateral breast cancer compared to unilateral breast cancer in siblings aged 55 years (one with diagnosis aged 45 years) the scores for BRCAPRO and BOADICEA are: 8.7% and 9.3% for a BRCA1/2 mutation in the double primary compared to 4.1% and 3.8% respectively for two siblings. Again this suggests at least a doubling of the likelihood. The Manchester score in this setting is 10 points (5 for each gene) [START_REF] Evans | A new scoring system for the chances of identifying a BRCA1/2 mutation, outperforms existing models including BRCAPRO[END_REF]. There was not a difference in the detection rate at this Manchester score.

There was only a significant difference in detection rates between bilateral and unilateral cases if the rest of the family history was significant. This may be explained by lower phenocopy rates in bilateral cases compared to unilateral cases [START_REF] Smith | The trouble with phenocopies: are those testing negative for a family BRCA1/2 mutation really at population risk?[END_REF]. The rate of phenocopies in our series in non index cases with breast cancer only is 4% (1/26) for bilateral patients and 17% (32/183) for unilateral cases, although this does not reach statistical significance (p=0.08). This data would suggest that bilateral breast cancer only requires an upward adjustment in mutation probability when there is a concurrent strong family history above the 10% threshold and this adjustment is probably still too high in existing models. Our findings are in contrast to a number of previous reports that do not indicate that bilateral disease per se is a strong indicator of the presence of a BRCA1/2 mutation [START_REF] Steinmann | Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer[END_REF][START_REF] Gershoni-Baruch | BRCA1 and BRCA2 founder mutations in patients with bilateral breast cancer[END_REF]. This would appear to be true in isolated cases or those with minimal family history, but in families with higher Manchester scores, reflecting a strong family history of breast cancer, the rates of detection rise markedly. This has been previously suggested by a Danish study of 119 patients with bilateral or multifocal disease aged <46 years [START_REF] Bergthorsson | BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age[END_REF].

One of the previous studies suggested that early age at onset of first primary (<42 years) was a relative predictor of mutation status [START_REF] Gershoni-Baruch | BRCA1 and BRCA2 founder mutations in patients with bilateral breast cancer[END_REF] but data from this study suggest that this is already accounted for in the Manchester score.

However, division by this age group is likely to be artificial as it introduces a confounding bias as women diagnosed <45 years who have developed bilateral disease will have longer follow up than unilateral cases.

A recent study has also questioned the significance of bilateral disease in BRCAPRO [START_REF] Ready | Accuracy of the BRCAPRO model among women with bilateral breast cancer[END_REF]. Ready et al demonstrated that for bilateral cases with a carrier probability >31%, the proportion of positive tests was significantly lower than predicted by the BRCAPRO model (P < .05). Taking the same cut off in our study, 120/168 bilateral breast cancers with BRCAPRO scores above 31% were predicted to have a BRCA1/2 mutation whereas only 75 did (Fishers exact p<0.0001). It may be that models are overestimating the effect of bilateral breast cancer even in families with a significant family history. The simple formula of doubling the chance of a mutation because presumably two breast cancers have occurred in only two breasts rather than 2 in four in a sibling pair may be incorrect. Non genetic factors such as hormonal factors, may increase the risk of bilateral disease in one sibling whilst being discordant in a sister. For example one sibling may have experienced menarche at 9 years and not had her first full term pregnancy until 35 years whilst the unaffected sister experienced menarche at 16 years and first pregnancy at 19 years.

A direct comparison of the discrimination between BRCAPRO and the Manchester score is not appropriate as a small part of the dataset was used to develop the Manchester score (48 breast ovary double primaries and 52 bilateral breast cancers in the 472 developmental set). Nonetheless it can be seen that the Manchester score continues to discriminate well at both the 10% and 20% threshold even in the specific sets of multiple primary tumours.

Whilst Myriad tables work reasonably well for breast-ovary double primaries they should not be used for bilateral breast cancer as the tables did not include this parameter. An intriguing component of the BRCAPRO overestimation can be seen from the fact that in 37 patients predicted to have >98% chance of a mutation only 27 (73% p=0.007) had one. Even allowing for a drop to a 90% mutation detection sensitivity this is still a large overestimate.

Both the Manchester score and Myriad tables can identify a subset with at least an 80% chance of a mutation (table 4). The various scoring systems and models are being improved with pathology now incorporated into BRCAPRO and the Manchester score [START_REF] Evans | Addition of pathology and biomarker information significantly improves the performance of the Manchester scoring system for BRCA1 and BRCA2 testing[END_REF]. Using the pathology adjusted score of 40+ the Manchester score identifies 35/39 (90%) of cases with ovarian cancer as having a BRCA1/2 mutation. There are possible further explanations for why BRCAPRO may overestimate in bilateral sample sets these include: misdiagnosis of metastatic disease rather than a second primary, selection of less aggressive breast cancers and lower population frequency of BRCA1/2 than in the BRCAPRO algorithm. As genetic testing cannot be offered in families where all the affected patients have died, this could create a selection bias against aggressive disease.

Our results for CHEK2 c.1100delC are in contrast to a number of previous studies that suggest that this mutation is strongly associated with bilateral disease [START_REF] Fletcher | Family history, genetic testing, and clinical risk prediction: pooled analysis of CHEK2 c.1100delC in 1,828 bilateral breast cancers and 7,030 controls[END_REF][START_REF] Broeks | Excess risk for contralateral breast cancer in CHEK2*c.1100delC germline mutation carriers[END_REF][START_REF] Johnson | Interaction between CHEK2*c.1100delC and other low-penetrance breast-cancer susceptibility genes: a familial study[END_REF][START_REF] Kilpivaara | Correlation of CHEK2 protein expression and c.c.1100delC mutation status with tumor characteristics among unselected breast cancer patients[END_REF]. The odds ratio for second primary for CHEK2 c.1100delC carriers was 6.43 [START_REF] Fletcher | Family history, genetic testing, and clinical risk prediction: pooled analysis of CHEK2 c.1100delC in 1,828 bilateral breast cancers and 7,030 controls[END_REF] and 6.5 [START_REF] Broeks | Excess risk for contralateral breast cancer in CHEK2*c.1100delC germline mutation carriers[END_REF] in two large studies of bilateral disease and significantly higher than for unilateral cases. The high contralateral rate was also suggested by the Peto group to have implications for relatives of CHEK2 c.1100delC carriers with bilateral disease although the 12 fold relative risk was based on only 8 breast cancers [START_REF] Johnson | Interaction between CHEK2*c.1100delC and other low-penetrance breast-cancer susceptibility genes: a familial study[END_REF]. It is not clear why this analysis did not detect any difference between unilateral and bilateral cases although the selection criteria for testing meant that nearly all unilateral cases had a family history of breast cancer. The 2% frequency for CHEK2 c.1100delC is at least 4 times our local population rate [24]. The largest series by Fletcher et al 2009 [START_REF] Fletcher | Family history, genetic testing, and clinical risk prediction: pooled analysis of CHEK2 c.1100delC in 1,828 bilateral breast cancers and 7,030 controls[END_REF] was ascertained from multiple sources including a UK series selected on family history. CHEK2 1100 delC was found in 10/321 (3.1%) compared to 6/1027 (0.6%) Controls OR=5.47 (1.78-18.43). The 3.1% rate is not statistically significantly greater than either our unilateral or bilateral rates. We feel it is still premature to use CHEK2 testing to guide patient management.

In summary, this study of over 2000 breast cancer cases tested for BRCA1/2 and CHEK2 c.1100delC mutations has identified a number of differences with previous reports. The effects of a case with double primary diagnoses do not appear to be as great in mutation prediction as currently utilised in prediction models, although bilateral breast cancer is still a strong predictor in large familial aggregations. CHEK2 does not appear to be as strongly linked to bilateral breast cancer as previously suggested in individuals who have a strong family history.

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Competing Interest: None to declare.

Acknowledgements

DGE and FL are supported by the Manchester NIHR Biomedical Research centre. We would also like to thank Prof Nazneen Rahman and her team at the Institute of Cancer Research, Margaret Warren-Perry, Sheila Seal, Anthony Renwick, Clare Turnbull, Deborah Hughes and Sarah Hines for screening samples for BRCA1/2 and CHEK2 as part of the Familial Breast Cancer Study, which is funded by Cancer Research UK. We would also like to thank Linda Ashcroft Biostatistician at Christie Hospital for her input.