Simple and rapid characterization of novel large germline deletions in SDHB, SDHC and SDHD-related paraganglioma.

Germline mutations in genes encoding subunits of succinate dehydrogenase (SDH) are associated with hereditary paraganglioma and pheochromocytoma. Although most mutations in SDHB, SDHC and SDHD are intraexonic variants, large germline deletions may represent up to 10% of all variants but are rarely characterized at the DNA sequence level. Additional phenotypic effects resulting from deletions that affect neighboring genes are also not understood. We performed multiplex ligation-dependent probe amplification, followed by a simple long-range PCR 'chromosome walking' protocol to characterize breakpoints in 20 SDHx-linked paraganglioma-pheochromocytoma patients. Breakpoints were confirmed by conventional PCR and Sanger sequencing. Heterozygous germline deletions of up to 104 kb in size were identified in SDHB, SDHC, SDHD and flanking genes in 20 paraganglioma-pheochromocytoma patients. The exact breakpoint could be determined in 16 paraganglioma-pheochromocytoma patients of which 15 were novel deletions. In six patients proximal genes were also deleted, including PADI2, MFAP2, ATP13A2 (PARK9), CFAP126, TIMM8B and C11orf57. These genes were either partially or completely deleted, but did not modify the phenotype. This study increases the number of known SDHx deletions by over 50% and demonstrates that a significant proportion of large gene deletions can be resolved at the nucleotide level using a simple and rapid method.

Rare mutations in XRCC2 increase the risk of breast cancer.

An exome-sequencing study of families with multiple breast-cancer-affected individuals identified two families with XRCC2 mutations, one with a protein-truncating mutation and one with a probably deleterious missense mutation. We performed a population-based case-control mutation-screening study that identified six probably pathogenic coding variants in 1,308 cases with early-onset breast cancer and no variants in 1,120 controls (the severity grading was p < 0.02). We also performed additional mutation screening in 689 multiple-case families. We identified ten breast-cancer-affected families with protein-truncating or probably deleterious rare missense variants in XRCC2. Our identification of XRCC2 as a breast cancer susceptibility gene thus increases the proportion of breast cancers that are associated with homologous recombination-DNA-repair dysfunction and Fanconi anemia and could therefore benefit from specific targeted treatments such as PARP (poly ADP ribose polymerase) inhibitors. This study demonstrates the power of massively parallel sequencing for discovering susceptibility genes for common, complex diseases.

Survival and contralateral breast cancer in CHEK2 1100delC breast cancer patients: impact of adjuvant chemotherapy.

BACKGROUND: We assessed the sensitivity to adjuvant chemotherapy in cell cycle checkpoint kinase 2 (CHEK2) vs non-CHEK2 breast cancer patients by comparing the contralateral breast cancer incidence and distant disease-free and breast cancer-specific survival between both groups, stratified for adjuvant chemotherapy. METHODS: One Dutch hereditary non-BRCA1/2 breast cancer patient cohort (n=1220) and two Dutch cohorts unselected for family history (n=1014 and n=2488, respectively) were genotyped for CHEK2 1100delC. Hazard ratios for contralateral breast cancer, distant disease-free and breast cancer-specific death for mutation carriers vs noncarriers were calculated using the Cox proportional hazard method, stratified for adjuvant chemotherapy. RESULTS: The CHEK2 mutation carriers (n=193) had an increased incidence of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59-6.07). Distant disease-free and breast cancer-specific survival were similar in the first 6 years in mutation carriers compared with noncarriers, but diverted as of 6 years after breast cancer diagnosis (multivariate hazard ratios and 95% confidence intervals 2.65 (1.79-3.93) and 2.05 (1.41-2.99), respectively). No significant interaction between CHEK2 and adjuvant chemotherapy was observed. CONCLUSIONS: The CHEK2 1100delC-associated breast cancer is associated with a higher contralateral breast cancer rate as well as worse survival measures beyond 6 years after diagnosis. No differential sensitivity to adjuvant chemotherapy was observed in CHEK2 patients.

The impact of next generation sequencing on the analysis of breast cancer susceptibility: a role for extremely rare genetic variation?

Women with a family history of breast cancer have an approximately twofold elevated risk of the disease. Even though an array of genes has been associated with breast cancer risk the past two decades, variants within these genes jointly explain at most 40% of this familial risk. Many explanations for this 'missing heritability' have been proposed, including the existence of many very rare variants, interactions between genetic and environmental factors and structural genetic variation. In this review, we discuss how next generation sequencing will teach us more about the genetic architecture of breast cancer, with a specific focus on very rare genetic variants. While such variants potentially explain a substantial proportion of familial breast cancer, assessing the breast cancer risks conferred by them remains challenging, even if this risk is relatively high. To assess more moderate risks, epidemiological approaches will require very large patient cohorts to be genotyped for the variant, only achievable through international collaboration. How well we will be able to eventually resolve the missing heritability for breast cancer in a clinically meaningful way crucially depends on the underlying complexity of the genetic architecture.

Familial male breast cancer is not linked to the BRCA1 locus on chromosome 17q.

Breast cancer in men is about a hundredfold less common than in women and this has hindered research into its genetic basis. We have examined 22 families with at least one case of male breast cancer for linkage to the hereditary breast and ovarian cancer locus, BRCA1, on chromosome 17q. We found strong evidence against linkage to BRCA1 (lod score-16.63) and the best estimate of the proportion of linked families was 0% (95% CI 0-18%). Our results indicate that there is a gene(s) other than BRCA1 which predisposes to early-onset breast cancer in women and which confers a higher risk of male breast cancer. Identification of additional pedigrees that include cases of male breast cancer may therefore facilitate the mapping and isolation of this gene.

Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus.

Women who carry a mutation in the BRCA1 gene (on chromosome 17q21), have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (ref. 1). The variable penetrance of BRCA1 suggests that other genetic and non-genetic factors play a role in tumourigenesis in these individuals. The HRAS1 variable number of tandem repeats (VNTR) polymorphism, located 1 kilobase (kb) downstream of the HRAS1 proto-oncogene (chromosome 11p15.5) is one possible genetic modifier of cancer penetrance. Individuals who have rare alleles of the VNTR have an increased risk of certain types of cancers, including breast cancer (2-4). To investigate whether the presence of rare HRAS1 alleles increases susceptibility to hereditary breast and ovarian cancer, we have typed a panel of 307 female BRCA1 carriers at this locus using a PCR-based technique. The risk for ovarian cancer was 2.11 times greater for BRCA1 carriers harbouring one or two rare HRAS1 alleles, compared to carriers with only common alleles (P = 0.015). The magnitude of the relative risk associated with a rare HRAS1 allele was not altered by adjusting for the other known risk factors for hereditary ovarian cancer (5). Susceptibility to breast cancer did not appear to be affected by the presence of rare HRAS1 alleles. This study is the first to show the effect of a modifying gene on the penetrance of an inherited cancer syndrome.

BRCA1 genomic deletions are major founder mutations in Dutch breast cancer patients.

To date, more than 300 distinct small deletions, insertions and point mutations, mostly leading to premature termination of translation, have been reported in the breast/ovarian-cancer susceptibility gene BRCA1. The elevated frequencies of some mutations in certain ethnic subpopulations are caused by founder effects, rather than by mutation hotspots. Here we report that the currently available mutation spectrum of BRCA1 has been biased by PCR-based mutation-screening methods, such as SSCP, the protein truncation test (PTT) and direct sequencing, using genomic DNA as template. Three large genomic deletions that are not detected by these approaches comprise 36% of all BRCA1 mutations found in Dutch breast-cancer families to date. A 510-bp Alu-mediated deletion comprising exon 22 was found in 8 of 170 breast-cancer families recruited for research purposes and in 6 of 49 probands referred to the Amsterdam Family Cancer Clinic for genetic counselling. In addition, a 3,835-bp Alu-mediated deletion encompassing exon 13 was detected in 4 of 170 research families, while an deletion of approximately 14 kb was detected in a single family [corrected]. Haplotype analyses indicated that each recurrent deletion had a single common ancestor.

High prevalence of founder mutations of the succinate dehydrogenase genes in the Netherlands.

Mutations in four genes encoding subunits or cofactors of succinate dehydrogenase (SDH) cause hereditary paraganglioma and pheochromocytoma syndromes. Mutations in SDHB and SDHD are generally the most common, whereas mutations in SDHC and SDHAF2 are far less frequently observed. A total of 1045 DNA samples from Dutch paraganglioma and pheochromocytoma patients and their relatives were analyzed for mutations of SDHB, SDHC, SDHD or SDHAF2. Mutations in these genes were identified in 690 cases, 239 of which were index cases. The vast majority of mutation carriers had a mutation in SDHD (87.1%). The second most commonly affected gene was SDHAF2 (6.7%). Mutations in SDHB were found in only 5.9% of samples, whereas SDHC mutations were found in 0.3% of samples. Remarkably, 69.1% of all carriers of a mutation in an SDH gene in the Netherlands can be attributed to a single founder mutation in SDHD, c.274G>T and p.Asp92Tyr. Moreover, 88.8% of all SDH mutation carriers carry one of just six Dutch founder mutations in SDHB, SDHD and SDHAF2. The dominance of SDHD mutations is unique to the Netherlands, contrasting with the higher prevalence of SDHB mutations found elsewhere. In addition, we found that most SDH mutation-related paragangliomas-pheochromocytomas in the Netherlands can be explained by only six founder mutations in SDHAF2, SDHB and SDHD. The findings underline the regional differences in the SDH mutation spectrum, differences that should be taken into account in the development of effective screening protocols. The results show the crucial role that demographic factors play in the frequency of gene mutations.

A non-BRCA1/2 hereditary breast cancer sub-group defined by aCGH profiling of genetically related patients.

Germline mutations in BRCA1 and BRCA2 explain approximately 25% of all familial breast cancers. Despite intense efforts to find additional high-risk breast cancer genes (BRCAx) using linkage analysis, none have been reported thus far. Here we explore the hypothesis that BRCAx breast tumors from genetically related patients share a somatic genetic etiology that might be revealed by array comparative genomic hybridization (aCGH) profiling. As BRCA1 and BRCA2 tumors can be identified on the basis of specific genomic profiles, the same may be true for a subset of BRCAx families. Analyses used aCGH to compare 58 non-BRCA1/2 familial breast tumors (designated BRCAx) to sporadic (non-familiar) controls, BRCA1 and BRCA2 tumors. The selection criteria for BRCAx families included at least three cases of breast cancer diagnosed before the age of 60 in the family, and the absence of ovarian or male breast cancer. Hierarchical cluster analysis was performed to determine sub-groups within the BRCAx tumor class and family heterogeneity. Analysis of aCGH profiles of BRCAx tumors indicated that they constitute a heterogeneous class, but are distinct from both sporadic and BRCA1/2 tumors. The BRCAx class could be divided into sub-groups. One subgroup was characterized by a gain of chromosome 22. Tumors from family members were classified within the same sub-group in agreement with the hypothesis that tumors from the same family would harbor a similar genetic background. This approach provides a method to target a sub-group of BRCAx families for further linkage analysis studies.

Mutations in SDHD are the major determinants of the clinical characteristics of Dutch head and neck paraganglioma patients.

OBJECTIVE: Head and neck paragangliomas (HNPGL) are associated with mutations in genes encoding subunits of succinate dehydrogenase (SDH). The aim of this study was to evaluate SDH mutations, family history and phenotypes of patients with HNPGL in the Netherlands. DESIGN: We evaluated the clinical data and the mutation status of 236 patients referred between 1950 and 2009 to Leiden University Medical Center. RESULTS: The large majority of the patients carried mutations in SDHD (83%), and the p.Asp92Tyr Dutch founder mutation in SDHD alone accounted for 72% of all patients with HNPGL. A mutation in SDHAF2 was found in 4%, mutations in SDHB in 3% and a mutation in SDHC was identified in a single patient (0·4%). Over 80% of patients presented with positive family history, of whom 99·5% carried a mutation in an SDH gene. SDH mutations were also found in 56% of isolated patients, chiefly in SDHD (46%), but also in SDHB (8%) and SDHC (2%). The clinical parameters of these different subgroups are discussed: including the age at diagnosis, associated pheochromocytomas, tumour multifocality and malignancy rate. CONCLUSION: The majority of Dutch patients with HNPGL present with a positive family history, in contrast to other European countries. The clinical characteristics of patients with HNPGL are chiefly determined by founder mutations in SDHD, the major causative gene in both familial and isolated patients with HNPGL. The high frequency of founder mutations in SDHD suggests a higher absolute prevalence of paraganglioma syndrome in the Netherlands.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma.

Hereditary paraganglioma (PGL) is characterized by the development of benign, vascularized tumors in the head and neck. The most common tumor site is the carotid body (CB), a chemoreceptive organ that senses oxygen levels in the blood. Analysis of families carrying the PGL1 gene, described here, revealed germ line mutations in the SDHD gene on chromosome 11q23. SDHD encodes a mitochondrial respiratory chain protein-the small subunit of cytochrome b in succinate-ubiquinone oxidoreductase (cybS). In contrast to expectations based on the inheritance pattern of PGL, the SDHD gene showed no evidence of imprinting. These findings indicate that mitochondria play an important role in the pathogenesis of certain tumors and that cybS plays a role in normal CB physiology.

Ever since Knudson.

Many publications have documented loss of heterozygosity (LOH) on many different chromosomes in a wide variety of tumours, implicating the existence of multiple tumour suppressor genes (TSGs). Knudson's two-hit hypothesis predicts that these LOH events are the second step in the inactivation of both alleles of a TSG. However, to date the number of TSGs identified that are inactivated mainly at the somatic level in cancers and are not inherited has remained disappointingly small. Here we postulate that the accurate mapping of LOH events in a series of tumours to define a common LOH region is greatly confounded by deficient LOH detection, genetic instability and intertumour heterogeneity. Finding the TSGs in chromosomal regions of frequent LOH might require 'brute-force' genomic approaches.

Genetic susceptibility for breast cancer: how many more genes to be found?

Today, breast cancer is the most commonly occurring cancer among women. It accounts for 22% of all female cancers and the estimated annual incidence of breast cancer worldwide is about one million cases. Many risk factors have been identified but a positive family history remains among the most important ones established for breast cancer, with first-degree relatives of patients having an approximately two-fold elevated risk. It is currently estimated that approximately 20-25% of this risk is explained by known breast cancer susceptibility genes, mostly those conferring high risks, such as BRCA1 and BRCA2. However, these genes explain less than 5% of the total breast cancer incidence, even though several studies have suggested that the proportion of breast cancer that can be attributed to a genetic factor may be as high as 30%. It is thus likely that there are still breast cancer susceptibility genes to be found. It is presently not known how many such genes there still are, nor how many will fall into the class of rare high-risk (e.g. BRCAx) or of common low-risk susceptibility genes, nor if and how these factors interact with each other to cause susceptibility (a polygenic model). In this review we will address this question and discuss the different undertaken approaches used in identifying new breast cancer susceptibility genes, such as (genome-wide) linkage analysis, CGH, LOH, association studies and global gene expression analysis.

Amplification of the neu (c-erbB-2) oncogene in human mammmary tumors is relatively frequent and is often accompanied by amplification of the linked c-erbA oncogene.

We investigated alterations in the structure and expression of oncogenes in mammary tumors and mammary tumor-derived cell lines. In 16 of 95 samples, we detected amplification of the human neu oncogene, also known as c-erB-2, accompanied by overexpression in the tumors from which intact RNA could be isolated. In 10 of these DNAs, the linked oncogene c-erbA was also amplified, whereas another gene on human chromosome 17, p53, was present in normal copy numbers. Overexpression of c-erbA could not be detected in the tumors analyzed. The relatively high frequency of neu amplification points to a functional role in human breast cancer. Coamplification of the c-erbA oncogene could contribute to this disease as well but is most likely fortuitous.

Multifactorial analysis of differences between sporadic breast cancers and cancers involving BRCA1 and BRCA2 mutations.

BACKGROUND: We have previously demonstrated that breast cancers associated with inherited BRCA1 and BRCA2 gene mutations differ from each other in their histopathologic appearances and that each of these types differs from breast cancers in patients unselected for family history (i.e., sporadic cancers). We have now conducted a more detailed examination of cytologic and architectural features of these tumors. METHODS: Specimens of tumor tissue (5-microm-thick sections) were examined independently by two pathologists, who were unaware of the case or control subject status, for the presence of cell mitosis, lymphocytic infiltration, continuous pushing margins, and solid sheets of cancer cells; cell nuclei, cell nucleoli, cell necrosis, and cell borders were also evaluated. The resulting data were combined with previously available information on tumor type and tumor grade and further evaluated by multifactorial analysis. All statistical tests are two-sided. RESULTS: Cancers associated with BRCA1 mutations exhibited higher mitotic counts (P = .001), a greater proportion of the tumor with a continuous pushing margin (P<.0001), and more lymphocytic infiltration (P = .002) than sporadic (i.e., control) cancers. Cancers associated with BRCA2 mutations exhibited a higher score for tubule formation (fewer tubules) (P = .0002), a higher proportion of the tumor perimeter with a continuous pushing margin (P<.0001), and a lower mitotic count (P = .003) than control cancers. CONCLUSIONS: Our study has identified key features of the histologic phenotypes of breast cancers in carriers of mutant BRCA1 and BRCA2 genes. This information may improve the classification of breast cancers in individuals with a family history of the disease and may ultimately aid in the clinical management of patients.

Detection of chromosome aneuploidy in interphase nuclei from human primary breast tumors using chromosome-specific repetitive DNA probes.

We have used in situ hybridization with chromosome specific repetitive DNA sequences as a probe to reveal particular chromosomes as distinct spots or clusters of signal within interphase nuclei. Using karyotypically defined cells and cell lines, we show that the number of signals obtained per nucleus correlates with the number of particular chromosomes present in that nucleus. Further, admixtures of karyotypically different cell lines could be detected. In situ hybridization of nuclei and metaphase spreads derived from the breast cancer cell line MCF-7 shows that a deviant number of spots/nucleus indicates a numerical and/or structural chromosomal aberration. In seven primary breast tumors studied, we detected numerical aberrations of the target sites of chromosomes 1 and/or 18. Although all had a single peak in DNA flow measurements, six of the cases appeared to be heterogeneous with respect to their spots/nucleus content.

Evidence for limited molecular genetic heterogeneity as defined by allelotyping and clonal analysis in nine metastatic breast carcinomas.

To investigate genetic intratumor heterogeneity, 42 samples of nine primary breast carcinomas and 29 related lymph node metastases were examined for DNA ploidy status, allelotype, and X chromosome inactivation pattern. Two primary breast carcinomas showed DNA index heterogeneity and five contained a single DNA aneuploid tumor stemline, whereas the two remaining primary tumors were solely DNA diploid. Most primary DNA tumor stemlines recurred in lymph node metastases (9 of 11). The allelotype, constructed with 31 different probes mapping to 23 different chromosome arms showed allelic imbalances on nearly all chromosome arms investigated. All tumors contained multiple allelic imbalances (range, 3-12). An allelic imbalance present in a primary tumor was consistently present in all DNA samples of that primary tumor and also in all DNA samples of related lymph node metastases, irrespective of DNA index heterogeneity. X chromosome inactivation pattern analysis with probe M27 beta (DXS255) confirmed the presence of clonal tumor cell populations in these tumors at the time of diagnosis. Densitometry of autoradiograms, which by eye showed retention of heterozygosity, revealed a narrow clustering of allelic imbalance factors between 1.0 and 1.4. In contrast, autoradiograms visually showing an allelic imbalance exhibited a marked interprobe, intertumor and intratumor variation in allelic imbalance factors. No relation between densitometry results and DNA ploidy status was found. Thus, at the time of diagnosis, an advanced primary breast carcinoma consists of a clonal tumor cell population with an established complement of allelic imbalances in all parts of the primary tumor and in the related lymph node metastases. Secondary to the establishment of allelic imbalances, intratumor heterogeneity for the copy number of involved alleles may develop, which in turn probably precedes metastasis.

Frequent somatic imbalance of marker alleles for chromosome 1 in human primary breast carcinoma.

Loss of heterozygosity at particular chromosomal loci in the tumor cell, as evidenced by restriction fragment length polymorphism analysis, has been taken as a hallmark of the presence of tumor suppressor genes. Recent studies of breast carcinoma have suggested that such genes might be located on the short as well as on the long arm of chromosome 1. We report here that comparison of constitutional and tumor genotypes of 84 breast tumors at 7 polymorphic chromosome 1 loci indicates a frequent imbalance of alleles on both 1p (12 of 61 informative patients) and 1q (37 of 71 informative patients). In about one-half of these cases, however, this imbalance was consistent with a gain in copy number of one allele in tumor DNA relative to normal DNA, rather than loss of the other. In 10 tumors we performed chromosome 1 enumeration in the interphase nucleus using in situ hybridization with a probe detecting the heterochromatin region at 1q12. These experiments confirmed the supernumerary presence of region 1q12 in those tumors showing an allelic copy number gain of 1q. We suggest that there are several genes on chromosome 1 serving as targets for these changes, some of them associated with breast cancer development through their deletion and others through an increase in copy number.