Early-onset Familial Breast Cancer Research Articles

Germline loss-of-function variants in the BARD1 gene are associated with early-onset familial breast cancer but not ovarian cancer

BackgroundThe role of the BARD1 gene in breast cancer (BC) and ovarian cancer (OC) predisposition remains elusive, as published case-control investigations have revealed controversial results. We aimed to assess the role of deleterious BARD1 germline variants in BC/OC predisposition in a sample of 4920 BRCA1/2-negative female BC/OC index patients of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC).MethodsA total of 4469 female index patients with BC, 451 index patients with OC, and 2767 geographically matched female control individuals were screened for loss-of-function (LoF) mutations and potentially damaging rare missense variants in BARD1. All patients met the inclusion criteria of the GC-HBOC for germline testing and reported at least one relative with BC or OC. Additional control datasets (Exome Aggregation Consortium, ExAC; Fabulous Ladies Over Seventy, FLOSSIES) were included for the calculation of odds ratios (ORs).ResultsWe identified LoF variants in 23 of 4469 BC index patients (0.51%) and in 36 of 37,265 control individuals (0.10%), resulting in an OR of 5.35 (95% confidence interval [CI] = 3.17–9.04; P < 0.00001). BARD1-mutated BC index patients showed a significantly younger mean age at first diagnosis (AAD; 42.3 years, range 24–60 years) compared with the overall study sample (48.6 years, range 17–92 years; P = 0.00347). In the subgroup of BC index patients with an AAD < 40 years, an OR of 12.04 (95% CI = 5.78–25.08; P < 0.00001) was observed. An OR of 7.43 (95% CI = 4.26–12.98; P < 0.00001) was observed when stratified for an AAD < 50 years. LoF variants in BARD1 were not significantly associated with BC in the subgroup of index patients with an AAD ≥ 50 years (OR = 2.29; 95% CI = 0.82–6.45; P = 0.11217). Overall, rare and predicted damaging BARD1 missense variants were significantly more prevalent in BC index patients compared with control individuals (OR = 2.15; 95% CI = 1.26–3.67; P = 0.00723). Neither LoF variants nor predicted damaging rare missense variants in BARD1 were identified in 451 familial index patients with OC.ConclusionsDue to the significant association of germline LoF variants in BARD1 with early-onset BC, we suggest that intensified BC surveillance programs should be offered to women carrying pathogenic BARD1 gene variants.

Mutations in RECQL Gene Are Associated with Predisposition to Breast Cancer.

The genetic cause for approximately 80% of familial breast cancer patients is unknown. Here, by sequencing the entire exomes of nine early-onset familial breast cancer patients without BRCA1/2 mutations (diagnosed with breast cancer at or before the age of 35) we found that two index cases carried a potentially deleterious mutation in the RECQL gene (RecQ helicase-like; chr12p12). Recent studies suggested that RECQL is involved in DNA double-strand break repair and it plays an important role in the maintenance of genomic stability. Therefore, we further screened the RECQL gene in an additional 439 unrelated familial breast cancer patients. In total, we found three nonsense mutations leading to a truncated protein of RECQL (p.L128X, p.W172X, and p.Q266X), one mutation affecting mRNA splicing (c.395-2A>G), and five missense mutations disrupting the helicase activity of RECQL (p.A195S, p.R215Q, p.R455C, p.M458K, and p.T562I), as evaluated through an in vitro helicase assay. Taken together, 9 out of 448 BRCA-negative familial breast cancer patients carried a pathogenic mutation of the RECQL gene compared with one of the 1,588 controls (P = 9.14×10-6). Our findings suggest that RECQL is a potential breast cancer susceptibility gene and that mutations in this gene contribute to familial breast cancer development.

Familial breast cancer: the first linkage

The identification of a new gene that affects cancer susceptibility is now almost a daily event, so the report in Science in 1990 from a team led by Mary-Claire King 1 Hall JM Lee MK Newman B et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science. 1990; 250: 1684-1689 Crossref PubMed Scopus (2020) Google Scholar might now seem rather mundane. However, the importance of this paper should not be underestimated: it was the turning point after which it could no longer be denied that genetic variation had an effect on breast cancer risk.

Degradation of BRCA2 in Alkyltransferase-Mediated DNA Repair and Its Clinical Implications

Germ-line mutations in BRCA2 have been linked to early-onset familial breast cancer. BRCA2 is known to play a key role in repairing double-strand breaks. Here, we describe the involvement of BRCA2 in O6-alkylguanine DNA alkyltransferase (AGT)-mediated repair of O6-methylguanine adducts. We show that BRCA2 physically associates and undergoes repair-mediated degradation with AGT. In contrast, BRCA2 with a 29-amino-acid deletion in an evolutionarily conserved domain does not bind to alkylated AGT; the two proteins are not degraded; and mouse embryonic fibroblasts are specifically sensitive to alkylating agents that result in O6-methylguanine adducts. We show that O6-benzylguanine (O6BG), a nontoxic inhibitor of AGT, can also induce BRCA2 degradation. BRCA2 is a viable target for cancer therapy because BRCA2-deficient cells are hypersensitive to chemotherapeutic DNA-damaging agents. We show a marked effect of O6BG pretreatment on cell sensitivity to cisplatin. We also show the efficacy of this approach on a wide range of human tumor cell lines, which suggests that chemosensitization of tumors by targeted degradation of BRCA2 may be an important consideration when devising cancer therapeutics.

Impaired Tamoxifen Metabolism Reduces Survival in Familial Breast Cancer Patients

Tamoxifen has been the mainstay adjuvant hormonal treatment for breast cancer for many years. Conversion of tamoxifen to its active metabolite, endoxifen, is reduced by low activity of the cytochrome P450 enzyme, CYP2D6. We examined the effect of reduced CYP2D6 activity on the response to tamoxifen in patients with familial early-onset breast cancer. We conducted a case note review and genotyping for the CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*41 alleles in 115 patients (47 BRCA1, 68 BRCA2) with familial breast cancer who had been treated with 20-mg tamoxifen following surgery. Eight (7%) individuals had genotypes consistent with poor metabolizer status, and 4 (3.5%) individuals took CYP2D6 inhibitor drugs concomitant with their tamoxifen and were also considered poor metabolizer. Time to tumor recurrence, disease-free survival, and overall survival were reduced in the patient group with poor metabolizer CYP2D6 activity. However, a significant effect was confined to patients with BRCA2 mutations with a worse overall survival (median survival, 7 versus 28 years; P = 0.008; adjusted hazard ratio, 9.7). Poor metabolizer status for CYP2D6 predicts worse overall survival in patients with familial breast cancer. Therefore, CYP2D6 inhibitor drugs should not be prescribed concomitantly with tamoxifen. Prospective studies should be undertaken to establish the effect of CYP2D6 status on outcome in familial breast cancer patients treated with tamoxifen.

P61 Investigation of three founder mutations in BRCA1 and BRCA2 in Iranian breast cancer patients

BRCA1 is known as a familial early onset breast cancer gene located in the long arm of chromosome 17. Alterations in this gene have been reported in different populations, some of which are population-specific mutations showing a founder effect. In this study, which is the first report on the genetic testing of Iranian women, exon-2 of the BRCA1 gene was sequenced in 80 Iranian breast cancer patients and none of the patients showed the 185del AG mutation or any changes in the sequences of this exon. Among this population, 22 patients having one or more of any kind of tumor in their first-degree relatives were selected and their entire BRCA1 gene coding region was sequenced by direct PCR-sequencing. A novel point mutation in exon-16, with unknown significance, was observed among the breast cancer patients and control subjects. This A/G mutation caused the substitution of Glutamine 1612 with Glycine, with an allele frequency of 38.6 and 52.8% in patients and controls, respectively. In addition, a point mutation in exon-15 and eight other polymorphic alterations were detected which have been reported previously. Three of these polymorphic sites were placed in the intronic part of the gene. To understand the significance of the contribution of the BRCA1 gene in the breast cancer among Iranian, further investigations are needed.

CHEK2 1100delC is prevalent in Swedish early onset familial breast cancer

BackgroundA truncating variant, 1100delC, in check point-kinase CHEK2, has been identified as a risk factor for familial and sporadic breast cancer. The prevalence in healthy non-breast cancer cases is low and varies between populations.MethodsWe analyzed the prevalence of CHEK2 1100delC in 763 breast cancer patients with a defined family history and 760 controls from the Stockholm region. The breast cancer patients originated from; a population-based cohort (n = 452) and from a familial cancer clinic (n = 311), the detailed family history was known in both groups.ResultsThe variant was found in 2.9% of the familial cases from the population-based cohort and in 1.9% from the familial cancer clinic. In total 2.2% of the patients with a family history of breast cancer carried the variant compared to 0.7% of the controls (p = 0.03). There was no increased prevalence in sporadic patients (0.3%). The variant was most frequent in young familial patients (5.1% of cases ≤45 years, p = 0.003). The mean age at diagnosis of variant carriers was 12 years lower than in non-carriers (p = 0.001).ConclusionIn conclusion, CHEK2 1100delC exists in the Swedish population. The prevalence is increased in familial breast cancer and the variant seems to influence age at onset.

MDM2 SNP309 accelerates familial breast carcinogenesis independently of estrogen signaling

A single nucleotide polymorphism (SNP309T>G) in the intronic promoter of MDM2 was recently found to accelerate carcinogenesis in early-onset cancer cases. This cancer acceleration presumably was due to increased SP1 binding, resulting in enhanced MDM2 transcriptional activation by estrogens. We evaluated MDM2 SNP309 in 343 familial breast cancer cases with known mutation status for CHEK2 1100delC, BRCA1 and BRCA2. Cancer acceleration was indeed observed in early-onset familial breast cancer cases (diagnosed <or= 51 years), with 16% of cases carrying the MDM2 SNP309 GG genotype as compared to 4% of late-onset cases (P = 0.029). The cancer acceleration was even more pronounced in the non-mutant familial breast cancer cases, with 17% of early-onset cases carrying MDM2 SNP309 GG as compared to 2% of late-onset cases (n = 214; P = 0.015). There was no evidence for an influence of estrogen signaling in the cancer acceleration by MDM2 SNP309, as there were no differences in the prevalence of MDM2 SNP309 GG among CHEK2 1100delC and BRCA2 mutant cases (with 90% ER-positive cancers) or BRCA1 mutant cases (10% ER-positive cancers). Nor did we observe differences in MDM2 SNP309 frequencies among 75 familial breast cancer cases of our cohort with known ER status. Overall, our data suggest that MDM2 SNP309 accelerates familial breast carcinogenesis, but that this acceleration is not influenced by estrogen signaling.

CYP17 promoter polymorphism and breast cancer risk in males and females in relation to BRCA2 status.

A T–C polymorphism in the promoter region of the CYP17 gene has been associated with male and female breast cancer risk as well as early-onset familial breast cancer. The potential role of this polymorphism was investigated in relation to breast cancer risk in Icelandic male and female carriers and noncarriers of a BRCA2 mutation. The study population consisted of 39 male and 523 female breast cancer cases and 309 male and 395 female controls. Of the cases, 15 males and 55 females carried a BRCA2 mutation. We did not find a significant association between male breast cancer risk and CYP17 genotypes. Among male breast cancer cases, the frequency of the CC genotype was higher among carriers of the 999del5 mutation (33.3%) than noncarriers (16.7%), although this difference also did not reach a statistical significance. No association was observed with breast cancer risk among females irrespective of menopausal status, stage of the disease or BRCA2 status. Our findings do not indicate a role for the CYP17 T–C polymorphism in female breast cancer, but a role in male carriers of a BRCA2 mutation could not be excluded because of the small sample size.

Germline BRCA1 mutations in Iranian women with breast cancer

BRCA1 is known as a familial early onset breast cancer gene located in the long arm of chromosome 17. Alterations in this gene have been reported in different populations, some of which are population-specific mutations showing a founder effect. In this study, which is the first report on the genetic testing of Iranian women, exon-2 of the BRCA1 gene was sequenced in 80 Iranian breast cancer patients and none of the patients showed the 185del AG mutation or any changes in the sequences of this exon. Among this population, 22 patients having one or more of any kind of tumor in their first-degree relatives were selected and their entire BRCA1 gene coding region was sequenced by direct PCR-sequencing. A novel point mutation in exon-16, with unknown significance, was observed among the breast cancer patients and control subjects. This A/G mutation caused the substitution of Glutamine 1612 with Glycine, with an allele frequency of 38.6 and 52.8% in patients and controls, respectively. In addition, a point mutation in exon-15 and eight other polymorphic alterations were detected which have been reported previously. Three of these polymorphic sites were placed in the intronic part of the gene. To understand the significance of the contribution of the BRCA1 gene in the breast cancer among Iranian, further investigations are needed.

Significantly lower rates of BRCA1/ BRCA2 founder mutations in Ashkenazi women with sporadic compared with familial early onset breast cancer

To delineate the clinical, genetic and family history attributes in Jewish Ashkenazi women with early onset (<42 years) breast cancer we genotyped such women for the three predominant Jewish Ashkenazi mutations in BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT). The study cohort was composed of 172 women diagnosed with breast cancer at or before the age of 42 years, obtained from the oncology department registry. Mutations were identified in 54 women (31%). Of 79 women with a positive family history for breast and/or ovarian cancer, and 93 with no such family history, 45 (57%) and 9 (10%), respectively, were mutation carriers (χ 2=46; P<0.001). Contralateral breast cancer occurred in 15 of 54 mutation carriers (28%) compared with 8 of 118 (7%) non-carriers (χ 2=14; P<0.001). Early onset breast cancer per se is a weak predictor of finding germ line mutation(s) in BRCA1 and BRCA2, unless associated with a positive family history and/or bilaterality.

Complex Segregation Analyses: Uses and Limitations

Complex segregation analysis (CSA) is a general method for evaluating the transmission of a trait within pedigrees. It proceeds by testing models of varying degrees of generality, both to determine whether a Mendelian locus is likely to exert a large effect on the phenotype of interest and to estimate the magnitude of genetic sources of variation in the trait. This information is valuable both as a prelude to linkage analysis, which generally assumes Mendelian transmission, and in providing a model on which to base parametric linkage methods.

Automated detection of prevalent mutations in BRCA1 and BRCA2 genes, using a fluorogenic PCR allelic discrimination assay.

Mutations in the genes BRCA1 and BRCA2 account for 5%-10% of familial early onset breast cancer. Identification of these mutations allows molecular diagnosis for breast cancer susceptibility. A high through-put automated PCR allelic discrimination assay (ADA) was developed to detect the prevalent mutations in these genes. Two allele specific oligonucleotides (ASO) are directly used in the PCR reaction, in both of which the fluorescent reporter and quencher dyes are attached to the 5' and 3' ends, respectively. During PCR, fluorescence is generated after cleavage of the annealed ASO by the 5' nuclease activity of Taq polymerase. The wild-type BRCA sequence is distinguished from the mutant sequence by the differential fluorescence emission of two different reporter dyes. The sensitivity of ADA is at the level of a single cell following a nested PCR. Eighty-six patient samples can be analyzed for each mutation in 15-min post-PCR without the need for radioactivity, gel electrophoresis, or membrane blotting/hybridization.

Cloning mammary cell cDNAs from 17q12–q23 using interspecific somatic cell hybrids and subtractive hybridization

We have cloned human genes that are encoded in the region 17q12–q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12–q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12–q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12–q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12–q23 region.

Characterization of 10 new polymorphic dinucleotide repeats and generation of a high-density microsatellite-based physical map of the BRCA1 region of chromosome 17q21.

A familial early onset breast cancer gene (BRCA1) has been localized to chromosome 17q21. To aid in the identification of this gene a number of new microsatellite markers from the D17S857 to D17S78 region were isolated and characterized. These markers, along with previously published markers from the region, were localized on a physical map by STS content mapping of cosmids from the BRCA1 interval. This high-density STS map of the BRCA1 region will be useful for linkage studies of families with apparent inherited breast cancer and for loss of heterozygosity analysis of breast tumor DNAs.

Multicolor FISH Mapping with Alu-PCR-Amplified YAC Clone DNA Determines the Order of Markers in the BRCA1 Region on Chromosome 17q12-q21

A gene designated BRCA1, implicated in the susceptibility to early-onset familial breast cancer, has recently been localized to chromosome 17q12-q21. To date, the order of DNA markers mapped within this region has been based on genetic linkage analysis. We report the use of multicolor fluorescence in situ hybridization to establish a physically based map of five polymorphic DNA markers and 10 cloned genes spanning this region. Three cosmid clones and Alu-PCR-generated products derived from 12 yeast artificial chromosome clones representing each of these markers were used in two-color mapping experiments to determine an initial proximity of markers relative to each other on metaphase chromosomes. Interphase mapping was then employed to determine the order and orientation of closely spaced loci by direct visualization of fluorescent signals following hybridization of three probes, each detected in a different color. Statistical analysis of the combined data suggests that the order of markers in the BRCA1 region is cen-THRA1-TOP2-GAS-OF2-17HSI)-248yg9-RNU2-OF3-PPY/p131-EPB3-Mfd188-WNT3-HOX2-GP3A-tel. This map is consistent with that determined by radiation-reduced hybrid mapping and will facilitate positional cloning strategies in efforts to isolate and characterize the BRCA1 gene.

High-resolution cytogenetic mapping of 342 new cosmid markers including 43 RFLP markers on human chromosome 17 by fluorescence in situ hybridization.

We have constructed a high-resolution cytogenetic map of human chromosome 17 with 342 cosmid markers, each newly isolated from a cosmid library constructed from a human-mouse hybrid cell line containing a single human chromosome 17. Direct mapping on R- and/or G-banded (pro)metaphase chromosomes by fluorescence in situ hybridization localized these markers throughout the chromosome, although density was highest in the R-band-dominant regions of 17p13, 17p11.2, 17q11.2-q12, 17q21.3, 17q23, and 17q25. By screening some of the cosmid clones, we identified 71 polymorphic systems with 43 markers; 11 of these are VNTRs. As our high-resolution cytogenetic map contains a large number of markers, it can provide useful landmarks for a contig map of chromosome 17. Furthermore, the map will contribute to positional cloning of aberrant genes responsible for inherited diseases such as Miller-Dieker syndrome (MDS), Smith-Magenis syndrome (SMS), and familial early-onset breast cancer, as well as putative tumor suppressor genes on this chromosome.

Closing in on the gene for early onset familial breast cancer: F. Collins, J. Chamberlain, K. Abel, W. Flejter, S. Chandrasekharappa, L. Brody, S. Wei-Guo, M. Boehnke, B. Weber. University of Michigan, Ann Arbor, MI

Closing in on the gene for early onset familial breast cancer: F. Collins, J. Chamberlain, K. Abel, W. Flejter, S. Chandrasekharappa, L. Brody, S. Wei-Guo, M. Boehnke, B. Weber. University of Michigan, Ann Arbor, MI

Genetic heterogeneity of early-onset familial breast cancer.

A gene for early-onset familial breast cancer has recently been mapped to the chromosome 17q12-23 region. In order to confirm the gene location, we have tested an extensive early-onset breast cancer family with 4 markers in this chromosome region. Linkage was negative with all 4 markers. This study suggests that there is genetic heterogeneity among early-onset breast cancer families.

Linkage of Early-Onset Familial Breast Cancer to Chromosome 17q21

Human breast cancer is usually caused by genetic alterations of somatic cells of the breast, but occasionally, susceptibility to the disease is inherited. Mapping the genes responsible for inherited breast cancer may also allow the identification of early lesions that are critical for the development of breast cancer in the general population. Chromosome 17q21 appears to be the locale of a gene for inherited susceptibility to breast cancer in families with early-onset disease. Genetic analysis yields a lod score (logarithm of the likelihood ratio for linkage) of 5.98 for linkage of breast cancer susceptibility to D17S74 in early-onset families and negative lod scores in families with late-onset disease. Likelihood ratios in favor of linkage heterogeneity among families ranged between 2000:1 and greater than 10(6):1 on the basis of multipoint analysis of four loci in the region.