5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints.

BACKGROUND: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. METHODS: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. RESULTS: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. CONCLUSIONS: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

Clinical relevance of 8q23, 15q13 and 18q21 SNP genotyping to evaluate colorectal cancer risk.

To determine if the at-risk single-nucleotide polymorphism (SNP) alleles for colorectal cancer (CRC) could contribute to clinical situations suggestive of an increased genetic risk for CRC, we performed a prospective national case-control study based on highly selected patients (CRC in two first-degree relatives, one before 61 years of age; or CRC diagnosed before 51 years of age; or multiple primary CRCs, the first before 61 years of age; exclusion of Lynch syndrome and polyposes) and controls without personal or familial history of CRC. SNPs were genotyped using SNaPshot, and statistical analyses were performed using Pearson's χ(2) test, Cochran-Armitage test of trend and logistic regression. We included 1029 patients and 350 controls. We confirmed the association of CRC risk with four SNPs, with odds ratio (OR) higher than previously reported: rs16892766 on 8q23.3 (OR: 1.88, 95% confidence interval (CI): 1.30-2.72; P=0.0007); rs4779584 on 15q13.3 (OR: 1.42, CI: 1.11-1.83; P=0.0061) and rs4939827 and rs58920878/Novel 1 on 18q21.1 (OR: 1.49, CI: 1.13-1.98; P=0.007 and OR: 1.49, CI: 1.14-1.95; P=0.0035). We found a significant (P<0.0001) cumulative effect of the at-risk alleles or genotypes with OR at 1.62 (CI: 1.10-2.37), 2.09 (CI: 1.43-3.07), 2.87 (CI: 1.76-4.70) and 3.88 (CI: 1.72-8.76) for 1, 2, 3 and at least 4 at-risk alleles, respectively, and OR at 1.71 (CI: 1.18-2.46), 2.29 (CI: 1.55-3.38) and 6.21 (CI: 2.67-14.42) for 1, 2 and 3 at-risk genotypes, respectively. Combination of SNPs may therefore explain a fraction of clinical situations suggestive of an increased risk for CRC.

Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes.

To optimize the molecular diagnosis of hereditary breast and ovarian cancer (HBOC), we developed a next-generation sequencing (NGS)-based screening based on the capture of a panel of genes involved, or suspected to be involved in HBOC, on pooling of indexed DNA and on paired-end sequencing in an Illumina GAIIx platform, followed by confirmation by Sanger sequencing or MLPA/QMPSF. The bioinformatic pipeline included CASAVA, NextGENe, CNVseq and Alamut-HT. We validated this procedure by the analysis of 59 patients' DNAs harbouring SNVs, indels or large genomic rearrangements of BRCA1 or BRCA2. We also conducted a blind study in 168 patients comparing NGS versus Sanger sequencing or MLPA analyses of BRCA1 and BRCA2. All mutations detected by conventional procedures were detected by NGS. We then screened, using three different versions of the capture set, a large series of 708 consecutive patients. We detected in these patients 69 germline deleterious alterations within BRCA1 and BRCA2, and 4 TP53 mutations in 468 patients also tested for this gene. We also found 36 variations inducing either a premature codon stop or a splicing defect among other genes: 5/708 in CHEK2, 3/708 in RAD51C, 1/708 in RAD50, 7/708 in PALB2, 3/708 in MRE11A, 5/708 in ATM, 3/708 in NBS1, 1/708 in CDH1, 3/468 in MSH2, 2/468 in PMS2, 1/708 in BARD1, 1/468 in PMS1 and 1/468 in MLH3. These results demonstrate the efficiency of NGS in performing molecular diagnosis of HBOC. Detection of mutations within other genes than BRCA1 and BRCA2 highlights the genetic heterogeneity of HBOC.

Multiple sequence variants of BRCA2 exon 7 alter splicing regulation.

BACKGROUND: Exonic variants of unknown biological significance (VUS) identified in patients can affect mRNA splicing, either by changing 5' or 3' splice sites or by modifying splicing regulatory elements. Bioinformatic predictions of these elements are still inaccurate and only few such elements have been functionally mapped in BRCA2. We studied the effect on splicing of eight exon 7 VUS, selected from the French UMD-BRCA2 mutation database. METHODS: We performed splicing minigene assays and analyses of patient RNA. We also developed a pyrosequencing-based quantitative assay, to measure, in patient RNA, the relative contribution of each allele to the production of exon 7-containing transcripts. Moreover, an exonic splicing enhancer (ESE)-dependent minigene assay was used to evaluate the splicing regulatory properties of wild-type and mutant segments. RESULTS: Six out of the eight variants induced splicing defects. In the minigene assay, c.517G>T and c.631G>A altered the natural splice sites, c.572A>G created a new 5' splice site, and c.520C>T, c.587G>A and c.617C>G induced exon 7 skipping (66%, 25% and 46%, respectively). Pyrosequencing of patient RNA confirmed these levels of exon skipping for c.520C>T and c.617C>G. Results from the ESE-dependent minigene assay indicated that c.520C>T and c.587G>A disturb splicing regulatory elements. CONCLUSIONS: BRCA2 exon 7 splicing is regulated by multiple exonic elements and is sensitive to disease-associated sequence variations. Measurements of allelic imbalance in patient-derived RNA and/or quantitative analyses using minigene assays provide valuable estimates of the extent of partial splicing defects. Assessment of pathogenicity of variants with partial splicing effect awaits additional evidence and especially the completion of segregation analyses.

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.

Assessment of SPAG9 Transcript in Fine Needle Aspirates of Thyroid Nodules.

OBJECTIVES: Sperm-associated antigen 9 (SPAG9) has been suggested as a possible biomarker in several malignancies including thyroid cancer. We investigated the expression of SPAG9 mRNA in fine needle aspiration (FNA) material from papillary thyroid carcinoma (PTC) and benign thyroid nodules. STUDY DESIGN: SPAG9 expression was assessed in 36 FNA samples corresponding to 16 PTC and 20 benign nodules using the original method detecting the SPAG9 transcript containing intron 21 (NCBI X91879). The presence of the BRAF V600E point mutation was also analyzed by pyrosequencing. RESULTS: Six of 16 (38%) PTC samples were positive for X91879 SPAG9 transcript compared to 8 of 20 (40%) benign samples (p = 0.88). Out of 12 BRAF-positive PTC, 3 (25%) also expressed the SPAG9 transcript compared to 3 out of 4 BRAF-negative PTC (75%; p = 0.12). CONCLUSIONS: The X91879 SPAG9 transcript originally described does not appear to be overexpressed in FNA material from PTC or to be clinically relevant in the diagnosis of thyroid nodules.

Contribution of bioinformatics predictions and functional splicing assays to the interpretation of unclassified variants of the BRCA genes.

A large fraction of sequence variants of unknown significance (VUS) of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 may induce splicing defects. We analyzed 53 VUSs of BRCA1 or BRCA2, detected in consecutive molecular screenings, by using five splicing prediction programs, and we classified them into two groups according to the strength of the predictions. In parallel, we tested them by using functional splicing assays. A total of 10 VUSs were predicted by two or more programs to induce a significant reduction of splice site strength or activation of cryptic splice sites or generation of new splice sites. Minigene-based splicing assays confirmed four of these predictions. Five additional VUSs, all at internal exon positions, were not predicted to induce alterations of splice sites, but revealed variable levels of exon skipping, most likely induced by the modification of exonic splicing regulatory elements. We provide new data in favor of the pathogenic nature of the variants BRCA1 c.212+3A>G and BRCA1 c.5194-12G>A, which induced aberrant out-of-frame mRNA forms. Moreover, the novel variant BRCA2 c.7977-7C>G induced in frame inclusion of 6 nt from the 3' end of intron 17. The novel variants BRCA2 c.520C>T and BRCA2 c.7992T>A induced incomplete skipping of exons 7 and 18, respectively. This work highlights the contribution of splicing minigene assays to the assessment of pathogenicity, not only when patient RNA is not available, but also as a tool to improve the accuracy of bioinformatics predictions.

Two novel variants in the 3'UTR of the BRCA1 gene in familial breast and/or ovarian cancer.

For the majority of breast and/or ovarian cancer patients tested for BRCA1/2 genes, mutation screening of the coding regions remains negative. MicroRNAs which negatively regulate mRNA translation by binding to 3' untranslated region (3'UTR) are implicated in cancer. Genetic changes in the 3'UTR of several genes were reported to be associated with higher susceptibility to particular tumor types. The aim of this study was to analyze the BRCA1 3'UTR in patients tested negative for BRCA1/2 deleterious mutations, in order to find variants implicated in the decrease of BRCA1 expression through modification of miRNA binding. Genotyping analyses were performed on genomic DNA of 70 BRCA negatives index cases, selected among patients with breast or ovarian cancer, less than 50 years old, with a strong family history. The co-occurrence of the identified variants with deleterious BRCA1 mutations was then determined in a control population of 210 patients. A luciferase gene reporter assay was used to investigate the impact of the variants on the BRCA1 gene expression. Two novel variants, c.*750A>G and c.*1286C>A, were identified in the 3'UTR of BRCA1 gene, in two patients. The former was found three times in the control population, whereas the latter was absent. The used functional assay did not reveal any effect on the luciferase expression. This study reveals a weak genomic variability in the 3'UTR of the BRCA1 gene. All together, the results led us to classify the variant c.*750A>G as probably neutral, the variant c.*1286C>A remaining unclassified.

One-year longitudinal study of fatigue, cognitive functions, and quality of life after adjuvant radiotherapy for breast cancer.

PURPOSE: Most patients with localized breast cancer (LBC) who take adjuvant chemotherapy (CT) complain of fatigue and a decrease in quality of life during or after radiotherapy (RT). The aim of this longitudinal study was to compare the impact of RT alone with that occurring after previous CT on quality of life. METHODS AND MATERIALS: Fatigue (the main endpoint) and cognitive impairment were assessed in 161 CT-RT and 141 RT patients during RT and 1 year later. Fatigue was assessed with Functional Assessment of Cancer Therapy-General questionnaires, including breast and fatigue modules. RESULTS: At baseline, 60% of the CT-RT patients expressed fatigue vs. 33% of the RT patients (p <0.001). Corresponding values at the end of RT were statistically similar (61% and 53%), and fatigue was still reported at 1 year by more than 40% of patients in both groups. Risk factors for long-term fatigue included depression (odds ratio [OR] = 6), which was less frequent in the RT group at baseline (16% vs. 28 %, respectively, p = 0.01) but reached a similar value at the end of RT (25% in both groups). Initial mild cognitive impairments were reported by RT (34 %) patients and CT-RT (24 %) patients and were persistent at 1 year for half of them. No biological disorders were associated with fatigue or cognitive impairment. CONCLUSIONS: Fatigue was the main symptom in LBC patients treated with RT, whether they received CT previously or not. The correlation of persistent fatigue with initial depressive status favors administering medical and psychological programs for LBC patients treated with CT and/or RT, to identify and manage this main quality-of-life-related symptom.

A one-step prescreening for point mutations and large rearrangement in BRCA1 and BRCA2 genes using quantitative polymerase chain reaction and high-resolution melting curve analysis.

High-resolution melting (HRM) of DNA is a versatile method for mutation scanning that monitors the fluorescence of double-strand DNA with saturating dye. Performing HRM on a real-time thermocycler enables semiquantitative analysis (quantitative polymerase chain reaction, qPCR) to be associated to HRM analysis for detection of both large gene rearrangements and point mutations (qPCR-HRM). We evaluated this method of mutation screening for the two major breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. Screening of these two genes is time-consuming and must include exploration of large rearrangements that represent 5% to 15% of the alterations observed in these genes. To assess the reliability of the HRM technology, 201 known nucleotide variations scattered over all amplicons were tested. The sensitivity of qPCR was evaluated by analyzing seven large rearrangements. All previously identified variants tested were detected by qPCR-HRM. A retrospective study was done with 45 patients: qPCR-HRM allowed all the variants previously tested by denaturing high-performance liquid chromatography to be identified. qPCR analysis showed three cases of allele dropout (due to a 104-bp deletion, SNP primer mismatch, and an Alu insertion). A prospective study was done with 165 patients allowing 22 deleterious mutations, 16 unclassified variants, and 2 rearrangements to be detected. qPCR-HRM is a simple, sensitive, and fast method that does not require modified PCR primers. Thus, this method allows in one step the detection of point mutation, gene rearrangements, and prevention of missing a mutation due to primer mismatch.

A locus on 19p13 modifies risk of breast cancer in BRCA1 mutation carriers and is associated with hormone receptor-negative breast cancer in the general population.

Germline BRCA1 mutations predispose to breast cancer. To identify genetic modifiers of this risk, we performed a genome-wide association study in 1,193 individuals with BRCA1 mutations who were diagnosed with invasive breast cancer under age 40 and 1,190 BRCA1 carriers without breast cancer diagnosis over age 35. We took forward 96 SNPs for replication in another 5,986 BRCA1 carriers (2,974 individuals with breast cancer and 3,012 unaffected individuals). Five SNPs on 19p13 were associated with breast cancer risk (P(trend) = 2.3 × 10⁻9 to P(trend) = 3.9 × 10⁻7), two of which showed independent associations (rs8170, hazard ratio (HR) = 1.26, 95% CI 1.17-1.35; rs2363956 HR = 0.84, 95% CI 0.80-0.89). Genotyping these SNPs in 6,800 population-based breast cancer cases and 6,613 controls identified a similar association with estrogen receptor-negative breast cancer (rs2363956 per-allele odds ratio (OR) = 0.83, 95% CI 0.75-0.92, P(trend) = 0.0003) and an association with estrogen receptor-positive disease in the opposite direction (OR = 1.07, 95% CI 1.01-1.14, P(trend) = 0.016). The five SNPs were also associated with triple-negative breast cancer in a separate study of 2,301 triple-negative cases and 3,949 controls (P(trend) = 1 × 10⁻7) to P(trend) = 8 × 10⁻5; rs2363956 per-allele OR = 0.80, 95% CI 0.74-0.87, P(trend) = 1.1 × 10⁻7

The BRCA1 c.5434C->G (p.Pro1812Ala) variant induces a deleterious exon 23 skipping by affecting exonic splicing regulatory elements.

BACKGROUND A large fraction of the sequence variants of unknown significance or unclassified variants (UVs) could be pathogenic by affecting mRNA splicing. The breast and ovarian cancer susceptibility gene BRCA1 exhibits a large spectrum of sequence variation but only two variants, both located in exon 18, have been shown experimentally to affect splicing regulatory elements. The present study investigated the impact on splicing of the variant BRCA1 c.5434C-->G (p.Pro1812Ala), identified in an ovarian cancer patient. This variant has previously been studied at the protein level with inconclusive results concerning its pathogenic role. METHODS Analysis of RNA from patient peripheral blood was performed by RT-PCR. The effect of the variant was tested by using splicing reporter hybrid minigene assays. RESULTS Using patient RNA analyses and hybrid minigene assays, we showed that this variant induces a major splicing defect, with skipping of exon 23, resulting in frameshift and predicted protein termination within the second BRCT domain. Moreover, we showed that the segment c.5420-5449 of BRCA1, in the centre of exon 23, exhibits splicing enhancer properties. This enhancement is abolished by the c.5434C-->G mutation, indicating that the nucleotide change, in this highly conserved region, affects a splicing regulatory element. Bioinformatics analyses predict that the mutation c.5434C-->G creates an hnRNPA1 dependent splicing silencer. CONCLUSION These data, together with segregation data, argue for the classification of BRCA1 c.5434C-->G as a pathogenic splicing mutation. These results also suggest that UVs in highly conserved nucleotide sequences of short exons may be good candidates for detecting functionally relevant splicing regulatory elements.

AURKA F31I polymorphism and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a consortium of investigators of modifiers of BRCA1/2 study.

The AURKA oncogene is associated with abnormal chromosome segregation and aneuploidy and predisposition to cancer. Amplification of AURKA has been detected at higher frequency in tumors from BRCA1 and BRCA2 mutation carriers than in sporadic breast tumors, suggesting that overexpression of AURKA and inactivation of BRCA1 and BRCA2 cooperate during tumor development and progression. The F31I polymorphism in AURKA has been associated with breast cancer risk in the homozygous state in prior studies. We evaluated whether the AURKA F31I polymorphism modifies breast cancer risk in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. Consortium of Investigators of Modifiers of BRCA1/2 was established to provide sufficient statistical power through increased numbers of mutation carriers to identify polymorphisms that act as modifiers of cancer risk and can refine breast cancer risk estimates in BRCA1 and BRCA2 mutation carriers. A total of 4,935 BRCA1 and 2,241 BRCA2 mutation carriers and 11 individuals carrying both BRCA1 and BRCA2 mutations was genotyped for F31I. Overall, homozygosity for the 31I allele was not significantly associated with breast cancer risk in BRCA1 and BRCA2 carriers combined [hazard ratio (HR), 0.91; 95% confidence interval (95% CI), 0.77-1.06]. Similarly, no significant association was seen in BRCA1 (HR, 0.90; 95% CI, 0.75-1.08) or BRCA2 carriers (HR, 0.93; 95% CI, 0.67-1.29) or when assessing the modifying effects of either bilateral prophylactic oophorectomy or menopausal status of BRCA1 and BRCA2 carriers. In summary, the F31I polymorphism in AURKA is not associated with a modified risk of breast cancer in BRCA1 and BRCA2 carriers.

Influence of CA 15-3 blood level and doubling time on diagnostic performances of 18F-FDG PET in breast cancer patients with occult recurrence.

AIM: To evaluate the influence of CA 15-3 blood level and doubling time on diagnostic performances of 18F-FDG PET in breast cancer patients with occult recurrence. MATERIALS AND METHODS: Thirty-five 18F-FDG PET examinations in 32 patients with CA 15-3 blood level above the normal range, and negative conventional imaging within 3 months before PET examination were included in this retrospective study. PET examinations were reviewed blindly by two experienced nuclear medicine physicians who were unaware of any clinical, biological or radiological information. CA 15-3 assays performed prior to the PET examinations and all using the same technique were collected and used for doubling time calculation if (1) no therapeutic modification occurred in the meantime, and (2) the delay between assays was less than 6 months. RESULTS: Median CA 15-3 blood levels were higher in the positive PET group (100 U x ml(-1)) than in the negative group (65 U x ml(-1)) (P=0.04). The likelihood of depicting recurrence was higher in patients with a short doubling time (<180 days) (P=0.05), a CA 15-3 blood level >60 U x ml(-1) (P=0.05), and when a short doubling time was associated with a CA 15-3 blood level >60 U x ml(-1) (P=0.03). CONCLUSIONS: The likelihood of depicting recurrence was influenced by CA 15-3 blood level and doubling time. Further studies are required to confirm that selections of patients based on those criteria could improve the sensitivity of positron emission tomography in the detection of breast cancer recurrence, particularly in the case of low CA 15-3 blood level.

Breast cancer risk in BRCA1 and BRCA2 mutation carriers and polyglutamine repeat length in the AIB1 gene.

Marked variation in phenotypic expression among BRCA1 and BRCA2 mutation carriers may be partly explained by modifier genes that influence mutation penetrance. Variation in CAG/CAA repeat lengths coding for stretches of glutamines in the C-terminus of the AIB1 protein (amplified in breast cancer 1, a steroid receptor coactivator) has been proposed to modify the breast cancer risk in women carrying germline BRCA1 mutations. We genotyped the AIB1 repeat length polymorphism from the genomic DNA of a group of 851 BRCA1 and 324 BRCA2 female germline mutation carriers to estimate an association with breast cancer risk modification. Hazard ratios (HR) were calculated using a Cox proportional hazards model. For BRCA1 and BRCA2 mutation carriers, analyzed separately and together, we found that women who carried alleles with 28 or more polyglutamine repeats had no increased risk of breast cancer compared to those who carried alleles with fewer repeats (HR for BRCA1/2 carriers = 0.88, 95% CI [confidence interval] = 0.75-1.04). Analyzing average repeat lengths as a continuous variable showed no excess risk of breast cancer (BC) in BRCA1 or BRCA2 mutation carriers (HR for average repeat length in BRCA1/2 carriers = 1.01, 95% CI = 0.92-1.11). These results strongly suggest that contrary to previous studies, there is no significant effect of AIB1 genetic variation on BC risk in BRCA1 mutation carriers and provide an indication that there is also no strong risk modification in BRCA2 carriers.

Common BRCA2 variants and modification of breast and ovarian cancer risk in BRCA1 mutation carriers.

The HH genotype of the nonconservative amino acid substitution polymorphism N372H in the BRCA2 gene was reported to be associated with a 1.3- to 1.5-fold increase in risk of both breast and ovarian cancer. As these studies concerned sporadic cancer cases, we investigated whether N372H and another common variant located in the 5'-untranslated region (203G > A) of the BRCA2 gene modify breast or ovarian cancer risk in BRCA1 mutation carriers. The study includes 778 women carrying a BRCA1 germ-line mutation belonging to 403 families. The two BRCA2 variants were analyzed by the TaqMan allelic discrimination technique. Genotypes were analyzed by disease-free survival analysis using a Cox proportional hazards model. We found no evidence of a significant modification of breast cancer penetrance in BRCA1 mutation carriers by either polymorphism. In respect of ovarian cancer risk, we also saw no effect with the N372H variant but we did observe a borderline association with the 5'-untranslated region 203A allele (hazard ratio, 1.43; CI, 1.01-2.00). In contrast to the result of Healey et al. on newborn females and adult female controls, we found no departure from Hardy-Weinberg equilibrium in the distribution of N372H alleles for our female BRCA1 carriers. We conclude that if these single-nucleotide polymorphisms do modify the risk of cancer in BRCA1 mutation carriers, their effects are not significantly larger than that of N372H previously observed in the general population.

Significant contribution of germline BRCA2 rearrangements in male breast cancer families.

Although screening for large deletions or duplications of the BRCA1 gene is becoming a routine component of the molecular diagnosis of familial breast cancer, little is known about the occurrence of such rearrangements in the BRCA2 gene. Because of the high frequency of BRCA2 mutations in breast cancer families with at least one case of male breast cancer, we selected a cohort of 39 such families, tested negative for mutations in the coding regions of BRCA1 and BRCA2, and developed an assay for BRCA2 rearrangements, based on quantitative multiplex PCR of short fluorescent fragments (QMPSF). We found three rearrangements: (1) a deletion of exons 12 and 13; (2) a duplication of exons 1 and 2; and (3) a complete deletion of BRCA2. We determined the boundaries of the deletion of exons 12 and 13, showing that it resulted from an unequal recombination between Alu sequences. We mapped the complete BRCA2 deletion, which extends over at least 298 kb and showed that it does not affect APRIN/AS3, previously characterized as a tumor suppressor gene, but it comprises several loci corresponding to proven or putative transcripts of unknown functional significance. These data suggest that screening for BRCA2 rearrangements should be done, especially in male breast cancer families tested negative for BRCA1 and BRCA2 mutations.

BCL-2/JH translocation in peripheral blood lymphocytes of unexposed individuals: lack of seasonal variations in frequency and molecular features.

BCL-2/J(H) rearrangement has been proposed as a biomarker for evaluating the genotoxicity of occupational and environmental exposures. Available data on time-related modification of this rearrangement in peripheral blood lymphocytes in unexposed healthy individuals is scarce. We investigated the characteristics of BCL-2/J(H) rearrangements in 33 adults unexposed to genotoxins at 2 seasonal time points: winter and spring. BCL-2/J(H) rearrangement was detected in 79% of individuals (detection limit = 8.48 x 10(-8)). Its frequency ranged from <1 to 40 translocations per million lymphocytes with a significant (p = 0.04) positive correlation with age. No significant modifications of BCL-2/J(H) rearrangement frequency or in the number of clones harboring this rearrangement were observed according the 2 time points. No obvious influence of season-related environmental factors on frequency or molecular features of BCL-2/J(H) rearrangements was found in this population suggesting that this would not be a confounding factor.

BRCA1 wild-type allele modifies risk of ovarian cancer in carriers of BRCA1 germ-line mutations.

Strong inter- and intrafamilial variation of penetrance of breast and ovarian cancer is observed in BRCA1 mutation carriers. The wild-type copy of the BRCA1 gene is a plausible candidate as a cancer risk modifier given that the residual function corresponding to the intact BRCA1 allele may influence the process of tumor formation in BRCA1 carriers. Indeed, growing evidence is now becoming available on impaired reparation of double-strand DNA breaks in cells heterozygous for BRCA1 mutations, implying an enhanced mutability of BRCA1(+/-) cells. To determine whether certain variant forms of the wild-type BRCA1 allele are implicated in variation of the BRCA1-related cancer risk, their effect was studied in a panel of 591 women with BRCA1 germ-line mutations. We found that BRCA1 carriers with the wild-type BRCA1 copy bearing a common Gly1038 variant were at increased risk of ovarian cancer (hazards ratio, 1.50; 95% confidence interval, 1.03-2.19). The results of our study imply that a quite significant proportion of the interindividual variability in ovarian cancer penetrance in BRCA1 carriers may be explained by a common BRCA1 Gly1038 wild-type allele, given its high frequency (0.27).