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Identification of a novel splice-site mutation of the BRCA1 gene in two breast cancer families: Screening reveals low frequency in Icelandic breast cancer patients
Human Mutation
Abstract
Identification of a novel splice-site mutation of the BRCA1 gene in two breast cancer families: screening reveals low frequency in Icelandic breast cancer patients.
... The study group consisted of 1031 patients (women) diagnosed between 1976 and 2007 previously screened for the local BRCA1 c.5074G>A and BRCA2 c.767-771delCAAAT-germline mutations (22,23). In addition to the two BRCA founder mutations, BRCA2 c.767-771delCAAAT and the much rarer BRCA1 c.5074G>A, the only other BRCA mutation of some frequency, c.9976A>T, is not found to be associated with risk of breast or ovarian cancer but rather risk of small cell lung cancer and squamous cell carcinoma of the skin (24). ...
Background:
Breast Cancer 1 gene (BRCA1) is known to be inactivated in breast tumors by promoter methylation. Tumor cells in patients carrying a germline mutation in BRCA1 are sensitive to cytotoxic drugs that cause DNA double strand breaks. However, very little is known on whether patients with BRCA1 promoter methylated tumors are similarly sensitive to cytotoxic drugs. In this study, we address this by making use of extensive follow-up data on patients treated with cyclophosphamide, methotrexate, and fluorouracil in Iceland between 1976 and 2007.
Methods:
We analyzed BRCA1 promoter methylation by pyrosequencing DNA from tumor samples from 1031 patients with primary breast cancer. Of those, 965 were sporadic cases, 61 were BRCA2, and five were BRCA1 germline mutation carriers. All cases were examined with respect to clinicopathological parameters and breast cancer-specific survival in patients treated with cytotoxic drugs. Information on chemotherapy treatment in noncarriers was available for 26 BRCA1 methylated tumors and 857 unmethylated tumors.
Results:
BRCA1 was promoter methylated in 29 sporadic tumors or in 3.0% of cases (29 of 965), whereas none of the tumors derived from BRCA germline mutation carriers were promoter methylated. Important to note, patients with BRCA1 promoter methylation receiving chemotherapeutic drug treatment show highly improved breast cancer-specific survival compared with unmethylated controls (hazard ratio = 0.10, 95% confidence interval = 0.01 to 0.75, two-sided P = .02).
Conclusions:
BRCA1 promoter methylation is predictive of improved disease outcome in patients receiving cyclophosphamide, methotrexate, and fluorouracil drug treatment. Our results support the use of markers indicative of "BRCAness" in sporadic breast cancers to identify patients that are likely to benefit from the use of DNA-damaging agents.
... Other mutations that are relatively common in specific populations, referred to as founder mutations, can be used to in limited screening tests. For example, in Iceland, only two mutations have been reported: the common founder mutation BRCA2 c.771_775del and the rarer BRCA1 c.5074G > A [226]. Despite having a higher risk for developing ovarian cancer, BRCA1/2 carriers have a better clinical outcome in terms of survival, with BRCA2 carriers having a more favorable outcome than BRCA1 carriers [54]. ...
... BRCA2-stökkbreytingin finnst í um 7% kvenna sem greinast með brjóstakrabbamein á Íslandi 8 en stökkbreytingar í BRCA1geni eru aftur á móti fátíðar og finnast í minna en 1% kvenna með brjóstakrabbamein á Íslandi. 9 Íslensk erfðagreining ásamt öðrum vísindamönnum innan heilbrigðisþjónustunnar hefur í gegnum rannsóknir sínar aflað erfðaupplýsinga um stóran hluta þjóðarinnar 7 en mikið hefur verið deilt um hvort og þá hvernig megi nýta þessar upplýsingar til heilbrigðisþjónustu eða forvarna. Þar skarast viðhorf um rétt kvenna til að fá vitneskju um eigið heilsufar og að geta haft áhrif þar á, við rétt kvenna til þess að hafna slíkum upplýsingum. ...
... Hungarian ancestry(Ramus et al., 1997b;van der Looij et al., 2000).In Iceland, only two mutations were reported: the founder mutation BRCA2 c.771_775del and the rarer BRCA1 c.5074G > A(Bergthorsson et al., 1998). A number of other situations can be identified in which specific mutations explain a large proportion of the total mutations observed in a population. ...
The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on Caucasians in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on 6 continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.
... However, this basic test may be supplemented with screening for BRCA1 c.181T>G, as the second most common mutation of the region, and for some special cases, to include most common Hungarian BRCA2 founder mutation c.9097dup (9326insA) for those with Hungarian ancestry (van der Looij, et al., 2000, Ramus, et al., 1997b. In Iceland, only two mutations were reported: the founder mutation BRCA2 c.771_775del and the rarer BRCA1 c.5074G>A (Bergthorsson, et al., 1998). A number of other situations can be identified in which specific mutations explain a large proportion of the total mutations observed in a population. ...
The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on Caucasians in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on 6 continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.
... In the current study, only sam- ples that had gone through exactly the same blood handling and DNA isolation methods (phenol-chloroform) were included. All samples had been screened for the only two known local BRCA founder mutations, i.e., BRCA2 999del5 (c.771_775del5) and a much less frequent BRCA1 5193G!A (2,35). Carriers of this rare BRCA1 founder mutation were excluded from the study. ...
Background:
Germline BRCA2 mutations increase risk of breast cancer and other malignancies. BRCA2 has been shown to play a role in telomere protection and maintenance. Telomere length (TL) has been studied as a modifying factor for various diseases, including breast cancer. Previous research on TL in BRCA mutation carriers has produced contradicting results.
Methods:
We measured blood TL, using a high-throughput monochrome multiplex qPCR method, in a well-defined Icelandic cohort of female BRCA2 mutation carriers (n=169), sporadic breast cancer patients (n=561) and healthy controls (n=537).
Results:
Breast cancer cases had significantly shorter TL than unaffected women (p<0.0001), both BRCA2 mutation carriers (p=0.0097) and non-carriers (p=0.00006). Using exclusively samples acquired before breast cancer diagnosis, we found that shorter telomeres were significantly associated with increased breast cancer risk in BRCA2 mutation carriers (HR = 3.60, 95% CI 1.17-11.28, p=0.025) but not in non-carriers (HR = 1.40, 95% CI 0.89-2.22, p=0.15). We found no association between TL and breast cancer specific survival.
Conclusions:
Blood TL is predictive of breast cancer risk in BRCA2 mutation carriers. Breast cancer cases have significantly shorter TL than unaffected women, regardless of BRCA2 status, indicating that samples taken after breast cancer diagnosis should not be included in evaluations of TL and breast cancer risk.
Impact:
Our study is built on a well-defined cohort, highly accurate methods and long follow-up and can therefore help to clarify some previously published, contradictory results. Our findings also suggest that BRCA2 has an important role in telomere maintenance, even in normal blood cells.
Breast cancer (BC) is a complex and heterogeneous disease caused by interaction of both genetic and nongenetic risk factors. The biological diversity of sporadic BCs consists in the development of several BC subtypes, which are systematically different from one another and which present specific genetic and phenotypic features. Recently, with the advent of cDNA microarrays it has been possible to associate a distinctive “molecular portrait” to a single BC subtype and, consequently, improve BC taxonomy. From a clinical point of view, the gene expression profiles could supply the classic pathological experiment with the aim to select patients with a better prognosis and that could have a benefit from a specific chemotherapy treatment. Recently a new role in BC progression has been identified in a group of small, noncoding RNAs, the MicroRNA (miRNA), which regulate gene expression. Of particular interest is the identification of different miRNA expression levels according to the five molecular BC subtypes identified by experiments on microarray gene expression. About 3–8% of all breast cancers are hereditary, and it is estimated that the main contributors to this type of cancer are mutations in autosomal dominant genes segregating with the disease. Nowadays, BRCA1 and BRCA2 are considered as the two main BC susceptibility genes, although the involvement of additional low penetrance genes implicated in particular syndromes should not be overlooked. Epidemiologic studies reported that women who are BRCA1 mutation carriers have a 45–60% cumulative risk to develop BC before age 35–40, and the average cumulative risk in BRCA1-mutation carriers by age 70 years is 65% whereas the corresponding risk to develop this neoplasm for BRCA2 mutation carriers is estimated to be 25–40% and 45%, respectively. Oncogenetic counseling requires a “multidisciplinary approach,” involving geneticists, oncologists, and psychologists and is offered by many diagnostic clinical genetic services. Nowadays, risk evaluation regarding BC patients is performed with the use of specific mathematical models, such as BRCAPro, the Couch and the Myriad models.
Iceland's founder population provides advantages for researchers analyzing complex disorders such as many of the age-associated diseases. Iceland is one of the least densely populated countries in the world, with a total population of 290,500 people in 2003, averaging 2.9 inhabitants per square kilometer. The nutritional value of diet in Iceland improved significantly during the last century, and it now comes closer in most respects to the targets set by the Icelandic Nutrition Council. Obesity is an increasing problem in Iceland, especially among children. In a recent study, body mass index was found to have increased considerably between 1938 and 1998 among nine-year olds. During the past decade, there has been a remarkable increase in expenditure on research and development work. Icelanders spent 3% of their gross domestic product on research and development undertakings in 2001, compared to 1.1% in 1990, thereby reaching the goal that the European Union has set for itself to achieve by 2010.
Objective
To estimate the risk of malignant diseases in families of probands with the same mutation in the BRCA2 gene.
Design
A cohort study using record linkage of a breast cancer family resource and the Icelandic Cancer Registry.
Setting
Iceland.
Subjects
Families of 995 breast cancer patients, from which 887 were tested for a single founder 999del5 mutation; 90 had the mutation and 797 did not.
Results
Relatives of probands with the mutation had significantly increased relative risk (RR) of breast cancer. For first degree relatives, the RR was 7.55 (95% CI 6.04 to 9.03) but was 1.72 (95% CI 1.49 to 1.96) in first degree relatives of probands without the mutation. For prostate and ovarian cancer, the first and second degree relatives of probands with the mutation had a significantly increased RR, but in families of probands without the mutation no significant familial risk was found.
Conclusions
The 999del5 mutation in the BRCA2 gene explains a substantial proportion of familial risk of breast cancer in Iceland, but significant familial risk remains in relatives of probands without the mutation. For prostate and ovarian cancer, the mutation accounts for most of the familiality observed in families of breast cancer patients.
In Western Europe and the United States approximately 1 in 12 women develop breast cancer. A small proportion of breast cancer cases, in particular those arising at a young age, are attributable to a highly penetrant, autosomal dominant predisposition to the disease. The breast cancer susceptibility gene, BRCA2, was recently localized to chromosome 13q12-q13. Here we report the identification of a gene in which we have detected six different germline mutations in breast cancer families that are likely to be due to BRCA2. Each mutation causes serious disruption to the open reading frame of the transcriptional unit. The results indicate that this is the BRCA2 gene.
In Western Europe and the United States approximately 1 in 12 women develop breast cancer. A small proportion of breast cancer cases, in particular those arising at a young age, are attributable to a highly penetrant, autosomal dominant predisposition to the disease. The breast cancer susceptibility gene, BRCA2, was recently localized to chromosome 13q12-q13. Here we report the identification of a gene in which we have detected six different germline mutations in breast cancer families that are likely to be due to BRCA2. Each mutation causes serious disruption to the open reading frame of the transcriptional unit. The results indicate that this is the BRCA2 gene.
This paper reports the results of a collaborative linkage study involving 214 breast cancer families, including 57 breast-ovarian cancer families; this represents almost all the known families with 17q linkage data. Six markers on 17q, spanning approximately 30 cM, were typed in the families. The aims of the study were to define more precisely the localization of the disease gene, the extent of genetic heterogeneity and the characteristics of linked families and to estimate the penetrance of the 17q gene. Under the assumption of no genetic heterogeneity, the strongest linkage evidence was obtained with D17S588. Multipoint linkage analysis allowing for genetic heterogeneity provided evidence that the predisposing gene lies between the markers D17S588 and D17S250, an interval whose genetic length is estimated to be 8.3 cM in males and 18.0 cM in females. This position was supported over other intervals by odds of 66:1. The location of the gene with respect to D17S579 could not be determined unequivocally. Under the genetic model used in the analysis, the best estimate of the proportion of linked breast-ovarian cancer families was 1.0 (lower LOD -- 1 limit 0.79). In contrast, there was significant evidence of genetic heterogeneity among the families without ovarian cancer, with an estimated 45% being linked. These results suggest that a gene(s) on chromosome 17q accounts for the majority of families in which both early-onset breast cancer and ovarian cancer occur but that other genes predisposing to breast cancer exist. By examining the fit of the linkage data to different penetrance functions, the cumulative risk associated with the 17q gene was estimated to be 59% by age 50 years and 82% by age 70 years. The corresponding estimates for the breast-ovary families were 67% and 76%, and those for the families without ovarian cancer were 49% and 90%; these penetrance functions did not differ significantly from one another. 42 refs., 5 figs., 2 tabs.
A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5' splice sites, 26 at 3' splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5' splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions -1 and -2. Of the 3' splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly over-represented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5' splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a 'consensus value', both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5' splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a "potential for cryptic splice site usage" value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.