Carol Chu

Common Breast Cancer-Predisposition Alleles Are Associated with Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Oral Contraceptives and Breast Cancer Risk in the International BRCA1/2 Carrier Cohort Study: A Report From EMBRACE, GENEPSO, GEO-HEBON, and the IBCCS Collaborating Group

PURPOSE Earlier studies have shown that endogenous gonadal hormones play an important role in the etiology of breast cancer among BRCA1/2 mutation carriers. So far, little is known about the safety of exogenous hormonal use in mutation carriers. In this study, we examined the association between oral contraceptive use and risk of breast cancer among BRCA1/2 carriers. PATIENTS AND METHODS In the International BRCA1/2 Carrier Cohort study (IBCCS), a retrospective cohort of 1,593 BRCA1/2 mutation carriers was analyzed with a weighted Cox regression analysis. Results We found an increased risk of breast cancer for BRCA1/2 mutation carriers who ever used oral contraceptives (adjusted hazard ratio [HR] = 1.47; 95% CI, 1.16 to 1.87). HRs did not vary according to time since stopping use, age at start, or calendar year at start. However, a longer duration of use, especially before first full-term pregnancy, was associated with an increased risk of breast cancer for both BRCA1 and BRCA2 mutation carriers (4 or more years of use before first full-term pregnancy: HR = 1.49 [95% CI, 1.05 to 2.11] for BRCA1 carriers and HR = 2.58 [95% CI, 1.21 to 5.49] for BRCA2 carriers). CONCLUSION No evidence was found among BRCA1/2 mutation carriers that current use of oral contraceptives is associated with risk of breast cancer more strongly than is past use, as is found in the general population. However, duration of use, especially before first full-term pregnancy, may be associated with an increasing risk of breast cancer among both BRCA1 and BRCA2 mutation carriers.

Genetic diagnosis of familial breast cancer using clonal sequencing

Using conventional Sanger sequencing as a reference standard, we compared the sensitivity, specificity, and capacity of the Illumina GA II platform for the detection of TP53, BRCA1, and BRCA2 mutations in established tumor cell lines and DNA from patients with germline mutations. A total of 656 coding variants were identified in four cell lines and 65 patient DNAs. All of the known pathogenic mutations (including point mutations and insertions/deletions of up to 16 nucleotides) were identified, using a combination of the Illumina data analysis pipeline with custom and commercial sequence alignment software. In our configuration, clonal sequencing outperforms current diagnostic methods, providing a reduction in analysis times and in reagent costs compared with conventional sequencing. These improvements open the possibility of BRCA1/2 testing for a wider spectrum of at‐risk women, and will allow the genetic classification of tumors prior to the use of novel PARP inhibitors to treat BRCA‐deficient breast cancers. Hum Mutat 31:1–8, 2010.

Characterization of a 3;6 Translocation Associated with Renal Cell Carcinoma

The most frequent cause of familial clear cell renal cell carcinoma (RCC) is von Hippel–Lindau disease and the VHL tumor suppressor gene (TSG) is inactivated in most sporadic clear cell RCC. Although there is relatively little information on the mechanisms of tumorigenesis of clear cell RCC without VHL inactivation, a subset of familial cases harbors a balanced constitutional chromosome 3 translocation. To date nine different chromosome 3 translocations have been associated with familial or multicentric clear cell RCC; and in three cases chromosome 6 was also involved. To identify candidate genes for renal tumorigenesis we characterized a constitutional translocation, t(3;6)(q22;q16.1) associated with multicentric RCC without evidence of VHL target gene dysregulation. Analysis of breakpoint sequences revealed a 1.3‐kb deletion on chromosome 6 within the intron of a 2 exon predicted gene (NT_007299.434). However, RT‐PCR analysis failed to detect the expression of this gene in lymphoblast, fibroblast, or kidney tumor cell lines. No known genes were disrupted by the translocation breakpoints but several candidate TSGs (e.g., EPHB1, EPHA7, PPP2R3A RNF184, and STAG1) map within close proximity to the breakpoints.

Evidence for SMAD3 as a modifier of breast cancer risk in BRCA2 mutation carriers.

IntroductionCurrent attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies.MethodsWe successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers.ResultsSNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r2 = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, Ptrend = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, Ptrend = 0.018).ConclusionsThis study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.

Exon Deletions and Duplications in BRCA1 Detected by Semiquantitative PCR

rearrangements have recently been identified in the BRCA1 gene. Inclusion of a method for identifying such rearrangements should now be a prerequisite for providing a comprehensive mutation detection strategy. We have developed a semiquantitative PCR-based fluorescent assay for the detection of previously identified deletions. This method avoids the need for long PCR or Southern blotting and is suitable for large-scale epidemiological studies. The assay was used to screen 44 high-risk families within the U.K. Yorkshire Health Region. No deletions were detected, but five cases (11%) with an apparent duplication of exon 13 in BRCA1 were identified. The presence of this mutation was confirmed by long PCR. Further developments include extending the assay to include all exons of BRCA1.

Investigations on a clinically and functionally unusual and novel germline p53 mutation

This report describes an individual with a rare choroid plexus papilloma in adulthood (age 29) after earlier having an osteosarcoma (age 22). The results from this study, and others, suggest that it may be advisable to consider the possibility of a germline p53 mutation in adults presenting with choroid plexus tumours. In the current study automated DNA sequencing of genomic DNA detected a novel germline 7 base pair insertion in exon 5 of the p53 gene in this patient. The alteration in frame would produce amino acid substitutions beginning with alanine to glycine at position 161 and a stop codon at position 182 in the mutated protein. Surprisingly two assays of p53 function gave apparently wild-type results on peripheral blood lymphocytes from this individual. These results led us to carry out more detailed functional tests on the mutant protein. The mutant allele was expressed either at very low levels or not at all in phytohaemagglutinin stimulated lymphocytes. Further, the mutant protein was completely non-functional in terms of its ability to transactivate a series of p53-responsive genes (p21WAF1, bax, PIG3), to transrepress a target gene and to inhibit colony growth in transfected Saos-2 cells. However, surprisingly, data from irradiated peripheral blood lymphocytes and transfected Saos-2 cells, suggested that this truncated, mutant protein retains significant ability to induce apoptosis.

Submicroscopic deletions of the APC gene: a frequent cause of familial adenomatous polyposis that may be overlooked by conventional mutation scanning

Editor—Familial adenomatous polyposis (FAP) is an autosomal dominant colon cancer predisposition syndrome in which patients develop hundreds to thousands of precancerous colonic polyps that have a high risk of becoming malignant. Despite the fact that deletions of the APC locus were originally used to map1 2 and identify3 4 the APC gene, most studies of mutations in APC have used techniques which would not detect deletions.5-7 Molecular analysis of the APC gene in FAP patients has found the pathogenic mutation in 70% of cases,8 but a substantial minority have no mutation identified. Interstitial deletions of chromosome 5q, which include the APC locus, have been reported in patients with polyposis coli and mental retardation.9Submicroscopic deletions have been reported in only a few cases.10 11 In one family,10 linkage analysis with flanking and intragenic markers followed by in situ hybridisation with intragenic cosmid clones showed that the deletion was approximately 200 kb and included more than the 3′ half of the APC gene and the 3′ adjacent D5S346 microsatellite. A recent report11 described a quantitative PCR assay to detect submicroscopic deletions which included the entire APC gene and the adjacent D5S346 microsatellite in three unrelated Italian FAP families. These submicroscopic deletions do not appear to be associated with mental retardation. Microsatellite analysis of families with FAP in our region showed one family with apparent non-maternity at D5S346 in one of the affected offspring of an affected mother, suggesting the possibility of an APC deletion. A quantitative PCR assay was therefore developed to detect submicroscopic deletions of the APC gene. Previous analysis of 68 FAP families from the Yorkshire region found the pathogenic mutation in 46 cases (67%). These cases were referred for testing (with informed consent) from the Yorkshire Regional Clinical …

Evaluation by magnetic resonance imaging of aortic dilatation and coarctation in adult Turner syndrome patients

Objective  Turner syndrome has well‐recognized cardiovascular complications that appear in up to 40% of the patients and are more common in monosomy X. Left‐sided obstructive lesions are relatively more frequent and predispose to aortic root dilatation and life‐threatening aortic dissection. However, bicuspid aortic valves, hypertension, coarctation and aortic stenosis are also at risk of aortic dissection. Currently there is no clear guideline regarding the best single test for detection or monitoring aortic disease progression.

Y-chromosome mosaicism in girls with Turner's syndrome†

Turner’s syndrome (TS) is very common with as many as 1 in 50 conceptuses affected. However, only 1% of Turners conceptuses survive to be born. This very high intrauterine loss, coupled with the observation that there is a higher ratio of mosaic karyotypes to monosomy X in liveborns compared with aborted fetuses, has led to speculation that all liveborn patients must be mosaic for a critical cell line (Hook & Warburton, 1983). Since 70–80% of individuals with TS retain the maternal X chromosome, Y mosaicism could theoretically be present in 35–40% of cases (Ostrer & Clayton, 1989). The importance of identifying Y mosaicism in girls with TS is the high risk of gonadal tumour formation in the dysgenetic gonad. This has been quoted to be as high as 20–30% (Verp & Simpson, 1987). The most common tumour seen in these cases is a gonadoblastoma which is found almost exclusively (96%) in dysgenetic gonads. Gonadoblastoma is seen most commonly in the second decade but can develop in much younger children and has been reported in children as young as 14 months (Lukusaet al, 1986). Pure gonadoblastomas are usually small to moderate in size and histologically contain nests of mixed germ cells and smaller epithelial cells resembling immature Sertoli and granulosa cells. Gonadoblastomas do not metastasize and can regress spontaneously. However, overgrowth by other germ cell elements frequently occurs and 50% are associated with dysgerminomas and 10% with other malignant tumours such as embryonal carcinomas and choriocarcinomas. The gonadoblastoma may synthesise oestrogen or testosterone so that both feminisation and virilisation have been seen in different cases. The development of gonadoblastoma almost exclusively in individuals with dysgenetic gonads and either whole or structurally abnormal Y chromosomes had led Page (1987) to postulate that there may be a gonadoblastoma gene (GBY) located on the Y chromosome which, in the context of a dysgenetic gonad, acts as an oncogene. Other unrecognised factors must also predispose to tumour formation since not all individuals with Y sequences develop tumours. The GBY locus has been mapped to interval 4B (which contains the Y centromere) to interval 5E on the proximal Y long arm (Salo et al, 1995). Since low-level mosaicism can be missed by conventional cytogenetic techniques, many studies have utilised molecular genetic techniques to look for Y mosaicism in patients with TS. The majority of studies have concentrated on screening blood although some have also looked at gonadal tissue. A variety of techniques have been used ranging from Southern blotting using Y specific probes, conventional PCR, nested PCR and Southern blotting of PCR products. The percentage of cases positive for one or more Y sequence ranges from 2–30% in various studies. The reason for this wide range is not clear but probably depends partly on the region or regions of the Y chromosome studied and the sensitivity of the approach. Some techniques will detect 0 .0001% male DNA on a female background (Binder et al., 1995). However, the more sensitive the technique, the more likely it is that false positive results can be obtained because of contamination particularly if consecutive experiments take place in one laboratory (Page, 1994). Also, the clinical significance of Y chromosomal material outside the GBY region with respect to gonadal tumours is not clear. The majority of molecular studies of Y mosaicism in TS have been on very small numbers of individuals with bias towards those with small marker chromosomes or signs of virilization. Several reports have confirmed the utility of using molecular techniques to identify the small marker chromosomes in this situation (Saloet al., 1995; Nagafuchi et al., 1992). A paper in this issue (Mendeset al., 1998) reinforces these previous reports since the only case with gonadoblastoma in their series actually had a small ringy chromosome. Virilization in a TS patient is another indication for detailed studies looking for Y mosaicism. Such studies should ideally be carried out on more than one tissue. Osipova et l. (1998) and Bisat et al. (1993) reported cases with virilization and Y sequences present in gonads but not in blood. It is less clear why some girls with Turner’s syndrome are virilized with no Y material present. Menderset al. (1998) documented virilization in their patients with no evidence of Y mosaicism and raise some interesting questions about the cause of virilization in these patients. The current practice in most units is to actively seek Y mosaicism in individuals with TS only if there is a small marker chromosome or there is virilization. If Y material is found then prophylactic gonadectomy is offered. The question Mendes et al. (1998) and others raise is what should be the management if Commentary