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.

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.

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.

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.

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.

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.

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.

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.

Association between the CHEK2*1100delC germ line mutation and estrogen receptor status.

Already published data were further analyzed regarding the association between the CHEK2*1100delC germ line mutation and estrogen receptor (ER) status in patients with breast cancer. The CHEK2*1100delC mutation was more prevalent among the patients with a positive ER status (4.2% versus 1.0%). An ER-negative status was beside CHEK2*1100delC mutation and independently associated with an earlier of age onset of breast cancer. There was a trend that an ER-negative status, beside the presence of a CHEK2*1100delC mutation, was associated with a worse disease-free survival. There might be an association between ER status and a CHEK2*1100delC mutation. More studies with larger number of patients are needed to further investigate the relation between CHEK2*1100delC and ER status.

Tumour characteristics and prognosis of breast cancer patients carrying the germline CHEK2*1100delC variant.

BACKGROUND: The germline CHEK2*1100delC variant has been associated with breast cancer in multiple case families where involvement of BRCA1 and BRCA2 has been excluded. METHODS: We have investigated the tumour characteristics and prognosis of carriers of this germline variant by means of a prospective cohort study in an unselected cohort of 1084 consecutive patients with primary breast cancer. Data were collected for 34 patients with a germline CHEK2*1100delC mutation and for 102 patients without this mutation, stratified by age and date of diagnosis of the first primary breast cancer (within 1 year). RESULTS: Carriers developed steroid receptor positive tumours (oestrogen receptor (ER): 91%; progesterone receptor (PR): 81%) more frequently than non-carriers (ER: 69%; PR: 53%; p = 0.04). Mutation carriers more frequently had a female first or second degree relative with breast cancer (p = 0.03), or had any first or second degree relative with breast or ovarian cancer (p = 0.04). Patients with the CHEK2 variant had a more unfavourable prognosis regarding the occurrence of contralateral breast cancer (relative risk (RR) = 5.74; 95% confidence interval (CI) 1.67 to 19.65), distant metastasis-free survival (RR = 2.81; 95% CI 1.20 to 6.58), and disease-free survival (RR = 3.86; 95% CI 1.91 to 7.78). As yet, no difference with respect to overall survival has been found at a median follow up of 3.8 years. CONCLUSION: We conclude that carrying the CHEK2*1100delC mutation is an adverse prognostic indicator for breast cancer. If independently confirmed by others, intensive surveillance, and possibly preventive measures, should be considered for newly diagnosed breast cancer cases carrying the CHEK2*1100delC variant.

Possible consequences of applying guidelines to healthy women with a family history of breast cancer.

Possible effects of consistently applying published guidelines on healthy women with breast cancer in their family history were analysed. We investigated 1060 unrelated breast cancer patients and calculated the numbers of first-degree relatives that would be referred to a familial cancer clinic if the guidelines were consistently applied. A first-degree relative was considered a candidate for referral if she was female, without breast cancer at the moment of the interview, alive and over the age of 24. The criteria for referral were based on one Dutch and two British guidelines. According to the Dutch guideline, for one affected woman with breast cancer, 0.25 (95% CI 0.22-0.28) healthy first-degree female relatives should be offered a consultation at a familial cancer clinic (FCC). Application of the British guidelines would lead to a similar number of referrals. Of all healthy first-degree female relatives, who should be referred to an FCC, 34-37% had an index case among their family who was already known at a genetic department. If current guidelines are consistently applied, a sharp increase in referrals to FCCs may be expected. These guidelines, however, are arbitrary and only limited data are available on the efficacy of this surveillance for high-risk healthy women.

Interpreting epidemiological research: blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1.

While sequence analysis is considered by many to be the most sensitive method of detecting unknown mutations in large genes such as BRCA1, most published estimates of the prevalence of mutations in this gene have been derived from studies that have used other methods of gene analysis. In order to determine the relative sensitivity of techniques that are widely used in research on BRCA1, a set of blinded samples containing 58 distinct mutations were analysed by four separate laboratories. Each used one of the following methods: single strand conformational polymorphism analysis (SSCP), conformation sensitive gel electrophoresis (CSGE), two dimensional gene scanning (TDGS), and denaturing high performance liquid chromatography (DHPLC). Only the laboratory using DHPLC correctly identified each of the mutations. The laboratory using TDGS correctly identified 91% of the mutations but produced three apparent false positive results. The laboratories using SSCP and CSGE detected abnormal migration for 72% and 76% of the mutations, respectively, but subsequently confirmed and reported only 65% and 60% of mutations, respectively. False negatives therefore resulted not only from failure of the techniques to distinguish wild type from mutant, but also from failure to confirm the mutation by sequence analysis as well as from human errors leading to misreporting of results. These findings characterise sources of error in commonly used methods of mutation detection that should be addressed by laboratories using these methods. Based upon sources of error identified in this comparison, it is likely that mutations in BRCA1 and BRCA2 are more prevalent than some studies have previously reported. The findings of this comparison provide a basis for interpreting studies of mutations in susceptibility genes across many inherited cancer syndromes.

A partial BRCA1 sequence homology mapping to 4q28.

Using a BRCA1 cDNA probe in Southern analysis, we detected a sequence of 348 bp on 4q28 that is homologous to the 3' end of BRCA1. A 28-kb sequence contig has been assembled spanning the homologous region, which we designated BRCA1-h. An open reading frame was identified encoding a sequence of 82 amino acids; 22 of the last 23 amino acids are identical to the last 23 residues of BRCA1. BLAST-searches, RT-PCR and RACE-experiments have been unable to provide evidence that BRCA1-h is part of an expressed gene.

Clinical and pathological features of BRCA1 associated carcinomas in a hospital-based sample of Dutch breast cancer patients.

Thus far, studies investigating the differences in tumour characteristics between breast cancer in BRCA1-carriers and other patients, have focused on highly selected groups of patients, potentially limiting the conclusions that can be drawn. Previously, we had identified 10 patients with BRCA1 germline mutations in a hospital-based series of 642 breast cancer patients not selected for age or family history. The aim of this analysis is to investigate the clinical and pathological features of these BRCA1 associated carcinomas as compared to other breast cancers in this representative sample. Tumours from patients with BRCA1 germline mutations (n = 10) were compared to an age-matched sample of other patients (n = 50) from the same cohort. The following characteristics were considered: axillary nodal status and tumour size, histologic parameters (tumour type, histologic grade, mitotic rate, tubule formation, nuclear grade, CIS and lymphangio invasion) and expression of several proteins (oestrogen and progesterone receptors, cyclin D1, p53, HER2/neu, E-cadherin). In BRCA1 associated tumours receptors for oestrogen and progesterone were expressed less frequently (respectively, P = 0.001 and P = 0.002) than in controls, which is in line with findings from other studies. Other differences were also in accordance with findings from other studies, although not statistically significant. We conclude that the features of BRCA1 associated tumours detected in a hospital-based series of breast cancer patients not selected for family history of age at diagnosis are similar to tumours in cases selected for family history or age at diagnosis.

Large regional differences in the frequency of distinct BRCA1/BRCA2 mutations in 517 Dutch breast and/or ovarian cancer families.

In 517 Dutch families at a family cancer clinic, we screened for BRCA1/2 alterations using the Protein Truncation Test (PTT) covering approximately 60% of the coding sequences of both genes and direct testing for a number of previously identified Dutch recurrent mutations. In 119 (23%) of the 517 families, we detected a mutation in BRCA1 (n=98; 19%) or BRCA2 (n=21; 4%). BRCA1/2 mutations were found in 72 (52%) of 138 families with breast and ovarian cancer (HBOC), in 43 (13%) of the 339 families with breast cancer only (HBC), in 4 (36%) of 11 families with ovarian cancer only (HOC), and in nine of 29 families with one single young case (<40 years) of breast cancer. Between the different subgroups of families (subdivided by the number of patients, cancer phenotype and age of onset) the proportion of BRCA1/2 mutations detected, varied between 6 and 82%. Eight different mutations, each encountered in at least six distinct families, represented as much as 61% (73/119 families) of all mutations found. The original birthplaces of the ancestors of carriers of these eight recurrent mutations were traced. To estimate the relative contribution of two important regional recurrent mutations (BRCA1 founder mutation IVS12-1643del3835 and BRCA2 founder mutation 5579insA) to the overall occurrence of breast cancer, we performed a population-based study in two specific small regions. The two region-specific BRCA1 and BRCA2 founder mutations were detected in 2.8% (3/106) and 3.2% (3/93) of the unselected breast tumours, respectively. Of tumours diagnosed before the age of 50 years, 6.9% (3/43) and 6.6% (2/30) carried the region-specific founder mutation. Thus, large regional differences exist in the prevalence of certain specific BRCA1/BRCA2 founder mutations, even in very small areas concerning populations of approximately 200000 inhabitants.

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.

Men at risk of being a mutation carrier for hereditary breast/ovarian cancer: an exploration of attitudes and psychological functioning during genetic testing.

Males with a BRCA1/BRCA2 mutation are not at greatly increased risk for cancer, whereas their (grand)daughters, and other female relatives who carry the mutation, are. Males from BRCA1/BRCA2 families may opt for genetic testing to confirm whether or not they may have transmitted the mutation to their children and, if so, to inform them at an appropriate age about the genetic risk and its implications. The psychological implications of genetic testing for men at risk of being a BRCA1/BRCA2 mutation carrier have received little attention. We report on 28 men requesting BRCA1 or BRCA2 testing, and their partners. Men were at 25% (n =4) or 50% risk (n =24) of being a mutation carrier, the majority with daughters and half of them with daughters aged over 20 years. Levels of psychological distress were assessed several weeks before and after disclosure of the test result. In addition, we investigated the level of intrusive thoughts and feelings about breast and ovarian cancer and the tendency to avoid these. By means of interviews and questionnaires, participants could report on (expected) emotional implications of genetic testing for themselves and their children, on experiences with cancer in the family and on personality trait optimism. Distress levels prior to the result in tested men and their partners were low. Many men and partners expected the test result to affect their children's, but not their own level of problems. Men without daughters and those with an optimistic personality had especially low distress prior to disclosure. Most men reported that they did not actively avoid the issue. Only four of the 28 men were identified as mutation carriers. High distress after disclosure of the result was reported by one mutation carrier and by three non-mutation carriers. Verbatim transcripts from interviews showed a large variation of psychological reactions in male mutation carriers (eg regarding guilt feelings). Low pre-test distress in males does not necessarily indicate avoidance of the issue. Future studies may indicate which psychological reactions occur in male mutation carriers when the problem becomes more acute, eg when a daughter is found to carry the mutation and/or is diagnosed with breast or ovarian cancer.