Loss of heterozygosity atBRCA1/2 loci in hereditary and sporadic ovarian cancers

Loss of heterozygosity atBRCA1/2 loci in breast and ovarian tumors is a suggested risk factor for germlineBRCA1/2 mutation status. We evaluated the presence of losses of selected microsatellite markers localized on chromosomes 17 and 13q in hereditary and sporadic ovarian tumors. 151 consecutive primary ovarian tumors (including 21 withBRCA1/2 mutations and 130 without the mutations) were screened for loss of heterozygosity at loci on chromosomes 17 and 13q. Losses of heterozygosity of at least one microsatellite marker localized on chromosomes 17 and 13q were revealed in 123 (81.5%) and 104 (68.9%) tumors, respectively. Losses of all informative markers on chromosomes 17 and 13 occurred in 30 (19.9%) and 31 (20.5%) tumors, respectively. There was no difference in the frequency of losses atBRCA1 intragenic markers (D17S855 and D17S1323) between BRCA1-positive and BRCA1-negative patients. The frequency of losses on chromosome 17 was higher in high-grade than in low-grade carcinomas. Loss of heterozygosity on chromosomes 17 and 13q is a frequent phenomenon in both hereditary and sporadic ovarian cancers. The frequency of losses atBRCA1 intragenic markers in the ovarian tumor tissue is not strongly related to the presence ofBRCA1 germline mutations.     

BRCA1/2 mutation screening and LOH analysis of lung adenocarcinoma tissue in a multiple-cancer patient with a strong family history of breast cancer

BACKGROUND: Germline mutations in BRCA1/2 greatly elevate risks of breast and ovarian cancers, but the role of these genes in tumourigenesis of other cancer types is still being investigated. OBJECTIVE: We report on an investigation of BRCA1/2 mutations and their loss of heterozygosity (LOH) in a patient with a strong family history of breast cancer who was diagnosed with consecutive primary cervical, ovarian and lung carcinomas. METHODS AND RESULTS: BRCA1/2 mutation screening of the proband revealed a common familial breast- and ovarian cancer-associated germline BRCA2 mutation (3034del4bp). We then performed LOH analysis for BRCA2 in lung adenocarcinoma tissue of the patient. Using the laser-capture microdissection (LCM) technique, we obtained pure populations of neoplastic cells from which DNA could be extracted. Mutation analysis by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing revealed loss of the mutant allele in the adenocarcinoma tumour tissue. CONCLUSION: To our knowledge, this is the first report of investigation for LOH for BRCA2 in primary lung adenocarcinoma tissue of a patient with multiple primary tumours related to a familial germline BRCA2 mutation. Interestingly, it was the mutant, not the wild-type, allele which was lost in the lung adenocarcinoma tissue.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.     

Sex-specific effect of the <Emphasis Type="Italic">TP53</Emphasis> PIN3 polymorphism on cancer risk in a cohort study of <Emphasis Type="Italic">TP53</Emphasis> germline mutation carriers

Germline mutations in the tumor suppressor gene TP53 occur in the majority of families with Li-Fraumeni syndrome, who are at an increased risk for a wide spectrum of early onset cancers. Several genetic polymorphisms in TP53 modify its effect on cancer risk. While some studies indicate that the TP53 PIN3 deletion allele (D) accelerate tumor onset in carriers with TP53 germline mutations, other studies have shown that the TP53 PIN3 insertion allele (I) confers a significantly higher risk of developing cancer than D allele. To further determine the effects of the TP53 PIN3 polymorphism on cancer development among TP53 germline mutations and to evaluate if those are differenence between male and female carriers, we studied a total of 152 germline mutation carriers with available DNA samples that can be used for genotyping. Our results indicate that the TP53 PIN3 polymorphism has a sex-specific effect on the risk of cancer in TP53 mutation carriers, conferring cancer risk in men (P = 0.0041) but not women with DI or II genotypes.     

Hereditary breast cancer; Genetic penetrance and current status with BRCA

The most important cause of developing hereditary breast cancer is germline mutations occurring in breast cancer (BCs) susceptibility genes, for example, BRCA1, BRCA2, TP53, CHEK2, PTEN, ATM, and PPM1D. Many BC susceptibility genes can be grouped into two classes, high- and low-penetrance genes, each of which interact with multiple genes and environmental factors. However, the penetrance of genes can also be represented by a spectrum, which ranges between high and low. Two of the most common susceptibility genes are BRCA1 and BRCA2, which perform vital cellular functions for repair of homologous DNA. Loss of heterozygosity accompanied by hereditary mutations in BRCA1 or BRCA2 increases chromosomal instability and the likelihood of cancer, as well as playing a key role in stimulating malignant transformation. With regard to pathological features, familial breast cancers caused by BRCA1 mutations usually differ from those caused by BRCA2 mutations and nonfamilial BCs. It is essential to acquire an understanding of these pathological features along with the genetic history of the patient to offer an individualized treatment. Germline mutations in BRCA1 and BRCA2 genes are the main genetic and inherited factors for breast and ovarian cancer. In fact, these mutations are very important in developing early onset and increasing the risk of familial breast and ovarian cancer and responsible for 90% of hereditary BC cases. Therefore, according to the conducted studies, screening of BRCA1 and BRCA2 genes is recommended as an important marker for early detection of all patients with breast or ovarian cancer risk with family history of the disease. In this review, we summarize the role of hereditary genes, mainly BRCA1 and BRCA2, in BC.     

Germline mutations in the BRCA1 gene predisposing to breast and ovarian cancers in Upper Silesia population

Germline mutations in the BRCA1 or BRCA2 genes predispose their carriers to breast or/and ovary cancers during their lifetime. The most frequent mutations: 5382insC, 185delAG, C61G and 4153delA in BRCA1, and 6174delT and 9631delC in BRCA2 were studied in a group of 148 probands admitted for genetic counseling, using allele-specific amplification (ASA) PCR test. Fifteen carriers of three different mutations: 5382insC, 185delAG and C61G in BRCA1 were found. Two families carried the 185delAG mutation and additional two C61G in BRCA1. Nobody carried the mutation 4153delA in BRCA1 nor 6174delT or 9631delC in BRCA2. Most of the carriers of a germline mutation were observed among the patients who developed bilateral breast cancer (17%). The lowest frequency of the germline mutations was found in the healthy persons who had two or more relatives affected with breast or ovarian cancer.     

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.     

The Contribution of Germline BRCA1 and BRCA2 Mutations to Familial Ovarian Cancer: No Evidence for Other Ovarian Cancer–Susceptibility Genes

To establish the contribution of germline BRCA1 and BRCA2 mutations to familial ovarian cancer, we have analyzed both genes in DNA samples obtained from an affected individual in each of 112 families containing at least two cases of epithelial ovarian cancer. Germline mutations were found in 43% of the families; BRCA1 mutations were approximately four times more common than BRCA2 mutations. The extent of family history of ovarian and breast cancers was strongly predictive of BRCA1-mutation status. Segregation analysis suggests that a combination of chance clustering of sporadic cases and insensitivity of mutation detection may account for the remaining families; however, the contribution of other genes cannot be excluded. We discuss the implications for genetic testing and clinical management of familial ovarian cancer arising from the data presented in these studies.     

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.Key words: homologous recombination, PARP inhibitor, BRCA1, BRCA2, PTEN, PALB2, EMSY, double strand break repair     

Tumorigenesis in colorectal tumors from patients with hereditary non-polyposis colorectal cancer

Tumorigenesis of colorectal cancer in patients with hereditary non-polyposis colorectal cancer (HNPCC) has been postulated to follow a different pathway from that of sporadic colorectal tumors. A characteristic of HNPCC-associated tumors is the replication error phenotype. We studied tumorigenesis in 8 fresh-frozen and 67 paraffin-embedded colorectal tumors derived from 29 families with HNPCC or a familial aggregation of colorectal cancer. By using intragenic markers, inactivation of the wild-type allele of hMLH1 was shown to occur through loss of heterozygosity and not through a somatic point mutation. Microsatellite instability is very common and occurs early in almost all colorectal tumors from HNPCC patients. Transforming growth factor β type II receptor (TβRII) mutations occur in these tumors at a high frequency. Of colorectal cancers from families with HNPCC, 63% have frameshift mutations in TβRII, compared with 10% of sporadic colorectal cancers. APC and K-RAS mutations appear to be as frequent in the HNPCC tumors as in the sporadic counterpart. Received: 3 March 1997 / Accepted: 23 June 1997     

brca2 and tp53 Collaborate in Tumorigenesis in Zebrafish

Germline mutations in the tumor suppressor genes BRCA2 and TP53 significantly influence human cancer risk, and cancers from humans who inherit one mutant allele for BRCA2 or TP53 often display loss of the wildtype allele. In addition, BRCA2-associated cancers often exhibit mutations in TP53. To determine the relationship between germline heterozygous mutation (haploinsufficiency) and somatic loss of heterozygosity (LOH) for BRCA2 and TP53 in carcinogenesis, we analyzed zebrafish with heritable mutations in these two genes. Tumor-bearing zebrafish were examined by histology, and normal and neoplastic tissues were collected by laser-capture microdissection for LOH analyses. Zebrafish on a heterozygous tp53^M214K background had a high incidence of malignant tumors. The brca2^Q658X mutation status determined both the incidence of LOH and the malignant tumor phenotype. LOH for tp53 occurred in the majority of malignant tumors from brca2 wildtype and heterozygous mutant zebrafish, and most of these were malignant peripheral nerve sheath tumors. Malignant tumors in zebrafish with heterozygous mutations in both brca2 and tp53 frequently displayed LOH for both genes. In contrast, LOH for tp53 was uncommon in malignant tumors from brca2 homozygotes, and these tumors were primarily undifferentiated sarcomas. Thus, carcinogenesis in zebrafish with combined mutations in tp53 and brca2 typically requires biallelic mutation or loss of at least one of these genes, and the specific combination of inherited mutations influences the development of LOH and the tumor phenotype. These results provide insight into cancer development associated with heritable BRCA2 and TP53 mutations.     

Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer

A population-based series of 649 unselected incident cases of ovarian cancer diagnosed in Ontario, Canada, during 1995-96 was screened for germline mutations in BRCA1 and BRCA2. We specifically tested for 11 of the most commonly reported mutations in the two genes. Then, cases were assessed with the protein-truncation test (PTT) for exon 11 of BRCA1, with denaturing gradient gel electrophoresis for the remainder of BRCA1, and with PTT for exons 10 and 11 of BRCA2. No mutations were found in all 134 women with tumors of borderline histology. Among the 515 women with invasive cancers, we identified 60 mutations, 39 in BRCA1 and 21 in BRCA2. The total mutation frequency among women with invasive cancers, 11.7% (95% confidence interval [95%CI] 9.2%-14.8%), is higher than previous estimates. Hereditary ovarian cancers diagnosed at age <50 years were mostly (83%) due to BRCA1, whereas the majority (60%) of those diagnosed at age >60 years were due to BRCA2. Mutations were found in 19% of women reporting first-degree relatives with breast or ovarian cancer and in 6.5% of women with no affected first-degree relatives. Risks of ovarian, breast, and stomach cancers and leukemias/lymphomas were increased nine-, five-, six- and threefold, respectively, among first-degree relatives of cases carrying BRCA1 mutations, compared with relatives of noncarriers, and risk of colorectal cancer was increased threefold for relatives of cases carrying BRCA2 mutations. For carriers of BRCA1 mutations, the estimated penetrance by age 80 years was 36% for ovarian cancer and 68% for breast cancer. In breast-cancer risk for first-degree relatives, there was a strong trend according to mutation location along the coding sequence of BRCA1, with little evidence of increased risk for mutations in the 5' fifth, but 8.8-fold increased risk for mutations in the 3' fifth (95%CI 3.6-22.0), corresponding to a carrier penetrance of essentially 100%. Ovarian, colorectal, stomach, pancreatic, and prostate cancer occurred among first-degree relatives of carriers of BRCA2 mutations only when mutations were in the ovarian cancer-cluster region (OCCR) of exon 11, whereas an excess of breast cancer was seen when mutations were outside the OCCR. For cancers of all sites combined, the estimated penetrance of BRCA2 mutations was greater for males than for females, 53% versus 38%. Past studies may have underestimated the contribution of BRCA2 to ovarian cancer, because mutations in this gene cause predominantly late-onset cancer, and previous work has focused more on early-onset disease. If confirmed in future studies, the trend in breast-cancer penetrance, according to mutation location along the BRCA1 coding sequence, may have significant impact on treatment decisions for carriers of BRCA1-mutations. As well, BRCA2 mutations may prove to be a greater cause of cancer in male carriers than previously has been thought.     

Clinical and pathological characteristics of Chinese patients with BRCA related breast cancer

Breast cancers related to BRCA mutations are associated with particular biological features. Here we report the clinical and pathological characteristics of breast cancer in Chinese women with and without BRCA mutations and of carriers of BRCA1 mutations compared to BRCA2 mutations. Two hundred and 26 high-risk Hong Kong Chinese women were tested for BRCA mutations, medical information was obtained from medical records, and risk and demographic information was obtained from personal interviews. In this cohort, 28 (12.4%) women were BRCA mutation carriers and among these carriers, 39.3% were BRCA1 and 60.7% were BRCA2 mutations. Mutation carriers were more likely to have a familial history of breast and ovarian cancer, high-grade cancers, and triple negative (TN) cancers. Prevalence of TN was 48.3% in BRCA carriers and 25.6% in non-carriers and was 67.7% in BRCA1 and 35.3% in BRCA2 carriers. Estrogen receptor (ER) negative cancer was significantly associated with BRCA1 mutations, especially in those under 40 years of age. BRCA-related breast cancer in this Chinese population is associated with family history and adverse pathological/prognostic features, with BRCA2 mutations being more prevalent but BRCA1 carriers having more aggressive and TN cancers. Compared to Caucasian populations, prevalence of BRCA2 mutations and TN cancer in BRCA2 mutation carriers in Chinese population are elevated.     

BRCA1 Haploinsufficiency,but not Heterozygosity for a BRCA1-truncating Mutation,Deregulates Homologous Recombination

Humans heterozygous for BRCA1 mutations have a high risk of losing the remaining wild-type BRCA1 allele and developing breast/ovarian cancer, but a molecular basis for this has not yet been determined. It is thought that heterozygosity status — reduced wild-type BRCA1 protein dosage (haploinsufficiency) and/or the presence of a mutant BRCA1 protein — may affect BRCA1 functions and heighten the risk of cancer promoting mutations. BRCA1 maintains genome stability, at least in part, by regulating homologous recombination according to the type of DNA damage. To investigate whether this BRCA1 function is affected by heterozygosity status, we employed, as recombination reporters, human breast cancer MCF-7 cells known to have a single wild-type BRCA1 allele and reduced BRCA1 protein dosage. These cells revealed: 1) a spontaneous hyper-recombination phenotype; 2) reduced efficiency in homologous recombination repair of DNA double-strand breaks (DSBs); and 3) sensitivity to the DSB-inducing chemotherapeutic agent mitomycin C. Correction of BRCA1 protein dosage to the wild-type level reversed all these phenotypes, whereas physiological expression of the cancer-eliciting BRCA1 5382insC mutant allele had no effect on either phenotype. These findings implicate BRCA1 C-terminal domain in recombination control, and indicate that BRCA1 haploinsufficiency alone, which is also a feature of sporadic breast/ovarian cancer, is sufficient to compromise genome stability by triggering spontaneous recombination events that are likely to account for the loss of the remaining wild-type BRCA1 allele and increased cancer risk. Our observations may also have implications for the medical management of cancer patients and cancer prevention.     

Prevalence of BRCA1 Mutations in Familial and Sporadic Greek Ovarian Cancer Cases

Germline mutations in the BRCA1 and BRCA2 genes contribute to approximately 18% of hereditary ovarian cancers conferring an estimated lifetime risk from 15% to 50%. A variable incidence of mutations has been reported for these genes in ovarian cancer cases from different populations. In Greece, six mutations in BRCA1 account for 63% of all mutations detected in both BRCA1 and BRCA2 genes. This study aimed to determine the prevalence of BRCA1 mutations in a Greek cohort of 106 familial ovarian cancer patients that had strong family history or metachronous breast cancer and 592 sporadic ovarian cancer cases. All 698 patients were screened for the six recurrent Greek mutations (including founder mutations c.5266dupC, p.G1738R and the three large deletions of exon 20, exons 23–24 and exon 24). In familial cases, the BRCA1 gene was consequently screened for exons 5, 11, 12, 20, 21, 22, 23, 24. A deleterious BRCA1 mutation was found in 43/106 (40.6%) of familial cancer cases and in 27/592 (4.6%) of sporadic cases. The variant of unknown clinical significance p.V1833M was identified in 9/698 patients (1.3%). The majority of BRCA1 carriers (71.2%) presented a high-grade serous phenotype. Identifying a mutation in the BRCA1 gene among breast and/or ovarian cancer families is important, as it enables carriers to take preventive measures. All ovarian cancer patients with a serous phenotype should be considered for genetic testing. Further studies are warranted to determine the prevalence of mutations in the rest of the BRCA1 gene, in the BRCA2 gene, and other novel predisposing genes for breast and ovarian cancer.     

BRCA1: the enigma of tissue-specific tumor development

Recent evidence indicates that BRCA1, a gene product associated with breast and ovarian cancer susceptibility, is an important component of the cellular response to DNA damage. Despite being expressed ubiquitously in adult tissues, germline mutations in BRCA1 predispose individuals to breast and ovarian tumors with only minor effects on the predisposition to cancer in other sites. The reason for this tissue specificity of BRCA1 carcinomas must be found if we are to understand fully why these tumors occur and to enable us to design efficient preventive and therapeutic regimens. Here I propose that tissue-specific rates of loss of heterozygosity in the BRCA1 locus could contribute to tissue specificity in tumor development.     

Identification of a Comprehensive Spectrum of Genetic Factors for Hereditary Breast Cancer in a Chinese Population by Next-Generation Sequencing

The genetic etiology of hereditary breast cancer has not been fully elucidated. Although germline mutations of high-penetrance genes such as BRCA1/2 are implicated in development of hereditary breast cancers, at least half of all breast cancer families are not linked to these genes. To identify a comprehensive spectrum of genetic factors for hereditary breast cancer in a Chinese population, we performed an analysis of germline mutations in 2,165 coding exons of 152 genes associated with hereditary cancer using next-generation sequencing (NGS) in 99 breast cancer patients from families of cancer patients regardless of cancer types. Forty-two deleterious germline mutations were identified in 21 genes of 34 patients, including 18 (18.2%) BRCA1 or BRCA2 mutations, 3 (3%) TP53 mutations, 5 (5.1%) DNA mismatch repair gene mutations, 1 (1%) CDH1 mutation, 6 (6.1%) Fanconi anemia pathway gene mutations, and 9 (9.1%) mutations in other genes. Of seven patients who carried mutations in more than one gene, 4 were BRCA1/2 mutation carriers, and their average onset age was much younger than patients with only BRCA1/2 mutations. Almost all identified high-penetrance gene mutations in those families fulfill the typical phenotypes of hereditary cancer syndromes listed in the National Comprehensive Cancer Network (NCCN) guidelines, except two TP53 and three mismatch repair gene mutations. Furthermore, functional studies of MSH3 germline mutations confirmed the association between MSH3 mutation and tumorigenesis, and segregation analysis suggested antagonism between BRCA1 and MSH3. We also identified a lot of low-penetrance gene mutations. Although the clinical significance of those newly identified low-penetrance gene mutations has not been fully appreciated yet, these new findings do provide valuable epidemiological information for the future studies. Together, these findings highlight the importance of genetic testing based on NCCN guidelines and a multi-gene analysis using NGS may be a supplement to traditional genetic counseling.     

Somatic inactivation of the VHL gene in Von Hippel-Lindau disease tumors.

Von Hippel-Lindau (VHL) disease is a dominantly inherited disorder predisposing to retinal and CNS hemangioblastomas, renal cell carcinoma (RCC), pheochromocytoma, and pancreatic tumors. Interfamilial differences in predisposition to pheochromocytoma reflect allelic heterogeneity such that there is a strong association between missense mutations and risk of pheochromocytoma. We investigated the mechanism of tumorigenesis in VHL disease tumors to determine whether there were differences between tumor types or classes of germ-line mutations. Fifty-three tumors (30 RCCs, 15 hemangioblastomas, 5 pheochromocytomas, and 3 pancreatic tumors) from 33 patients (27 kindreds) with VHL disease were analyzed. Overall, 51% of 45 informative tumors showed loss of heterozygosity (LOH) at the VHL locus. In 11 cases it was possible to distinguish between loss of the wild-type and mutant alleles, and in each case the wild-type allele was lost. LOH was detected in all tumor types and occurred in the presence of both germ-line missense mutations and other types of germline mutation associated with a low risk of pheochromocytoma. Intragenic somatic mutations were detected in three tumors (all hemangioblastomas) and in two of these could be shown to occur in the wild-type allele. This provides the first example of homozygous inactivation of the VHL by small intragenic mutations in this type of tumor. Hypermethylation of the VHL gene was detected in 33% (6/18) of tumors without LOH, including 2 RCCs and 4 hemangioblastomas. Although hypermethylation of the VHL gene has been reported previously in nonfamilial RCC and although methylation of tumor-suppressor genes has been implicated in the pathogenesis of other sporadic cancers, this is the first report of somatic methylation in a familial cancer syndrome.