High Penetrance Breast Cancer Susceptibility Research Articles

BRCA1 detection by porous silicon double-layer via reflectometric interference Fourier transform spectroscopy

Porous silicon double-layer (PSiDL) was fabricated to reduce noise for biosensing measurements via reflectometric interference Fourier transform spectroscopy (RIFTS). The design of the top layer was performed to trap the bio-analyte molecules by their larger diameter pores. However, by constructing the smaller diameter pores on the bottom layer, attempts were made to prevent these molecules from entering the pores as much as possible. Thus, the top layer acted as the sensing layer, whereas the bottom layer played a reference layer role. The Field emission scanning electron microscopy (FE-SEM) revealed that the top and bottom layers had pores with the highest diameter frequency of about 19 and 7 nm, respectively. Surface modification was performed for both layers and was confirmed by Fourier transform infra-red spectroscopy (FTIR) and RIFTS. Modified PSiDL nanostructure was then used to detect the high penetrance breast cancer susceptibility gene, BRCA1, via RIFTS. Examination of both the peaks' location in fast Fourier transform (FFT) of reflection graphs and amplitude value of these peaks was performed. Both analyses showed that the top layer noise could be reduced by using the bottom layer signal.

Effect on Germline Mutation Rate in a High-Risk Chinese Breast Cancer Cohort after Compliance with The National Comprehensive Cancer Network (NCCN) 2023 v.1 Testing Criteria.

The National Comprehensive Cancer Network (NCCN) testing criteria for the high-penetrance breast cancer susceptibility genes, specifically BRCA1, BRCA2, CDH1, PALB2, PTEN, and TP53, have been recently modified in 2023 to 2023 v.1. The following criteria have been changed: (1) from a person diagnosed with breast cancer at ≤45 to ≤50; (2) from aged 45-50 of personal breast diagnosis to any age of diagnosis with multiple breast cancers; and (3) from aged ≥51 of personal breast diagnosis to any age of diagnosis with family history listed in NCCN 2022 v.2. High-risk breast cancer patients (n = 3797) were recruited from the Hong Kong Hereditary Breast Cancer Family Registry between 2007 and 2022. Patients were grouped according to NCCN testing criteria 2023 v.1 and 2022 v.2. A 30-gene panel for hereditary breast cancer was performed. The mutation rates on high-penetrance breast cancer susceptibility genes were compared. About 91.2% of the patients met the 2022 v.2 criteria, while 97.5% of the patients met the 2023 v.1 criteria. An extra 6.4% of the patients were included after the revision of the criteria, and 2.5% of the patients did not meet both testing criteria. The germline BRCA1/2 mutation rates for patients meeting the 2022 v.2 and 2023 v.1 criteria were 10.1% and 9.6%, respectively. The germline mutation rates of all 6 high-penetrance genes in these two groups were 12.2% and 11.6%, respectively. Among the additional 242 patients who were included using the new selection criteria, the mutation rates were 2.1% and 2.5% for BRCA1/2 and all 6 high-penetrance genes, respectively. Patients who did not meet both testing criteria were those with multiple personal cancers, a strong family history of cancers not listed in the NCCN, unclear pathology information, or the patient's voluntary intention to be tested. The mutation rates of BRCA1/2 and the 6 high-penetrance genes in these patients were 5.3% and 6.4%, respectively. This study provided a real-world application of the revision of NCCN guidelines and its effect on the germline mutation rate in the Chinese population. Applying the updated criteria for further genetic investigation would increase the positive detection rate, and potentially more patients would benefit. The balance between the resource and outcome requires careful consideration.

Abstract P5-04-01: Screening Patterns of Mammography and Breast Magnetic Resonance Imaging Following Cancer Genetic Testing in an Integrated Health Care System

Abstract Background Patients at high-risk for breast cancer with genetic mutations are recommended to receive annual breast MRI in adjunct to annual mammography. Despite 15 years of clinical guidelines, uptake of annual breast MRI remains low. Further, little is known about screening patterns across genetic test results in patients tested for hereditary cancer susceptibility as part of usual care. Methods We conducted a retrospective cohort study in an integrated health system among women aged ≥18 years old, without prior breast cancer who had genetic testing for high penetrance hereditary cancer susceptibility genes between January 1, 2010 and December 31, 2018. Genetic test orders and results (pathogenic or likely pathogenic (P/LP) variant(s), negative, or variant of uncertain significance (VUS)) were obtained from health system administrative and laboratory data. Mammogram and breast MRI use data were extracted from electronic health records and claims data. To characterize screening patterns, we calculated average proportion of time covered (PTC) as the number of days covered by mammography and/or breast MRI divided by the number of days from screening eligibility until the earliest censoring event: end of observation period, reached age 75 years, breast cancer diagnosis, death, bilateral mastectomy, or disenrollment in the health system. Per National Comprehensive Cancer Network (NCCN) guidelines, women with P/LP variants in BRCA1/2, TP53, PALB2, CHEK2, ATM, and NF1 became eligible for screening on either the day they received their genetic test result or reached age eligibility and received one year of covered time for each mammogram or MRI imaging procedure received. Per Healthcare Effectiveness Data and Information Set (HEDIS) performance measures, all other women became eligible for screening at age 50 years and received two years of covered time for each mammogram received. Average PTC was calculated overall, by genetic test results, and by screening type. Poisson regression was used to determine the association between average PTC and patient-level factors (age at test, race/ethnicity, and genetic mutations) and guideline factors (recommended screening age state). Results Of 1,167 women meeting the inclusion criteria, 140 (12%) had P/LP in high penetrance breast cancer susceptibility genes (Table 1). Average PTC for individuals with a high penetrance susceptibility gene was 34.4% for MRI, 47.6% for mammogram, 63.2% for either MRI or mammogram, and 19.0% for both screening tests. The average PTC for those who tested negative, had VUS only, and had P/LP variants in genes unrelated to breast cancer was 50.3%, 44.4%, and 40.6%, respectively. Poisson regression model showed that among those with P/LP variants in high penetrance breast cancer genes, average PTC for annual MRI was positively associated with recommended screening initiation age ≥40 years (incident rate ratio [IRR]=2.07, 95% confidence interval [CI]: 1.94-2.20), higher absolute lifetime risk of breast cancer (IRR=1.38, 95% CI: 1.31-1.49), being 40-59 years old at genetic testing (IRR=1.04, 95% CI: 1.01-1.06), and non-Hispanic White race/ethnicity (IRR=1.10, 95% CI: 1.06-1.14). Conclusions Use of breast screening in women who had a P/LP variant(s) in high penetrance breast cancer susceptibility genes was low and not consistent with clinical guidelines, but comparable to the use of screening in women with negative and VUS results. Our findings suggest the need to improve screening in high-risk women with known genetic mutations and women at average risk. Table 1. Average PTC by NCCN and HEDIS recommendation. Citation Format: Boya Guo, Sarah Knerr, Karen Wernli, Kathleen Mittendorf, Heather Feigelson, Marian Gilmore, Gail Jarvik, Tia Kauffman, Erin Keast, Beth Liles, Frances Lynch, Kristin Muessig, Sonia Sasaki, David Veenstra, Jamilyn Zepp, Ben Wilfond, Katrina Goddard, Beth Devine. Screening Patterns of Mammography and Breast Magnetic Resonance Imaging Following Cancer Genetic Testing in an Integrated Health Care System [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-04-01.

Abstract P5-03-17: BRCA1/2 gene mutations in patients with high-risk breast cancer in a tertiary-level hospital in Guatemala

Abstract Objectives, to stablish the frequency of BRCA1/2 mutation rate in high-penetrance breast cancer susceptibility population Methods Based on NCCN guidelines for testing criteria for high-penetrance breast cancer susceptibility genes genetic counseling was offered to 140 breast cancer patients in the hemato-oncology unit of Roosevelt Hospital at Guatemala City performing test with NGS and MLPA technology from 2019 to 2021. Results The overall BRCA1/2 mutation rate high-risk patients were 23% (33/140). Of the patients with mutations, 66.6% (22/33) had BRCA1 mutation, 33.3% (11/33) had BRCA2 mutation, of the mutated population the median age was 45 years. Regarding the phenotype in the mutated population, 75% were triple negative, 16% luminal and 9% with Her2 overexpression. Of the patients carrying the BRCA1 mutation, we identified the c.212+1G>A mutation in 40% of the patients, possibly a founder mutation. In the triple negative population and under 45 years of age, the percentage of patients with BRCA 1/2 mutation is 40.9 (88.8% BRCA1 and 11.1% BRCA2). Conclusions: we found a percentage of BRCA 1/2 mutations in the selected population (NCCN criteria) similar to that reported in other Latin American countries, highlighting the high percentage of BRCA mutations in women under 45 years with triple negative phenotype, previous reports have highlighted the frequency of the c.212+1G>A mutation of BRCA1 in breats cancer patient in Guatemala, in this study 40% of the BRCA1 mutations correspond to said mutation, considering it as a probable founder mutation. Citation Format: Juan Alvarado-Muñoz, Agatha Reyes-Morales, Marco Chivalan, Silvana Torselli, Hector Valenzuela, Gozalo Yalibat, Mario Ordoñez, Rosa León, Egly Alvarez. BRCA1/2 gene mutations in patients with high-risk breast cancer in a tertiary-level hospital in Guatemala [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-17.

BRCA and Breast Cancer-Related High-Penetrance Genes.

Genetic susceptibility explains 5-10% of all breast cancer cases. High-penetrance breast cancer susceptibility genes deliberate a greater than tenfold relative risk of breast cancer. BRCA1 and BRCA2 genes are the most common cause of hereditary breast cancer, and TP53, PTEN, and SKT11 (LKB1) are rarely present. The prevalence of BRCA1 and BRCA2 genetic alterations differ in various ethnic groups. The Korean Hereditary Breast Cancer (KOHBRA) Study, nationwide-scale study, was established to acquire evidence for the accurate risk assessment and management of hereditary breast and ovarian cancer (HBOC) in Korea prospectively since 2007. In this chapter, we review previous research related to hereditary breast cancer and summarize the present concepts and research results centered on theKorean Hereditary Breast Cancer Research at this time.

Abstract ES6-1: Who should have germline testing for hereditary breast cancer?

Abstract Who should have germline testing for hereditary breast cancer? Several guidelines exist for who should be offered germline testing for mutations in breast and ovarian cancer susceptibility genes. NCCN guidelines currently recommend germline BRCA testing for all patients with epithelial ovarian /fallopian tube/primary peritoneal cancer, pancreatic cancer, metastatic/advanced prostate cancer. Testing is also recommended for anyone for whom a BRCA mutation has been identified in the family or for whom a tumor somatic BRCA mutation has been identified (in any tumor type). Among breast cancer patients, NCCN testing criteria include: young age at diagnosis, triple negative disease, Ashkenazi Jewish heritage, male gender, and a personal or family history of ovarian, pancreatic cancer, aggressive prostate cancer or other breast cancer. This year, USPSTF updated their genetic testing guidelines for BRCA1/2. These updates will be reviewed. It is important not to forget testing for germline mutations in other high-penetrance breast cancer susceptibility genes and to retest individuals who previously had BRCA genetic testing and may be appropriate candidates for multi-gene testing. Recently, the American Society of Breast Surgeons has endorsed germline testing for mutations in BRCA1/2 and PALB2 for all breast cancer patients. Data that led to this recommendation will be reviewed as well as arguments for and against this recommendation. It is noteworthy that only 0.6% of breast cancer patients who did not meet current NCCN guidelines for genetic testing were found to have a germline mutation in BRCA1 or BRCA2 and 0.8% in any high-penetrance breast cancer susceptibility gene. It is important for oncologists to be able to recognize which findings on a tumor genomic profile should prompt a recommendation for germline testing. This includes the ability to recognize cancer susceptibility genes amongst the list of actionable findings endorsed by the ACMG, as well as cancer susceptibility genes for which there are published management recommendations. Recently, it has been recommended that germline BRCA testing be offered to all Ashkenazi Jewish individuals regardless of personal or family history of cancer since one in forty Ashkenazi individuals have a mutation in one of three BRCA founder mutations. BRCA mutations are 10 times more common in people with Ashkenazi Jewish ancestry, and up to half of BRCA mutations in this population occur in individuals without a known family history. Three-site Ashkenazi BRCA testing allows streamlined inexpensive BRCA testing without variants of unknown significance (VUS). One concern relates to how large population testing can be carried out, highlighting the need for new genetic testing service models. Finally, arguments for and against population genetic testing for non-Ashkenazi individuals will be discussed. As the likelihood of identifying a mutation and the penetrance of the mutations identified decreases, and the frequency of VUS increases, the potential for causing harm through unnecessary screening and prevention strategies can outweigh the benefit of providing cancer risk information through testing. Citation Format: N Tung. Who should have germline testing for hereditary breast cancer? [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr ES6-1.

Optimization of Porous Silicon Conditions for DNA-based Biosensing via Reflectometric Interference Spectroscopy.

Objective Substantial effort has been put into designing DNA-based biosensors, which are commonly used to detect presence of known sequences including the quantification of gene expression. Porous silicon (PSi), as a nanostructured base, has been commonly used in the fabrication of optimally transducing biosensors. Given that the function of any PSi-based biosensor is highly dependent on its nanomorphology, we systematically optimized a PSi biosensor based on reflectometric interference spectroscopy (RIS) detecting the high penetrance breast cancer susceptibility gene, BRCA1. Materials and Methods In this experimental study, PSi pore sizes on the PSi surface were controlled for optimum filling with DNA oligonucleotides and surface roughness was optimized for obtaining higher resolution RIS patterns. In addition, the influence of two different organic electrolyte mixtures on the formation and morphology of the pores, based on various current densities and etching times on doped p-type silicon, were examined. Moreover, we introduce two cleaning processes which can efficiently remove the undesirable outer parasitic layer created during PSi formation. Results of all the optimization steps were observed by field emission scanning electron microscopy (FE-SEM). Results DNA sensing reached its optimum when PSi was formed in a two-step process in the ethanol electrolyte accompanied by removal of the parasitic layer in NaOH solution. These optimal conditions, which result in pore sizes of approximately 20 nm as well as a low surface roughness, provide a considerable RIS shift upon complementary sequence hybridization, suggesting efficient detectability. ConclusionWe demonstrate that the optimal conditions identified here makes PSi an attractive solid-phase DNA-based biosensing method and may be used to not only detect full complementary DNA sequences, but it may also be used for detecting point mutations such as single nucleotide substitutions and indels.

RAD51C mutation screening in high-risk patients from Serbian hereditary breast/ovarian cancer families.

In 2010 an important finding was published showing that heterozygous mutations in RAD51C were highly penetrant and were able to confer an increased risk for breast and ovarian cancers. The role of possible third high penetrance breast cancer susceptibility gene was assigned to RAD51C. Because of its rising importance in breast cancer development and the lack of information about RAD51C in Slavic populations, our goal was to identify potential population specific mutations in this gene in order to determine more detailed genetic screening strategy and breast cancer risk assessment. The study included 55 females from Serbian hereditary breast/ovarian cancer families negative for sequence alterations and large genomic rearrangements in BRCA1/2 genes. Whole coding region and exon-intron boundaries of RAD51C were analyzed by dHPLC. All mutations were confirmed by Sanger sequencing. SIFT and Polyphen were used to predict possible impact of non-synonymous variants. We found 5 variants in RAD51C including two missense, one intronic, one in the 5'UTR and one variant in the promoter region of the gene. Three detected variants are common - c.1-118G>A (rs16943176, MAF = 0,203); c.1-26C>T (rs12946397, MAF = 0,207) and c.904+34T>C (rs28363318, MAF = 0,186). We detected two missense variants, c.790G>A (p.Gly264Ser) in exon 5 and c.859A>G (p.Thr287Ala) in exon 6. Both of them were previously shown to exhibit reduced protein function but their contribution to cancer risk is still unknown. Although the initial reports implied that RAD51C might be promising candidate for next high penetrance breast cancer susceptibility gene, lack of confirmation suggested that RAD51C mutations are not as common as expected. Our study did not reveal truncating mutations in RAD51C suggesting that other breast cancer susceptibility genes may account for the increased susceptibility in our cohort of high-risk BRCA1/2 negative families.

Prevalence of PALB2 mutations in Australian familial breast cancer cases and controls.

IntroductionPALB2 is emerging as a high-penetrance breast cancer predisposition gene in the order of BRCA1 and BRCA2. However, large studies that have evaluated the full gene rather than just the most common variants in both cases and controls are required before all truncating variants can be included in familial breast cancer variant testing.MethodsIn this study we analyse almost 2000 breast cancer cases sourced from individuals referred to familial cancer clinics, thus representing typical cases presenting in clinical practice. These cases were compared to a similar number of population-based cancer-free controls.ResultsWe identified a significant excess of truncating variants in cases (1.3 %) versus controls (0.2 %), including six novel variants (p = 0.0001; odds ratio (OR) 6.58, 95 % confidence interval (CI) 2.3–18.9). Three of the four control individuals carrying truncating variants had at least one relative with breast cancer. There was no excess of missense variants in cases overall, but the common c.1676A > G variant (rs152451) was significantly enriched in cases and may represent a low-penetrance polymorphism (p = 0.002; OR 1.24 (95 % CI 1.09–1.47).ConclusionsOur findings support truncating variants in PALB2 as high-penetrance breast cancer susceptibility alleles, and suggest that a common missense variant may also lead to a low level of increased breast cancer risk.

Clinical and molecular characterization of the BRCA2 p.Asn3124Ile variant reveals substantial evidence for pathogenic significance.

Variants of uncertain clinical significance (VUS) in the high-penetrance breast cancer susceptibility genes BRCA1 and BRCA2 represent a major obstacle in genetic counseling of high-risk breast cancer families. We analyzed a missense VUS located in BRCA2 (p.Asn3124Ile; HGVS: BRCA2 c.9371A>T) present in seven independent high-risk breast cancer families that were counseled and genetically tested in South-West Germany. The VUS was identified by DNA sequencing. We analyzed co-occurrence with deleterious BRCA1/2 mutations, segregation, evolutionary conservation, in silico impact prediction, and prevalence in the general population.All carriers of the VUS suffered from breast or ovarian cancer. In two families, an additional high burden of other cancers such as pancreatic, prostate, and gastric cancers was reported, one further family included two cases of male breast cancer. The VUS did not co-occur with deleterious BRCA1/2 mutations and segregated in two affected individuals of one family. In contrast to the 7/1,347 (0,5%) tested high-risk BC families without clearly pathogenic mutations in BRCA1/2, none of 3,126 healthy population controls sharing the same ethnic and geographical background were found to carry this VUS (p=0.0002). In-silico prediction revealed strong evolutionary conservation of the asparagine residue, residing in the C-terminal oligonucleotide-binding-fold-3 region, and a most likely damaging impact of this exchange on the protein structure.The BRCA2 p.Asn3124Ile (BRCA2 c.9371A>T) variant is a rare mutation with a damaging effect on the BRCA2 protein that is strongly associated with familial breast and ovarian cancer risk, indicating its most likely pathogenic nature and clinical relevance.

Breed-related differences in altered BRCA1 expression, phenotype and subtype in malignant canine mammary tumors

BRCA1 is a high-penetrance breast cancer susceptibility gene and BRCA1-associated breast cancer has a high familial prevalence that is more common among certain populations of humans. A similar high prevalence also exists for canine mammary tumors (CMTs) and the objective of this study was to determine the breed-related differences in malignant CMTs. Comparative analyses of the expression of various prognostic factors for CMTs, including BRCA1, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) were conducted on 139 malignant CMT cases from five breeds with the highest prevalence of CMTs in Korea. Significant breed-related differences were observed in the expression of BRCA1 (P=0.003), histological grade (P=0.038), and extensive lymphatic invasion (P=0.042). The Shih Tzu breed had the highest proportion of dogs with malignant CMT and strong overexpression of BRCA1. Cytoplasmic and membranous expression of BRCA1 was associated with the ER negative (P=0.004), PR negative (P=0.046), and triple negative (ER, PR, and HER-2 negative; P=0.016) phenotype and the basal-like molecular subtype (P=0.019) in Shih Tzu dogs. Since these features are similar to BRCA1-related human breast cancer, dogs with BRCA1-associated CMT, particularly Shih Tzu dogs, may serve as a suitable spontaneous model, although additional molecular studies are needed.

No evidence for breast cancer susceptibility associated with variants of BRD7, a component of p53 and BRCA1 pathways

BRD7 (bromodomain 7), a subunit of poly-bromo-associated BRG1-associated factor (PBAF)-specific Swi/Snf chromatin remodeling complexes, has been proposed as a tumour suppressor protein following its identification as an important component of both functional p53 and BRCA1 (breast cancer 1, early onset) pathways. As low BRD7 expression levels have been linked to p53-wild-type breast tumour cells, we hypothesized an implication of BRD7 germline alterations in the pathogenesis of hereditary breast cancer similar to that of TP53 in Li-Fraumeni syndrome. We performed sequence analysis of the BRD7 gene on 61 high-risk individuals with hereditary or very-early-onset breast cancer and 100 healthy controls. Four potentially disease-causing single-nucleotide alterations were detected within the cohort of breast cancer patients (one listed as a rare single-nucleotide polymorphism (SNP) in the NCBI (National Center for Biotechnology Information) SNP database). Two of the detected variants were also each found once within the control collective. Segregation analysis on both families of those carrying the remaining two variants revealed segregation of these BRD7 alterations independent of breast cancer. In conclusion, it seems that the BRD7 variants we detected represent rare polymorphisms and mainly rule out BRD7 as a frequent high-penetrance breast cancer susceptibility gene. However, further analyses in larger cohorts of women with hereditary breast cancer should clarify the role of BRD7 in breast cancer predisposition.

84P Frequency of 5382Insc and 185Delag Mutation of Brca1 Gene: An Experience from Eastern India

ABSTRACT Background The search for germline mutations within breast cancer susceptibility genes has been focused on the risk for developing malignancies in specific population. Mutations in the high penetrance breast cancer susceptibility gene BRCA1 account for a significant percentage of hereditary breast cancer cases. As reported earlier, carrier for the 5382insC and 185delAG BRCA1 mutation, lifetime risk of breast cancer for women is increased from 13% -81% in different population. Objectives The aims of the study was to evaluate the contribution of such mutation to the morbidity in eastern Indian breast cancer patients and their clinical manifestation and to study the BRCA1 gene positivity as prognostic factor in breast cancer patients. Methods Out of 92 patients (age range: 23-52) with family history (57 individuals) and without family history (35 individuals) were screened. 25 controls have been systematically investigated. Each mutation was detected separately using duplex PCR, PCR-SSCP. Results Studied mutations were identified in 14 patients (32%); 5382insC in 11 and 185delAG in 3 cases. All mutation carriers are below 45 years and had early onset of disease. They are in a follow up of 2 years at a stable condition. Conclusion 5382insC and 185delAG in BRCA1 gene are the first reported mutation in breast cancer in Eastern Indian origin. The presented data confirm a noticeable contribution of BRCA1 5382insC and 185delAG mutation in BC development in Eastern India that may justify an extended BRCA1 5382insC testing within this population. Further study is required to determine whether routine genetic testing should be offered to this population. The family members of mutation carrier are suggested to evaluate their mutation status to prevent the onset of disease at the earliest. Considering the importance of genetic counseling and recording, the adequate information for the pedigrees of cancer patients put forward the principle approaches in cancer clinics to facilitate early detection for preventing challenges. Disclosure All authors have declared no conflicts of interest.

RAD51C germline mutations in Chinese women with familial breast cancer

Germline mutations in high penetrance breast cancer susceptibility genes, BRCA1 and BRCA2, account for only approximately 10% of Chinese familial breast cancers [1], indicating other susceptibility genes related to Chinese familial breast cancer may exist. RAD51C gene (Rasrelated associated with diabetes) is located on chromosome 17q23, a region that was found to be frequently amplified in breast tumors [2, 3]. RAD51C gene is a member of the RAD51 family and plays an essential role in homologous recombination, DNA damage sensitivity, genomic integrity, embryonic development, activation of cell cycle checkpoint kinase 2 (CHK2), and cell cycle arrest in response to DNA damage [4–8]. Recently, a biallelic missense mutation in the RAD51C gene was found in a family from Pakistan with Fanconi anemia-like disorder [9]. In an accompanying article, six monoallelic deleterious mutations in the RAD51C gene were found in 480 BRCA1/2-negative breast/ovarian cancer families from Germany [10]. This study provides the first evidence to show that the RAD51C is a breast cancer susceptibility gene. Thus, the RAD51C gene may deserve to be comprehensively screened as a breast cancer susceptibility gene in other populations. In this study, we screened the entire coding regions and exon–intron boundaries of the RAD51C gene in 273 Chinese women with familial breast cancer who do not carry mutations in BRCA1 and BRCA2 genes by polymerase chain reaction (PCR)-sequencing. Familial breast cancer cases were from the Breast Center, Peking University Cancer Hospital from July 2006 to May 2010. The criteria of familial breast cancer are (1) patients have at least one or more firstor second-degree relatives affected with breast cancer and/or ovarian cancer regardless of age and (2) bilateral breast cancer regardless of age. Four hundreds seventy-five healthy Chinese women serve as controls. Both of the cases and controls are Han Chinese women and reside in the north of China. The whole coding sequences of RAD51C were amplified using nine sets of primers described elsewhere with a minor modification [10]. All fragments were sequenced using BigDye Terminator Cycle Sequencing Kit and ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA). Each mutation was confirmed by duplicate. In total, we detected eight germline sequence variants in the RAD51C gene in the 273 BRCA1/2-negative familial breast cancer cases. Among them, three were non-coding variants and five were coding variants (Table 1). None of them were previously reported. Of the coding variants, one was synomenous and four were non-synomenous. Among the four amino-acid substitution variants, 4C[G (R2G) was located in exon 1, 635G[A (R212H) and 644A[G (D215G) in exon 4, and 882G[C (Q294H) in exon 6. The missense variants R2G, D215G, and Q294H were detected in one index case, whereas R212H was detected in two unrelated cases. We then screened the four missense variants in 475 healthy controls. The variant R212H was found in two healthy individuals, indicating this variant was unlikely to be pathogenic. In contrast, none of the Zhiyuan Pang and Lu Yao contributed equally to this study.

Mutation analysis of RAD51L1 (RAD51B/REC2) in multiple-case, non-BRCA1/2 breast cancer families

Although a significant proportion of familial aggregation of breast cancer remains unexplained, many of the currently known breast cancer susceptibility genes, including BRCA1, BRCA2 and TP53, play a role in maintaining genome integrity by engaging in DNA repair. RAD51L1 is one of the five RAD51 paralogs involved in homologous recombination (HR) repair of DNA double-strand breaks (DSBs); it also interacts directly with p53. Deleterious mutations have been found in one RAD51 paralog, RAD51C (RAD51L2), in non-BRCA1/2 breast and ovarian cancer families, which suggests that all five paralogs are strong candidate breast cancer susceptibility genes. A genome-wide association study (GWAS) has already identified a single nucleotide polymorphism (SNP) deep within intron 10 of RAD51L1 as a risk locus for breast cancer. Based on its biological functions and association with RAD51C, there is reason to suggest that RAD51L1 (RAD51B/REC2) may also contain high risk mutations in the gene that give rise to multiple-case breast cancer families. In order to investigate this hypothesis, we have used high resolution melt (HRM) analysis to screen RAD51L1 for germline mutations in 188 non-BRCA1/2 multiple-case breast cancer families and 190 controls. We identified a total of seven variants: one synonymous, three intronic, and three previously identified SNPs, but no truncating or nonsense changes. Therefore, our results suggest that RAD51L1 is unlikely to represent a high-penetrance breast cancer susceptibility gene.

Absence of the BRCA1 del (exons 9–12) mutation in breast/ovarian cancer families outside of Mexican Hispanics

The frequency and spectrum of germ line mutations in the high-penetrance breast cancer susceptibility genes BRCA1 and BRCA2 shows considerable variation by ethnic group. Most genetic epidemiological studies of the BRCA genes have been performed among Caucasian populations [1], with the exception of a few studies involving other ethnic groups, such as Hispanics [2–4], Asians [5, 6], and African Americans [7–9]. In most of these studies only the frequencies of sequence detectable BRCA mutations were reported and large genomic rearrangements including deletions and insertions of one or more exons, which account for 6–15% of all deleterious mutations in these genes have infrequently been considered [10–12]. Most BRCA mutations are unique; however, few recurrent mutations with founder effects have been found in European, Hispanic, American and Asian populations [13]. In a recent study on Hispanic high-risk breast/ovarian cancer families of mainly Mexican descent from South California, Weitzel and colleagues reported the identification of a novel deletion of BRCA1 exons 9 through 12 using multiplex quantitative differential polymerase chain reaction (PCR) analysis. The deletion was detected in 3.8% of families negative for sequence detectable BRCA mutations [14]. The large genomic rearrangement mutation is considered deleterious as it results in loss of critical functional domains as well as premature truncation of the BRCA1 protein. Given the relatively high prevalence of the deletion in this cohort, the authors suggest to include the screening for this mutation in the genetic testing strategy in Hispanic women without sequence detectable BRCA mutations. The spectra of recurrent sequence detectable BRCA mutations, including some with founder effects in a cohort of Hispanic breast/ovarian cancer families of predominantly Mexican descent from the United States and in a cohort from Colombia have previously been shown to differ completely, with different common mutations being identified in the two populations [2, 4]. In order to investigate, whether the novel BRCA1 founder deletion is also specific for Hispanic families of Mexican descent or whether it also occurs in other Hispanic cohorts, we screened 229 cancer Diana Torres and Muhammad U. Rashid contributed equally to this work.

Hereditary breast cancer: from molecular pathology to tailored therapies

Hereditary breast cancer accounts for up to 5–10% of all breast carcinomas. Recent studies have demonstrated that mutations in two high-penetrance genes, namely BRCA1 and BRCA2, are responsible for about...

Evaluation of the BRCA1 interacting genes RAP80 and CCDC98 in familial breast cancer susceptibility

RAP80 and CCDC98 have arisen as new candidate breast cancer susceptibility genes, since they encode for two very recently identified BRCA1 interacting proteins. In this study we have performed the first mutational analysis of both genes in 168 multiple-case breast/ovarian cancer families, negative for mutations in BRCA1 or BRCA2. We have not found truncating mutations in any of the genes and only two missense variants, p.Tyr564His in RAP80, and p.Met299Ile in CCDC98 were found that could be suspected to have a pathogenic effect, although further analyses suggested that they were probably non deleterious. Our analysis suggests that RAP80 and CCDC98 do not play an important role as high penetrance breast cancer susceptibility genes.

BARD1 variants are not associated with breast cancer risk in Australian familial breast cancer

Several studies in various populations have suggested that non-synonymous BARD1 variants are associated with increased breast cancer risk. Using DHPLC analysis we screened the coding region of BARD1 for variants in 210 probands of breast cancer families including 129 families with no mutations in BRCA1 or BRCA2. These families were ascertained in Australia through the Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). Nine coding variants were detected among the kConFab families, including two novel variants (Thr598Ile and Ile692Thr). The frequency of five of these variants were evaluated in 258 non-cancer controls and 401 women with sporadic breast cancer. Three variants (1139del21, G1756C and A2285G) were detected in all three groups at a similar frequency suggesting that these do not represent BRCAX candidates. Two variants (Thr598Ile and Ile692Thr) were not detected in any of the 659 sporadic breast cancer cases and controls and were assessed for segregation with breast cancer in the families of the probands. However, neither variant was identified in any other breast cancer case in either family suggesting that these variants are non-pathogenic polymorphisms. We have found no evidence to support involvement of BARD1 in familial breast cancer risk in the Australian population. In addition, three variants previously reported to be pathogenic in other populations are likely to represent benign polymorphisms and therefore we conclude that BARD1 is unlikely to represent a high-penetrance breast cancer susceptibility gene.

027.Mapping novel breast cancer susceptibility genes by linkage analysis of Australian multiple case kindreds

We have been using the resources of the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) and of the Australian Breast Cancer Family Study (ABCFS) to identify kindreds suitable for mapping high penetrance breast cancer susceptibility loci other than BRCA1 and BRCA2. A 10 cM genomewide search was carried out in 40 families in which BRCA1 and BRCA2 mutations had been excluded with high probability. The highest LOD score under heterogeneity (HLOD) was 2.16 (non-parametric LOD 1.83, P = 0.04) at the 11p telomere; several other regions with HLODs = 1.5–2.0 also merited investigation using fine mapping but have so far neither been confirmed or rejected by these analyses. Subsets based on age of onset and presence of other cancers correlated to some extent with particular linkage peaks and several regions (notably 2q and 13q) corresponded to areas of suggestive linkage reported recently in more limited studies of other cohorts. A large collaborative analysis of these data together with those from similar studies undertaken by members of the international Breast Cancer Linkage Consortium (BCLC) is under way. It is therefore likely that further major breast cancer susceptibility loci will be localised in the near future. The complementarity of these studies with genetic association, candidate gene and tumour-based approaches will be discussed.