Candidate Breast Cancer Susceptibility Gene Research Articles

A joint transcriptome-wide association study across multiple tissues identifies candidate breast cancer susceptibility genes

Genome-wide association studies (GWASs) have identified more than 200 genomic loci for breast cancer risk, but specific causal genes in most of these loci have not been identified. In fact, transcriptome-wide association studies (TWASs) of breast cancer performed using gene expression prediction models trained in breast tissue have yet to clearly identify most target genes. To identify candidate genes, we performed a GWAS analysis in a breast cancer dataset from UK Biobank (UKB) and combined the results with the GWAS results of the Breast Cancer Association Consortium (BCAC) by a meta-analysis. Using the summary statistics from the meta-analysis, we performed a joint TWAS analysis that combined TWAS signals from multiple tissues. We used expression prediction models trained in 11 tissues that are potentially relevant to breast cancer from the Genotype-Tissue Expression (GTEx) data. In the GWAS analysis, we identified eight loci distinct from those reported previously. In the TWAS analysis, we identified 309 genes at 108 genomic loci to be significantly associated with breast cancer at the Bonferroni threshold. Of these, 17 genes were located in eight regions that were at least 1 Mb away from published GWAS hits. The remaining TWAS-significant genes were located in 100 known genomic loci from previous GWASs of breast cancer. We found that 21 genes located in known GWAS loci remained statistically significant after conditioning on previous GWAS index variants. Our study provides insights into breast cancer genetics through mapping candidate target genes in a large proportion of known GWAS loci and discovering multiple new loci.

Genome-wide investigations on regulatory functions of RECQ1 helicase.

DNA helicase RECQ1 (also known as RECQL or RECQL1) is a candidate breast cancer susceptibility gene significantly correlated with clinical outcomes of sporadic breast cancer patients. Prior studies have suggested that RECQ1 maintains genomic stability by regulating a wide variety of core cellular functions including DNA replication, DNA damage response, and transcription. However, it is unclear which, if any, of these are the primary functions of RECQ1 as related to its role in suppressing breast cancer. We describe here an unbiased integrative genomics approach that enabled us to discover a previously unknown regulatory role of RECQ1 in promoting Estrogen Receptor alpha (ERα) expression and the expression of specific ERα target genes in ER positive breast cancer cells. We discuss potential future applications of similar experimental strategies in advancing the mechanistic understanding and elucidating specific new details of genome-wide functions of RECQ1 and other RecQ helicases in maintaining genomic stability and preventing cancer.

Pleiotropy-guided transcriptome imputation from normal and tumor tissues identifies candidate susceptibility genes for breast and ovarian cancer.

SummaryFamilial, sequencing, and genome-wide association studies (GWASs) and genetic correlation analyses have progressively unraveled the shared or pleiotropic germline genetics of breast and ovarian cancer. In this study, we aimed to leverage this shared germline genetics to improve the power of transcriptome-wide association studies (TWASs) to identify candidate breast cancer and ovarian cancer susceptibility genes. We built gene expression prediction models using the PrediXcan method in 681 breast and 295 ovarian tumors from The Cancer Genome Atlas and 211 breast and 99 ovarian normal tissue samples from the Genotype-Tissue Expression project and integrated these with GWAS meta-analysis data from the Breast Cancer Association Consortium (122,977 cases/105,974 controls) and the Ovarian Cancer Association Consortium (22,406 cases/40,941 controls). The integration was achieved through application of a pleiotropy-guided conditional/conjunction false discovery rate (FDR) approach in the setting of a TWASs. This identified 14 candidate breast cancer susceptibility genes spanning 11 genomic regions and 8 candidate ovarian cancer susceptibility genes spanning 5 genomic regions at conjunction FDR < 0.05 that were >1 Mb away from known breast and/or ovarian cancer susceptibility loci. We also identified 38 candidate breast cancer susceptibility genes and 17 candidate ovarian cancer susceptibility genes at conjunction FDR < 0.05 at known breast and/or ovarian susceptibility loci. The 22 genes identified by our cross-cancer analysis represent promising candidates that further elucidate the role of the transcriptome in mediating germline breast and ovarian cancer risk.

Abstract 4175: Spectrum of heritable mutations in 43 known and candidate breast cancer susceptibility genes in African American women with breast cancer

Abstract Mutations in twenty genes have been associated with heritable breast cancer (BCA), yet the prevalence and clinical implications of most of these genes have not been well described in African American women (AAW). AAW who harbor inherited mutations in BCA susceptibility (BCS) genes are less likely to be identified and thus less likely to receive standard of surgical and preventive care for heritable BCA. A better understanding of heritable mutations in AAW with BCA would ultimately help to define guidelines for clinical management of high risk AAW. We performed targeted sequencing for 288 AAW diagnosed with invasive BCA unselected for family history, age, or BCA subtype. Participants were enrolled in the Detroit Research on Cancer Survivors (ROCS) cohort study or the Karmanos Cancer Institute Biobank. A custom sequencing panel captured the coding sequence of 43 genes (Known: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MRE11A, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53; Candidate: MLH1, MSH2, PMS1, SEC23B, BLM, ATR, BAP1, BBC3, CDKN1A, FAM175A, FANCA, FANCC, FANCI, FANCL, FANCM, GEN1, RAD51B, RBBP8, RECQL, RINT1, TP53BP1, XRCC1, XRCC3). Samples were sequenced using the Illumina MiSeq platform multiplexed for 100X mean depth of coverage. Intronic or synonymous exonic variants and those with &amp;lt;40 reads, or alternative allele frequency &amp;gt;3% in African populations in the 1000 Genomes, ExAC, or NHLBI ESP6500SI databases were filtered. Only variants in known BCS genes could be considered definitively pathogenic (DP), defined as missense variants categorized as pathogenic in ClinVar and all frameshift, nonsense, and splice site variants. All other variants were defined as variants of uncertain significance (VUS). We identified 54 women harboring DP mutations in 15 known BCS genes. Mutations in BRCA1/2 accounted for 32% of all DP mutations. Notably, nearly 17% of DP mutations were in MSH6, a gene only recently described as associated with increased risk of BCA in the general population. Among women with these DP mutations, nearly 25% had no high-risk characteristics. Ten VUS in known BCS genes were identified, where 4 were reported in ClinVar (BARD1, RAD5C, BRCA2, PMS2) and 6 were novel (PMS2, BRCA1, MUTYH, MSH6, NBN, MRE11). Among the candidate BCS genes, 63 VUS were identified: 51 frameshift variants, 5 nonsense variants, 3 splice site variants, and 4 missense mutations predicted to be pathogenic by ≥8/9 algorithms. Nearly 60% of participants had neither a DP mutation or VUS. These data suggest that AAW with BCA have a unique mutation spectrum and may benefit from BCS gene sequencing regardless of family history, age, or subtype. Additional sequencing in AAW will lay the foundation for identification of germline factors associated with BCA risk and prognosis specifically in AAW and offer opportunities for personalized risk assessment. Citation Format: Kristen Purrington, Gregory Dyson, Douglas Craig, Julie Boerner, Julie Madden, Jennifer Beebe-Dimmer, Ann G. Schwartz, Michael Simon. Spectrum of heritable mutations in 43 known and candidate breast cancer susceptibility genes in African American women with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4175.

Assessment of PARP4 as a candidate breast cancer susceptibility gene.

PARP4 has been proposed as a candidate breast cancer susceptibility gene. However, its function and involvement in breast carcinogenesis is unclear. We sought to determine the variant frequency of PARP4 in BRCA-negative women referred for genetic testing from Singapore and to perform functional analyses of PARP4. Next-generation sequencing of PARP4 was conducted for 198 BRCA-negative cases from Singapore. Three independent case-control association analyses of PARP4 were performed for (1) our Singaporean cohort, (2) three dbGaP datasets, and (3) cases from TCGA, with controls from the Exome Aggregation Consortium (ExAC). PARP4 knockout cells were generated utilizing the CRISPR-Cas9 approach in MDA-MB-231 (breast cancer) and MCF10A (normal breast) cell lines, and colony formation, cell proliferation, and migration assays carried out. Candidate variants in PARP4 were identified in 5.5% (11/198) of our Singapore cohort. Case-control association studies for our cases and the dbGaP datasets showed no significant association. However, a significant association was observed for PARP4 variants when comparing 988 breast cancer cases from the TCGA provisional data and 53,105 controls from ExAC (ALL) (OR 0.249, 95% CI 0.139-0.414, P = 2.86 × 10-11). PARP4 knockout did not affect the clonogenicity, proliferation rate, and migration of normal breast cells, but appeared to decrease the proliferation rate and clonogenicity of breast cancer cells. Taken together, our results do not support that PARP4 functions as a cancer susceptibility gene. This study highlights the importance of performing functional analyses for candidate cancer predisposition genes.

Exome sequencing and case-control analyses identify RCC1 as a candidate breast cancer susceptibility gene.

Breast cancer is a genetic disease but the known genes explain a minority of cases. To elucidate the molecular basis of breast cancer in the Tunisian population, we performed exome sequencing on six BRCA1/BRCA2 mutation-negative patients with familial breast cancer and identified a novel frameshift mutation in RCC1, encoding the Regulator of Chromosome Condensation 1. Subsequent genotyping detected the 19-bp deletion in additional 5 out of 153 (3%) breast cancer patients but in none of 400 female controls (p = 0.0015). The deletion was enriched in patients with a positive family history (5%, p = 0.0009) and co-segregated with breast cancer in the initial pedigree. The mutant allele was lost in 4/6 breast tumors from mutation carriers which may be consistent with the hypothesis that RCC1 dysfunction provides a selective disadvantage at the stage of tumor progression. In summary, we propose RCC1 as a likely breast cancer susceptibility gene in the Tunisian population.

Rat Mcs1b Is Concordant to the Genome-Wide Association-Identified Breast Cancer Risk Locus at Human 5q11.2 and MIER3 is a Candidate Cancer Susceptibility Gene

Low-penetrance alleles associated with breast cancer risk have been identified in population-based studies. Most risk loci contain either no or multiple potential candidate genes. Rat mammary carcinoma susceptibility 1b (Mcs1b) is a quantitative trait locus on RN02 that confers decreased susceptibility when Copenhagen (COP)-resistant alleles are introgressed into a Wistar Furth (WF)-susceptible genome. Five WF.COP congenic lines containing COP RN02 segments were compared. One line developed an average of 3.4 ± 2.0 and 5.5 ± 3.6 mammary carcinomas per rat ± SD when females were Mcs1b-resistant homozygous and Mcs1b heterozygous, respectively. These phenotypes were significantly different from susceptible genotype littermates (7.8 ± 3.1 mean mammary carcinomas per rat ± SD, P = 0.0001 and P = 0.0413, respectively). All other congenic lines tested were susceptible. Thus, Mcs1b was narrowed to 1.8 Mb of RN02 between genetic markers ENSRNOSNP2740854 and g2UL2-27. Mammary gland-graft carcinoma susceptibility assays were used to determine that donor (P = 0.0019), but not recipient Mcs1b genotype (P = 0.9381), was associated with ectopic mammary carcinoma outcome. Rat Mcs1b contains sequence orthologous to human 5q11.2, a breast cancer susceptibility locus identified in multiple genome-wide association studies. Human/rat MAP3K1/Map3k1 and mesoderm induction early response (MIER; MIER3)/MIER3 are within these orthologous segments. We identified MIER3 as a candidate Mcs1b gene based on 4.5-fold higher mammary gland levels of MIER3 transcripts in susceptible compared with Mcs1b-resistant females. These data suggest that the human 5q11.2 breast cancer risk allele marked by rs889312 is mammary gland autonomous, and MIER3 is a candidate breast cancer susceptibility gene.

Human MCS5A1 candidate breast cancer susceptibility gene FBXO10 is induced by cellular stress and correlated with lens epithelium-derived growth factor (LEDGF).

Genetic variation and candidate genes associated with breast cancer susceptibility have been identified. Identifying molecular interactions between associated genetic variation and cellular proteins may help to better understand environmental risk. Human MCS5A1 breast cancer susceptibility associated SNP rs7042509 is located in F-box protein 10 (FBXO10). An orthologous Rattus norvegicus DNA-sequence that contains SNV ss262858675 is located in rat Mcs5a1, which is part of a mammary carcinoma susceptibility locus controlling tumor development in a non-mammary cell-autonomous manner via an immune cell-mediated mechanism. Higher Fbxo10 expression in T cells is associated with Mcs5a increased susceptibility alleles. A common DNA-protein complex bound human and rat sequences containing MCS5A1/Mcs5a1 rs7042509/ss262858675 in electrophoretic mobility shift assays (EMSAs). Lens epithelium-derived growth factor (LEDGF), a stress-response protein, was identified as a candidate to bind both human and rat sequences using DNA-pulldown and mass spectrometry. LEDGF binding was confirmed by LEDGF-antibody EMSA and chromatin immunoprecipitation (ChIP). Ectopic expression of LEDGF/p75 increased luciferase activities of co-transfected reporters containing both human and rat orthologs. Over-expressed LEDGF/p75 increased endogenous FBXO10 mRNA levels in Jurkat cells, a human T-cell line, implying LEDGF may be involved in increasing FBXO10 transcript levels. Oxidative and thermal stress of Jurkat cells increased FBXO10 and LEDGF expression, further supporting a hypothesis that LEDGF binds to a regulatory region of FBXO10 and increases expression during conditions favoring carcinogenesis. We conclude that FBXO10, a candidate breast cancer susceptibility associated gene, is induced by cellular stress and LEDGF may play a role in expression of this gene.

Early exploration of two candidate breast cancer susceptibility genes identified by whole-exome sequencing

Breast cancer (BC) is the most frequently diagnosed cancer in women around the world, and an estimated15-20% of BC cases present with a family history of disease. Genetic variants in known susceptibility genes explain a relatively small proportion of the heritable risk for BC. Genetic variants have been broadly classified into three categories with different levels of risk and prevalence: rare mutations in high-risk genes (e.g. BRCA1, BRCA2); rare mutations in intermediate-risk genes (e.g. CHEK2); and common very modest-risk genetic variants (identified throughout the genome). These categories currently account for 20%, 5% and ~15% of the familial risk, respectively, leaving about 60% of the familial BC risk to be determined. We have conducted whole-exome capture followed by massive parallel sequencing (XC-MPS)-based analysis on greater than third degree affected relatives from highly selected multiple-case BC families (at least four cases of invasive breast cancer diagnosed before 50 years) which had previously been screened and found not to carry identifiable BRCA1 or BRCA2 mutations. In this presentation, we focus on two candidate BC susceptibility genes identified by this analysis, each playing a key-role in DNA repair. In both cases, one individual from the pair of cousins was found to carry a predicted protein damaging genetic variant at a consensus splice site. The variants were confirmed and extended analysis within the respective pedigrees performed by Sanger sequencing, subject to DNA sample availability. Further characterization of these variants is being pursued by much larger scale case-control genotyping using the TaqMan platform. Our work further illustrates the complexities of human genetic variation and the technical and analytical challenges of identifying variation that is associated with inherited predisposition to complex diseases such as breast cancer.

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.

Mutation analysis of the gene encoding the PALB2-binding protein MRG15 in BRCA1/2-negative breast cancer families

MRG15 is a recently identified member of the BRCA multiprotein complex, essential for the maintenance of the genome integrity and DNA repair. The functional relationship between PALB2 and MRG15 makes MRG15 a strong candidate breast cancer susceptibility gene. We screened affected probands from 232 BRCA1/2-negative breast cancer families for mutations in MRG15. We identified seven previously unreported variants but in silico analyses revealed that none of these variants appears to modify the function of MRG15. Thus, it seems unlikely that any constitutional changes in MRG15 confer an increased risk for breast cancer.

Genetic Contribution of GADD45A to Susceptibility to Sporadic and Non-BRCA1/2 Familial Breast Cancers: A Systemic Evaluation in Chinese Populations.

Abstract GADD45A is a candidate breast cancer susceptibility gene because its product participates in DNA repair and it is a downstream gene of p53 and BRCA1, both of which are breast cancer susceptibility genes. The genetic contribution of GADD45A to sporadic and familial breast cancer is still ambiguous. We screened germline mutation of GADD45A in 185 non-BRCA1/2 familial breast cancer patients, but no deleterious mutation was found. Seven single-nucleotide-polymorphisms were identified in a sub-sample. Five common variants (minor allele frequency (MAF)&amp;gt;10%) were genotyped for association analyses to scrutinize the relationship between breast cancer and polymorphisms in GADD45A in two independent population sets. In the first case-control study, a comparison of genotype frequencies between sporadic breast cancer patients and controls indicated the CT/TT-genotypes of +1506C&amp;gt;T and CG/CC-genotypes of +3204G&amp;gt;C were associated with decreased breast cancer risk (adjusted odds ratio (OR), 0.77; 95% confidence interval (CI), 0.62-0.96; and adjusted OR, 0.71; 95%CI, 0.57-0.88, respectively) compared with their wild-type homozygotes. A common hyplotype CGTCC was also associated with reduced risk (P=1.0×10-4). In a second familial breast cancer patient-based case-control study, although +1506C&amp;gt;T and +3204G&amp;gt;C failed to be validated, the haplotype CGTCC showed a borderline significance. Notably, the combined P-values were robust for +3204G&amp;gt;C (P=3.1×10-4) and CGTCC (P=1.6×10-5). Moreover, CGTCC was correlated with a higher GADD45A expression in normal breast tissues. In conclusion, although germline mutation of GADD45A is not common in familial breast cancer patients, polymorphisms/haplotypes in GADD45A contribute to breast cancer risk, at least to sporadic breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5163.

Genetic contribution of GADD45A to susceptibility to sporadic and non-BRCA1/2 familial breast cancers: a systematic evaluation in Chinese populations

Growth arrest and DNA damage-induced 45, alpha (GADD45A) is a candidate breast cancer susceptibility gene because its product participates in DNA repair and it is a downstream gene of p53 and BRCA1, both of which are breast cancer susceptibility genes. We screened germline mutations of GADD45A in 185 non-BRCA1/2 familial breast cancer patients, but no deleterious mutation was found. Seven single-nucleotide-polymorphisms were identified in a subsample. Five common variants (minor allele frequency > 10%) were genotyped for association analyses to scrutinize the relationship between breast cancer and polymorphisms in GADD45A in two independent population sets (total n = 1,861). In the first case-control study (n = 1,457, cases 820, controls 637), a comparison of genotype frequencies between sporadic breast cancer patients and controls indicated the CT/TT-genotypes of +1506C>T and CG/CC-genotypes of +3204G>C were associated with decreased breast cancer risk (adjusted odds ratio (OR), 0.77; 95% confidence interval (CI), 0.62-0.96; and adjusted OR, 0.71; 95%CI, 0.57-0.88, respectively) compared with their wild-type homozygotes. A common haplotype CGTCC was also associated with reduced risk (P = 1.0 x 10(-4)). In a second familial breast cancer patient-based case-control study (n = 404, cases 185, controls 219), although +1506C>T and +3204G>C failed to be validated, the haplotype CGTCC showed a borderline significance. Notably, the combined P-values were robust for +3204G>C (P = 3.1 x 10(-4)) and CGTCC (P = 1.6 x 10(-5)). Moreover, CGTCC was correlated with a higher GADD45A expression in normal breast tissues. In conclusion, although germline mutations of GADD45A is not common in familial breast cancer patients, polymorphisms/haplotypes in GADD45A contribute to breast cancer risk, at least to sporadic breast cancer.

Gene-body hypermethylation of ATM in peripheral blood DNA of bilateral breast cancer patients

Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility; however, the molecular causes underlying this predisposition in the majority of cases is not known. We hypothesize that epigenetic misregulation of cancer-related genes could partially account for this predisposition. We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared with 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulphite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared with 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared with controls (P = 0.0017), with the highest quartile of methylation associated with a 3-fold increased risk of breast cancer (OR 3.20, 95% CI 1.78–5.86, P = 0.000083). Increased methylation of this locus is associated with lower steady-state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age–phenotype-related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer-related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk.Accession numbers: The microarray data and associated .BED and .WIG files can be accessed through Gene Expression Omnibus accession number: GSE14603.

Aberrations of the MRE11–RAD50–NBS1 DNA damage sensor complex in human breast cancer: MRE11 as a candidate familial cancer-predisposing gene

The MRE11, RAD50, and NBS1 genes encode proteins of the MRE11–RAD50–NBS1 (MRN) complex critical for proper maintenance of genomic integrity and tumour suppression; however, the extent and impact of their cancer-predisposing defects, and potential clinical value remain to be determined. Here, we report that among a large series of approximately 1000 breast carcinomas, around 3%, 7% and 10% tumours showed aberrantly reduced protein expression for RAD50, MRE11 and NBS1, respectively. Such defects were more frequent among the ER/PR/ERBB2 triple-negative and higher-grade tumours, among familial (especially BRCA1/BRCA2-associated) rather than sporadic cases, and the NBS1 defects correlated with shorter patients’ survival. The BRCA1-associated and ER/PR/ERBB2 triple-negative tumours also showed high incidence of constitutively active DNA damage signalling (γH2AX) and p53 aberrations. Sequencing the RAD50, MRE11 and NBS1 genes of 8 patients from non- BRCA1/2 breast cancer families whose tumours showed concomitant reduction/loss of all three MRN-complex proteins revealed two germline mutations in MRE11: a missense mutation R202G and a truncating mutation R633STOP (R633X). Gene transfer and protein analysis of cell culture models with mutant MRE11 implicated various destabilization patterns among the MRN complex proteins including NBS1, the abundance of which was restored by re-expression of wild-type MRE11. We propose that germline mutations qualify MRE11 as a novel candidate breast cancer susceptibility gene in a subset of non-BRCA1/2 families. Our data have implications for the concept of the DNA damage response as an intrinsic anti-cancer barrier, various components of which become inactivated during cancer progression and also represent the bulk of breast cancer susceptibility genes discovered to date.

Mutation analysis and characterization of HSD17B2 sequence variants in breast cancer cases from French Canadian families with high risk of breast and ovarian cancer

Estrogen exposure is a risk factor for breast cancer. Given that HSD17B2 gene encodes an enzyme that catalyses estradiol inactivation, it appears as a good candidate breast cancer susceptibility gene. This study was designed to screen for HSD17B2 germline mutations potentially involved in breast cancer predisposition. Our re-sequencing analysis did not identify any deleterious germline mutations, and therefore mutations in HSD17B2 do not explain the clustering of breast cancer cases in non-BRCA1/2 high-risk French Canadian families. However, six sequence variants were identified, including two novel missense variants. Expression assays revealed that p.Ala111Asp and p.Gly160Arg did not alter the catalytic properties of 17beta-hydroxysteroid dehydrogenase type 2 enzyme, although p.Ala111Asp appears to affect protein stability resulting in significant decreases in the protein levels, providing valuable information on structure-function relationship.

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.

Mutation analysis of five candidate genes in familial breast cancer

Most of the known breast cancer susceptibility genes (BRCA1, BRCA2, CHEK2 and ATM) are involved in the damage response pathway. Other members of this pathway are therefore good candidates for additional breast cancer susceptibility genes. ATR, along with ATM, plays a central role in DNA damage recognition and Chk1 relays checkpoint signals from both ATR and ATM. PPP2R1B and PPP2R5B code for subunits of protein phosphatase 2A (PP2A), which regulates autophosphorylation of ATM. In addition, EIF2S6/Int-6, which was originally identified as a common integration site for the mouse mammary tumour virus in virally induced mouse mammary tumours, is a candidate breast cancer susceptibility gene because of its putative role in maintaining chromosome stability. To investigate the role of ATR, CHK1, PPP2R1B, PPP2R5B and EIF2S6/Int-6, we carried out mutation analysis of these genes in the index cases from non-BRCA1/BRCA2 breast cancer families. We also screened sporadic breast tumours for somatic mutations in PPP2R1B and PPP2R5B. Although we identified many novel variants, we found no evidence that highly penetrant germline mutations in these five genes contribute to familial breast cancer susceptibility.

A Systematic Assessment of Common Genetic Variation in CYP11A and Risk of Breast Cancer

CYP11A catalyzes the rate-limiting step in the biosynthesis of sex-steroid hormones. In this study, we employed a systematic approach that involved gene resequencing and a haplotype-based analysis to investigate the relationship between common variation in CYP11A and breast cancer risk among African-Americans, Latinas, Japanese-Americans, Native Hawaiians, and Whites in the Multiethnic Cohort Study. Resequencing in a multiethnic panel of 95 advanced breast cancer cases revealed no common missense variant (> or =5% frequency). Common haplotype patterns were assessed by genotyping 36 densely spaced single nucleotide polymorphisms (SNPs) spanning 67 kb of the CYP11A locus in a multiethnic panel of subjects (n = 349; 1 SNP/1.86 kb on average). We identified one to two regions of strong linkage disequilibrium in these populations. Twelve tagging SNPs were selected to predict the common haplotypes (> or =5% frequency) in these regions with high probability (average R(h)(2) = 0.94) and were examined in a breast cancer case-control study in the Multiethnic Cohort Study (1,615 cases and 1,962 controls). A global test for differences in risk according to common haplotypes over the locus was statistically significant (P = 0.006), as were associations with haplotypes in each block (block 1 global test, P = 0.008; haplotype 1D, effect per haplotype copy, odds ratios, 1.23; 95% confidence interval, 1.03-1.48) and block 2 (global test, P = 0.016; haplotype 2F odds ratios, 1.52; 95% confidence interval, 1.15-2.01). These haplotypes were most common in Japanese-Americans and Native Hawaiians, followed by Whites then Latinas, and were rare in African-Americans (<5% frequency); the haplotype effects on risk across each group were homogeneous. Based on these findings, CYP11A deserves further consideration as a candidate breast cancer susceptibility gene.

Mutation analysis and characterization of ATR sequence variants in breast cancer cases from high-risk French Canadian breast/ovarian cancer families

BackgroundAtaxia telangiectasia-mutated and Rad3-related (ATR) is a member of the PIK-related family which plays, along with ATM, a central role in cell-cycle regulation. ATR has been shown to phosphorylate several tumor suppressors like BRCA1, CHEK1 and TP53. ATR appears as a good candidate breast cancer susceptibility gene and the current study was designed to screen for ATR germline mutations potentially involved in breast cancer predisposition.MethodsATR direct sequencing was performed using a fluorescent method while widely available programs were used for linkage disequilibrium (LD), haplotype analyses, and tagging SNP (tSNP) identification. Expression analyses were carried out using real-time PCR.ResultsThe complete sequence of all exons and flanking intronic sequences were analyzed in DNA samples from 54 individuals affected with breast cancer from non-BRCA1/2 high-risk French Canadian breast/ovarian families. Although no germline mutation has been identified in the coding region, we identified 41 sequence variants, including 16 coding variants, 3 of which are not reported in public databases. SNP haplotypes were established and tSNPs were identified in 73 healthy unrelated French Canadians, providing a valuable tool for further association studies involving the ATR gene, using large cohorts. Our analyses led to the identification of two novel alternative splice transcripts. In contrast to the transcript generated by an alternative splicing site in the intron 41, the one resulting from a deletion of 121 nucleotides in exon 33 is widely expressed, at significant but relatively low levels, in both normal and tumoral cells including normal breast and ovarian tissue.ConclusionAlthough no deleterious mutations were identified in the ATR gene, the current study provides an haplotype analysis of the ATR gene polymorphisms, which allowed the identification of a set of SNPs that could be used as tSNPs for large-scale association studies. In addition, our study led to the characterization of a novel Δ33 splice form, which could generate a putative truncated protein lacking several functional domains. Additional studies in large cohorts and other populations will be needed to further evaluate if common and/or rare ATR sequence variants can be associated with a modest or intermediate breast cancer risk.