High proportion of missense mutations of the BRCA1 and BRCA2 genes in Japanese breast cancer families

Mutations in either of two recently identified genes, BRCA1 and BRCA2, are thought to be responsible for approximately two-thirds of all cases of autosomal-dominantly inherited breast cancer. To examine the nature and frequency of BRCA1 and BRCA2 mutations in Japanese families exhibiting a high incidence of breast cancer, we screened 78 unrelated families in this category for mutations of these two genes. Examining the entire coding sequences as well as exon–intron boundaries of both genes by polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and multiplex-SSCP analysis, we identified possible disease-causing alterations in BRCA1 among affected members of 15 families and in BRCA2 in another 14 families. In 15 of those 29 families, the affected individuals carried missense mutations, although most germline mutations reported worldwide have been deletions or nonsense mutations. Our results, indicating that missense mutations of BRCA1 and BRCA2 tend to predominate over frameshifts or nonsense mutations in Japanese breast cancer families, will contribute signifi-cantly to an understanding of mammary tumorigenesis in Japan, and will be of vital importance for future genetic testing. Received: September 4, 1997 / Accepted: October 28, 1997     

Screening for BRCA1 and BRCA2 mutations in Eastern Finnish breast/ovarian cancer families

Familial aggregation is thought to account for 5-10% of all breast cancer cases, and high penetrance breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 explain < or =20% of these. Hundreds of mutations among breast/ovarian cancer families have been found in these two genes. The mutation spectrum and prevalence, however, varies widely among populations. Thirty-six breast/ovarian cancer families were identified from a population sample of breast and ovarian cancer cases among a relatively isolated population in Eastern Finland, and the frequency of BRCA1/BRCA2 germline mutations were screened using heteroduplex analysis, protein truncation test and sequencing. Five different mutations were detected in seven families (19.4%). Two mutations were found in BRCA1 and three in BRCA2. One of the mutations (BRCA2 4088insA) has not been detected elsewhere in Finland while the other four, 4216-2nt A-->G and 5370 C-->T in BRCA1 and 999del5 and 6503delTT in BRCA2, are recurrent Finnish founder mutations. These results add to the evidence of the geographical differences in distribution of Finnish BRCA1/BRCA2 mutations. This screen also provides further evidence for the presumption that the majority of Finnish BRCA1/BRCA2 founder mutations have been found and that the proportion of BRCA1/BRCA2 mutations in Finnish breast/ovarian cancer families is around 20%.     

Novel germline mutations in the BRCA1 and BRCA2 genes in Indian breast and breast-ovarian cancer families

The two major hereditary breast/ovarian cancer predisposition tumor suppressor genes, BRCA1 and BRCA2 that perform apparently generic cellular functions nonetheless cause tissue-specific syndromes in the human population when they are altered, or mutated in the germline. However, little is known about the contribution of BRCA1 and BRCA2 mutations to breast and/or ovarian cancers in the Indian population. We have screened for mutations the entire BRCA1 and BRCA2 coding sequences, and intron-exon boundaries, as well as their flanking intronic regions in sixteen breast or breast and ovarian cancer families of Indian origin. We have also analyzed 20 female patients with sporadic breast cancer regardless of age and family history, and 69 unrelated normal individuals as control. Thus a total of 154 samples were screened for BRCA1 and BRCA2 mutations using a combination of polymerase chain reaction-mediated site directed mutagenesis (PSM), polymerase chain reaction-single stranded conformation polymorphism assay (PCR-SSCP) and direct DNA sequencing of PCR products (DS). Twenty-one sequence variants including fifteen point mutations were identified. Five deleterious pathogenic, protein truncating frameshift and non-sense mutations were detected in exon 2 (c.187_188delAG); and exon 11 (c.3672G>T) [p.Glu1185X] of BRCA1 and in exon 11 (c.5227dupT, c.5242dupT, c.6180dupA) of BRCA2 (putative mutations - four novel) as well as fourteen amino acid substitutions were identified. Twelve BRCA1 and BRCA2 missense variants were identified as unique and novel. In the cohort of 20 sporadic female patients no mutations were found.     

High frequency of recurrent mutations in BRCA1 and BRCA2 genes in Polish families with breast and ovarian cancer

Germ-line mutations in BRCA1 and BRCA2 genes result in a significantly increased risk of breast and ovarian cancer. Other genes involved in an increased predisposition to breast cancer include the TP53 gene, mutated in Li-Fraumeni syndrome. To estimate the frequency of germ-line mutations in these three genes in Upper Silesia, we have analyzed 47 breast/ovarian cancer families from that region. We found five different disease predisposing mutations in 17 (36%) families. Twelve families (25.5%) carried known BRCA1 mutations (5382insC and C61G), four families (8.5%) carried novel BRCA2 mutations (9631delC and 6886delGAAAA), and one family (2%) harbored novel mutation 1095del8 in the TP53 gene, which is the largest germline deletion in coding sequence of this gene identified thus far. The 5382insC mutation in BRCA1 was found in 11 families and the 9631delC mutation in BRCA2 occurred in three families. These two mutations taken together contribute to 82% of all mutations found in this study, and 30% of the families investigated harbor one of these mutations. The very high frequency of common mutations observed in these families can only be compared to that reported for Ashkenazi Jewish, Icelandic, and Russian high-risk families. This frequency, however, may not be representative for the entire Polish population. The observed distribution of mutations will favor routine pre-screening of predisposed families using a simple and cost-effective test.     

Analysis of BRCA1 and BRCA2 genes in Spanish breast/ovarian cancer patients: a high proportion of mutations unique to Spain and evidence of founder effects

We screened index cases from 410 Spanish breast/ovarian cancer families and 214 patients (19 of them males) with breast cancer for germ-line mutations in the BRCA1 and BRCA2 genes, using SSCP, PTT, CSGE, DGGE, and direct sequencing. We identified 60 mutations in BRCA1 and 53 in BRCA2. Of the 53 distinct mutations observed, 11 are novel and 12 have been reported only in Spanish families (41.5%). The prevalence of mutations in this set of families was 26.3%, but the percentage was higher in the families with breast and ovarian cancer (52.1%). The lowest proportion of mutations was found in the site-specific female breast cancer families (15.4%). Of the families with male breast cancer cases, 59.1% presented mutations in the BRCA2 gene. We found a higher frequency of ovarian cancer associated with mutations localized in the 5' end of the BRCA1 gene, but there was no association between the prevalence of this type of cancer and mutations situated in the ovarian cancer cluster region (OCCR) region of exon 11 of the BRCA2 gene. The mutations 187_188delAG, 330A>G, 5236G>A, 5242C>A, and 589_590del (numbered after GenBank U14680) account for 46.6% of BRCA1 detected mutations whereas 3036_3039del, 6857_6858del, 9254_9258del, and 9538_9539del (numbered after GenBank U43746) account for 56.6% of the BRCA2 mutations. The BRCA1 330A>G has a Galician origin (northwest Spain), and BRCA2 6857_6858del and 9254_9258del probably originated in Catalonia (northeast Spain). Knowledge of the spectrum of mutations and their geographical distribution in Spain will allow a more effective detection strategy in countries with large Spanish populations.     

Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian cancer families

Germline mutations in the BRCA1 and BRCA2 genes are responsible for the predisposition and development of familial breast and/or ovarian cancer. Most mutations of BRCA1 and BRCA2 associated with breast and/or ovarian cancer result in truncated proteins. To investigate the presence of BRCA1 and BRCA2 germline mutations in Korean breast and/or ovarian cancer families, we screened a total of 27 cases from 21 families including two or more affected first- or second-degree relatives with breast and/or ovarian cancer. PTT, PCR-SSCP, and DHPLC analysis, followed by sequencing were used in the screening process. In nine families, we found BRCA1 and BRCA2 germline mutations that comprised four frameshift mutations and five nonsense mutations. All nine mutations led to premature termination producing shortened proteins. Among the nine mutations, three novel BRCA1 mutations (E1114X, Q1299X, 4159delGA) and two novel BRCA2 mutations (K467X, 8945delAA) were identified in this work.     

Phenocopies in BRCA1 and BRCA2 families: evidence for modifier genes and implications for screening

Background

The identification of BRCA1 and BRCA2 mutations in familial breast cancer kindreds allows genetic testing of at‐risk relatives. Those who test negative are usually reassured and additional breast cancer surveillance is discontinued. However, we postulated that in high‐risk families, such as those seen in clinical genetics centres, the risk of breast cancer might be influenced not only by the BRCA1/BRCA2 mutation but also by modifier genes. One manifestation of this would be the presence of phenocopies in BRCA1/BRCA2 kindreds.

Methods

277 families with pathogenic BRCA1/BRCA2 mutations were reviewed and 28 breast cancer phenocopies identified. The relative risk of breast cancer in those testing negative was assessed using incidence rates from our cancer registry based on local population.

Results

Phenocopies constituted up to 24% of tests on women with breast cancer after the identification of the mutation in the proband. The standardised incidence ratio for women who tested negative for the BRCA1/BRCA2 family mutation was 5.3 for all relatives, 5.0 for all first‐degree relatives (FDRs) and 3.2 (95% confidence interval 2.0 to 4.9) for FDRs in whose family all other cases of breast and ovarian cancer could be explained by the identified mutation. 13 of 107 (12.1%) FDRs with breast cancer and no unexplained family history tested negative.

Conclusion

In high‐risk families, women who test negative for the familial BRCA1/BRCA2 mutation have an increased risk of breast cancer consistent with genetic modifiers. In light of this, such women should still be considered for continued surveillance.Epidemiological studies suggest that approximately 5% of breast cancer in Western countries is caused by high‐risk dominantly inherited susceptibility genes.^1,2 However, twin studies suggested a genetic component in 27% of breast cancer cases.³ High‐risk dominantly inherited breast cancer susceptibility was first described in 1866 by Broca,⁴ but proof of dominant inheritance was obtained 120 years later with the localisation and identification of BRCA1 and BRCA2.^5,6,7,8 Mutations in these genes account for most high‐risk families with ⩾4 breast cancers in members aged <60 years.⁹ Studies to find other genes associated with breast cancer continue; the elusive BRCA3 gene has not yet been identified.¹⁰ In the mean time, many high‐risk familial breast cancer kindreds are offered BRCA1/BRCA2 mutation analysis. If this is successful, predictive genetic testing is offered to at‐risk relatives. In some centres, relatives who test negative are reassured that their breast cancer risk is at population levels, as recommended by the recently published UK National Institute for Clinical Excellence (NICE) guidelines.¹¹ This policy assumes that, with few exceptions, the risk of a second high‐risk familial mutation is minimal. However, breast cancer risks in high‐risk kindreds with BRCA1/BRCA2 mutations are substantially higher than risks derived from population‐based studies.^9,12,13,14 In high‐risk clusters in the Breast Cancer Linkage Consortium, BRCA1 and BRCA2 mutations were estimated to cause cumulative lifetime risks of breast cancer of 85–87% and 77–84% by 70 years, respectively.^9,15,16 However, estimates of breast cancer risks by 70 years from population‐based studies are much lower (28–60%)^12,13,14 for BRCA1 and still lower for BRCA2. It has been suggested that even these studies may overestimate the effect of BRCA1/BRCA2 alone.¹⁷ It is therefore feasible that a substantial proportion of the risk in familial clusters with a BRCA1/BRCA2 mutation (the group of families that are usually seen by a Cancer Genetics Service) might be due to modifier genes.^18,19 Such a hypothesis would predict that some members of high‐risk families might be at increased risk of breast cancer even if they tested negative for the pathogenic BRCA1/BRCA2 mutation.²⁰ Phenocopies may be due to chance or environmental effects, another high‐risk mutation in a family, ascertainment bias or the presence of modifier genes. We have identified a large number of phenocopies from 277 families with a pathogenic BRCA1/BRCA2 mutation and have assessed breast cancer risks for people testing negative for a familial pathogenic mutation.     

Exclusion of large deletions and other rearrangements in BRCA1 and BRCA2 in Finnish breast and ovarian cancer families

In the Finnish population, identified mutations in BRCA1 and BRCA2 account for a less than expected proportion of hereditary breast and ovarian cancer. All previous studies performed in our country have concentrated on finding germ-line mutations in the coding and splice-site regions of these two genes. Therefore, we wanted to use a different methodological approach and search for large genomic rearrangements, to exclude the possibility of biased BRCA1 and BRCA2 mutation spectra due to known limitations of the previously used PCR-based detection methods. Our results support earlier notions that other genes than BRCA1 and BRCA2 will explain a majority of the still unexplained cases of hereditary susceptibility to breast and ovarian cancer.     

Prevalence of mutations in a panel of breast cancer susceptibility genes in BRCA1/2-negative patients with early-onset breast cancer

PurposeClinical testing for germ-line variation in multiple cancer susceptibility genes is available using massively parallel sequencing. Limited information is available for pretest genetic counseling regarding the spectrum of mutations and variants of uncertain significance in defined patient populations.MethodsWe performed massively parallel sequencing using targeted capture of 22 cancer susceptibility genes in 278 BRCA1/2-negative patients with early-onset breast cancer (diagnosed at younger than 40 years of age).ResultsThirty-one patients (11%) were found to have at least one deleterious or likely deleterious variant. Seven patients (2.5% overall) were found to have deleterious or likely deleterious variants in genes for which clinical guidelines exist for management, namely TP53 (4), CDKN2A (1), MSH2 (1), and MUTYH (double heterozygote). Twenty-four patients (8.6%) had deleterious or likely deleterious variants in a cancer susceptibility gene for which clinical guidelines are lacking, such as CHEK2 and ATM. Fifty-four patients (19%) had at least one variant of uncertain significance, and six patients were heterozygous for a variant in MUTYH.ConclusionThese data demonstrate that massively parallel sequencing identifies reportable variants in known cancer susceptibility genes in more than 30% of patients with early-onset breast cancer. However, only few patients (2.5%) have definitively actionable mutations given current clinical management guidelines.Genet Med17 8, 630–638.     

Frequency of BRCA1 and BRCA2 mutations in a clinic-based series of breast and ovarian cancer families

Mutations in BRCA1 and BRCA2 account for a significant proportion of hereditary breast and ovarian cancer cases. In this study, we sought to determine the frequency of BRCA1- and BRCA2-mutation carrier families in a hospital-based cancer family registry. The frequency of families with germline truncating mutations in BRCA1 and BRCA2 was 17.3% (18/104) and 1.9% (2/104), respectively. Two novel truncating mutations, BRCA1 1848delGA and BRCA2 5694insT, were identified. We also sought to determine the carrier frequency of other affected family members for which the mutation lineage could be established within these families. Not including the probands, 72% (18/25) of the affected family members within the BRCA1 mutation-associated families were carriers, and all four affected members of the BRCA2 families were carriers. These data imply that risk evaluation based on cancer family history alone may result in inaccurate estimates, and where possible, mutation testing should be considered in other affected family members to verify carrier status.     

Analysis of breast cancer susceptibility genes BRCA1 and BRCA2 in Thai familial and isolated early-onset breast and ovarian cancer

Here we report the study on BRCA1 and BRCA2 mutations in 12 Thai breast and/or ovarian cancer families and 6 early-onset breast or breast/ovarian cancer cases without a family history of cancer. Five distinct rare alterations were identified in each gene: four introducing premature stop codons, one in-frame deletion, two missense changes, two intronic alterations and one silent rare variant. The BRCA1 or BRCA2 truncating mutations were detected in four of seven patients with familial or personal history of breast and ovarian cancer, in one of four isolated early onset breast cancer cases and in none of seven breast cancer site specific families. The BRCA1 and BRCA2 mutation yield in Thai patients is consistent with that reported from Europe and North America in similar groups of patients, being particularly high in individuals with personal or family history of breast and ovarian cancer. The BRCA1 and BRCA2 alterations found in this series are different from those identified in other Asian studies, and all but two have never been reported before. We report at least three novel deleterious mutations, the BRCA1 3300delA, BRCA1 744ins20 and BRCA2 6382delT. One in-frame deletion was also found, the BRCA2 5527del9, which seggregated within family members of breast-only cancer patients and was thought to be a cancer-related mutation. BRCA1 3300delA and Asp67Glu alterations were detected each in at least two families and thus could represent founder mutations in Thais.     

BRCA1 and BRCA2 mutations in Russian familial breast cancer

We have screened index cases from 25 Russian breast/ovarian cancer families for germ‐line mutations in all coding exons of the BRCA1 and BRCA2 genes, using multiplex heteroduplex analysis. In addition we tested 22 patients with breast cancer diagnosed before age 40 without family history and 6 patients with bilateral breast cancer. The frequency of families with germline mutations in BRCA was 16% (4/25). One BRCA1 mutation, 5382insC, was found in three families. The results of present study, and those of a separate study of 19 breast‐ovarian cancer families, suggest that BRCA1 5382insC is a founder mutation in the Russian population. Three BRCA2 mutations were found in patients with breast cancer without family history: two in young patients and one in patients with bilateral breast cancer. Four novel BRCA2 mutations were identified: three frameshift (695insT, 1528del4, 9318del4) and one nonsense (S1099X). © 2002 Wiley‐Liss, Inc.     

Low frequency of BRCA1 germline mutations in 45 German breast/ovarian cancer families.

In this study we investigated 45 German breast/ovarian cancer families for germline mutations in the BRCA1 gene. We identified four germline mutations in three breast cancer families and in one breast-ovarian cancer family. among these were one frameshift mutation, one nonsense mutation, one novel splice site mutation, and one missense mutation. The missense mutation was also found in 2.8% of the general population, suggesting that it is not disease associated. The average age of disease onset in those families harbouring causative mutations was between 32.3 and 37.4 years, whereas the family harbouring the missense mutation had an average age of onset of 51.2 years. These findings show that BRCA1 is implicated in a small fraction of breast/ovarian cancer families suggesting the involvement of another susceptibility gene(s).     

Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high‐risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non‐radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high‐risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761‐3762delGA and 2616‐2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073‐5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA‐binding protein. © 2000 Wiley‐Liss, Inc.     

BRCA1 and BRCA2 sequence variants in Chinese breast cancer families

A series of 45 high-risk breast cancer patients, consisting of 25 affected individuals from 16 families in China with at least two cases of breast cancer and 20 cases of breast cancer diagnosed under age 35 without reported family history, were studied for germline mutations of the BRCA1 and BRCA2 genes. Thirteen of the 16 families contained at least one case diagnosed under age 50. Three distinct protein truncating sequence variants, likely to be disease-associated, were identified: two novel mutations in BRCA1 (1584G>T and 5028delC), and a previously reported mutation in BRCA2 (7883delTTAA). Additional sequence variants identified included common polymorphisms, and several variants of unknown clinical significance, including a novel BRCA1 alteration. Based on models for predictive testing using allele frequencies and risks estimated in Western populations, our results suggest that BRCA1/2 mutations account for a somewhat smaller fraction of breast cancer cases in Tianjin than in the Caucasian populations studied. This difference could be the result of a lower penetrance of BRCA1/2 mutations due to the surrounding environmental and hormonal milieu, or a lower frequency of mutations in this population. Larger, more detailed, studies will be necessary to determine which factors underlie this difference.     

Germline mutations in the BRCA1 and BRCA2 genes in Turkish breast/ovarian cancer patients

In this study we genotyped Turkish breast/ovarian cancer patients for BRCA1/BRCA2 mutations: protein truncation test (PTT) for exon 11 BRCA1 of and, multiplex PCR and denaturing gradient gel electrophoresis (DGGE) for BRCA2, complemented by DNA sequencing. In addition, a modified restriction assay was used for analysis of the predominant Jewish mutations: 185delAG, 5382InsC, Tyr978X (BRCA1) and 6174delT (BRCA2). Eighty three breast/ovarian cancer patients were screened: twenty three had a positive family history of breast/ovarian cancer, ten were males with breast cancer at any age, in eighteen the disease was diagnosed under 40 years of age, one patient had ovarian cancer in addition to breast cancer and one patient had ovarian cancer. All the rest (n=30) were considered sporadic breast cancer cases. Overall, 3 pathogenic mutations (3/53-5.7%) were detected, all in high risk individuals (3/23-13%): a novel (2990insA) and a previously described mutation (R1203X) in BRCA1, and a novel mutation (9255delT) in BRCA2. In addition, three missense mutations [two novel (T42S, N2742S) and a previously published one (S384F)] and two neutral polymorphisms (P9P, P2532P) were detected in BRCA2. Notably none of the male breast cancer patients harbored any mutation, and none of the tested individuals carried any of the Jewish mutations. Our findings suggest that there are no predominant mutations within exon 11 of the BRCA1 and in BRCA2 gene in Turkish high risk families.     

Adjusting the estimated proportion of breast cancer cases associated with BRCA1 and BRCA2 mutations: public health implications.

PURPOSE: Mutations in BRCA1 or BRCA2 genes increase breast cancer risk. Assuring reliability of information about these mutations is increasingly important to the health care community; mutation testing is becoming more widespread. We describe a methodology for assessing such information. METHODS: Our approach integrates four interdependent epidemiologic parameters: (1) the probability of developing breast cancer, (2) the proportion of breast cancer cases with a BRCA1 or BRCA2 mutation, (3) the proportion of women that carries a mutation, and (4) the proportion of women with a mutation that develops cancer. We assess the plausibility of estimates of these parameters from published reports and commonly accessed information sources. RESULTS: Assuming a fixed probability of developing breast cancer, the following estimates for the other three epidemiologic parameters are derived for women by age 70: 1% to 2% of all breast cancer cases are associated with a BRCA1 or BRCA2 mutation; 1 in 300 to 1 in 465 women carry a mutation; and 35 to 65% of mutation carriers develop breast cancer. Within these ranges, however, only selected combinations are plausible. The proportion of mutation-related breast cancer is lower than listed in some common information sources (1 to 2% vs 6%). Also, penetrance is somewhat lower and the carrier rate somewhat higher. CONCLUSIONS: The four epidemiologic parameters can be integrated to test their plausibility. BRCA1 and BRCA2 mutations are associated with only one-third as many breast cancer cases in the general population as reported by commonly accessed information sources.     

Ten novel BRCA1 and BRCA2 mutations in breast and/or ovarian cancer families from northern Germany

Germline mutations in the BRCA1 and BRCA2 gene account for the majority of high-risk breast/ovarian cancer families. We have screened such families from Northern Germany by using DHPLC analysis and subsequent direct sequencing techniques. In ten families we identified six novel BRCA1 and 4 novel BRCA2 mutations comprising four frame shift mutations, one nonsense and one splice site mutation in the BRCA1 gene as well as three frameshift mutations and one nonsense mutation in the BRCA2 gene. Our analysis contributes to the further characterisation of the mutational spectrum of BRCA1 and BRCA2.