BRCA1 and BRCA2 mutations in women with familial or early-onset breast/ovarian cancer in the Czech Republic

MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #697 (2004) Online

© 2004WILEY-LISS, INC. DOI: 10.1002/humu.9226

Received 11 August 2003; accepted revised manuscript 26 January 2004.

BRCA1 and BRCA2 Mutations in Women With Familial or Early-onset Breast/Ovarian Cancer in the Czech Republic Lenka Foretova 1†*, Eva Machackova1 †, Marie Navratilova1, Hana Pavlu1, Marcela Hruba1, Miroslava Lukesova1, and Dalibor Valik2

1Department of Cancer Epidemiology and Genetics, and 2Laboratory Medicine, Masaryk Memorial Cancer Institute, Zluty kopec 7, Brno, 656 53 Czech Republic

*Correspondence to: Lenka Foretova MD, Department of Cancer Epidemiology and Genetics, Masaryk Memorial Cancer Institute, Zluty kopec 7, Brno 656 53, Czech Republic; Tel/Fax: +420 543 136 907; E-mail: [email protected] †These authors contributed equally to this work. Grant sponsor: Internal Grant Agency of the Ministry of Health of the Czech Republic; Grant number: NC 5561-3 and NC 6396-3; Grant sponsor: Ministry of Health of the Czech Republic; Grant number: MZ 00020980501 Communicated by Mark H. Paalman

Germline mutations in BRCA1 and BRCA2 account for majority of hereditary breast and ovarian cancer. The complete coding sequence analysis of both genes was carried out in 197 breast/ovarian cancer patients from high-risk families and 53 patients with sporadic breast/ovarian cancer. In summary, 59 mutations (16 different) in BRCA1 and 29 mutations (17 different) in BRCA2 were identified in unrelated breast and/or ovarian index cases. Using the BIC Database numbering, the most frequently found mutations in BRCA1 were c.5385dupC (22 cases), c.3819_3823delGTAAA (8 cases) and c.300T>G (6 cases). The most frequently found mutations in BRCA2 were c.8138_8142delCCTTT (7 cases) and c.8765_8766delAG (7 cases). Altogether, these 5 mutations represented 56.8% of all detected mutations. A broad spectrum of other mutations was detected including four novel mutations (c.2881delA in BRCA1; and c. 6677_6678delAA, c.6982dupT and c.8397_8400dupTGGG in BRCA2). Deleterious mutations were found in 80 (40.6%) of 197 high risk-families, in 6 (37.5%) of 16 patients with sporadic bilateral breast, ovarian or both cancers and in 2 (6.2%) of 32 women with sporadic early-onset unilateral breast cancer. No mutation was detected in 5 cases of sporadic early-onset unilateral ovarian cancer. © 2004 Wiley-Liss, Inc.

KEY WORDS: BRCA1; BRCA2, mutation analysis; protein truncation test; heteroduplex analysis; direct sequencing; breast cancer; ovarian cancer; Czech

2 Foretova et al.

INTRODUCTION

Breast cancer is the second most frequent malignant disease in women in the Czech Republic accounting for 5,013 newly diagnosed cases in the year 1999 (94.9 cases per 100,000 women). The complete data from the National Cancer Registry of the Czech Republic shows a yearly increase in incidence of 8.4% and an increase of 44% over 10 years.

In a familial form breast cancer occurs at a young age and the risk of bilateral breast cancer and ovarian cancer is increased. The existence of an autosomal dominant pattern of inheritance accounting for 5-10% of breast cancer cases was demonstrated (Newman et al., 1988). Germline mutations in BRCA1 (MIM# 113705) and BRCA2 (MIM# 600185) are responsible for most of the familial breast and ovarian cancers (Ford et al., 1998).

The Breast Cancer Information Core Internet Webside (BIC Database) describes hundreds of mutations in BRCA1 and BRCA2 found in families with hereditary breast and ovarian cancer (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/index.html). The proportion of high-risk families with breast and/or ovarian cancer cases attributable to BRCA1 or BRCA2 mutations varies widely among populations. In some populations only one or a few founder mutations can explain a majority of inherited breast and ovarian cancer cases (for example, in Iceland or Israel), whereas in other populations a wide spectrum of different mutations throughout both genes is present with no evidence of a founder effect (Szabo et al., 1997, Neuhausen et al., 1999).

Only a relatively small amount of data about the Czech population has been published so far. In our pilot study, we presented a 57% detection rate of BRCA1 and BRCA2 deleterious mutations in 21 families with hereditary breast and breast/ovarian cancer (Machackova et al., 2000). Four novel mutations (two in BRCA1 and two in BRCA2) were recently reported by our group (Machackova et al., 2001).

The results of the screening of the whole coding region of the BRCA1 and BRCA2 in 197 breast/ovarian cancer patients from high-risk families, in 16 patients with sporadic bilateral breast, ovarian or both cancers, and in 37 women with sporadic early-onset unilateral breast or ovarian cancers are described in this article.

METHODS

Patients and criteria for testing

Patients were referred for genetic counseling to the Masaryk Memorial Cancer Institute in Brno by physicians or were sent for testing by other medical geneticists from various parts of the Czech Republic. All tested individuals gave signed informed consent. Genetic testing was offered to index patients with breast/ovarian cancer meeting any of the following criteria:

Familial cases

I. At least 3 diagnoses of breast and/or ovarian cancer in the family diagnosed at any age (bilateral cancer counted as two cases) - 103 families;

II. Only two diagnoses of breast and/or ovarian cancer in the family, at least one of them diagnosed before the age of 50 - 94 families;

Sporadic cases

III. Patient with bilateral breast/ovarian cancer, or with both breast and ovarian cancer without a family history of these cancers:

III.A. Patient with bilateral breast cancer diagnosed before the age of 50 - 9 patients; III.B. Patient with bilateral ovarian cancer diagnosed before the age of 50 - 3 patients; III.C. Patient with both breast and ovarian cancer diagnosed before the age of 60 - 4 patients; IV. Early-onset sporadic unilateral breast (32 patients) or ovarian (5 patients) cancer cases diagnosed before

the age of 40. In familial cancer cases, inclusion criteria I. comprised 103 families: 4 families had a history positive for

ovarian cancer only (hereditary ovarian cancer syndrome-HOC), 36 families had a history positive for breast and ovarian cancer (hereditary breast and ovarian syndrome-HBOC), and 63 families had a history positive for breast cancer only syndrome (hereditary breast syndrome-HBC). Inclusion criteria II. comprised 94 families: 1 family

BRCA1 and 2 Mutations in the Czech Republic3

had a history positive for HOC, 12 families had a history positive for HBOC, and 81 families had a history positive for HBC.

Mutation screening

Genomic DNA was isolated from blood samples by QIAamp DNA blood purification kit (Qiagen, Hilden, Germany). The non-radioactive protein truncation test (PTT) for exon 11 of BRCA1 and for exons 10 and 11 of BRCA2 were used as screening tests (Promega, Madison, USA). The remaining exons with their splice sites were screened by the Heteroduplex analysis (HA). The mutation detection approach, primers and their sources used for PTT and HA, were described in detail by Claes et al. (1999). Fragments with aberrant mobility detected on PTT or HA gels were sequenced on a new PCR product by direct sequencing using the Thermo sequenase fluorescent-labeled primer cycle sequencing kit (Amersham Biosciences, Buckinghamshire, UK) and analyzed on the ALF express DNA sequencer (Amersham Biosciences, Buckinghamshire, UK). Each detected sequence alteration was confirmed on an independently drawn blood sample or, in case of a deceased patient, a germline mutation was confirmed on DNA isolated from non-tumor paraffin embedded tissue. Mutations are described at the cDNA level according to the Human BRCA1 reference sequence: accession U14680; and the Human BRCA2 reference sequence: accession U43746; from the GenBank sequence database. The BRCA1/BRCA2 numbering for the traditional mutation nomenclature used in BIC Database is based on reference sequences as stated above where the A of the ATG translation initiation codon is at the position of 120 of BRCA1 and at the position of 229 of BRCA2. The approved systematic nomenclature follows the rule where the A of the ATG translation initiation codon is +1. The approved systemic nomenclature is used in parenthesis. The HUGO-approved systematic nomenclature for the description of sequence variants in DNA and protein sequences was published by Antonarakis et al. (1998) and further extended by den Dunnen and Antonarakis. (2000) and by den Dunnen and Paalman (2003).

RESULTS

In summary, 59 mutations (16 different) in BRCA1 (Table 1a) and 29 mutations (17 different) in BRCA2 were identified (Table 1b) in Czech breast and/or ovarian cancer patients. Tables 1a and 1b show both the HUGO-approved systematic nomenclature and the nomenclature used in the BIC database (see Methods). The mutations most frequently found in BRCA1 were: c.5385dupC (c.5266dupC by the approved systematic nomenclature) - 22 cases, c.3819_3823delGTAAA (c.3700_3704del5) - 8 cases and c.300T>G (c.181T>G) - 6 cases. The mutations most frequently found in BRCA2 gene were: c.8138_8142delCCTTT (c.7910_7914del5) - 7 cases and c.8765_8766delAG (c.8537_8538del2) - 7 cases. Altogether, these 5 mutations represent 56.8% of all detected mutations. A broad spectrum of other detected mutations demonstrates diversity of the population in the Czech Republic.

Four novel mutations were found: one in BRCA1 (c.2762delA) and three in BRCA2 (c.6449_6450del2, c.6754dupT and c.8169_8172dup4). Another four mutations were only seen in the Czech population: c.2488_2497dup10 and c.3642_3643del2 in BRCA1; and c.4845_4846del2 and c.6638delC in BRCA2 (Machackova et al. 2001). The spectrum of variants with unknown significance was not investigated and so is not included.

The efficiency of mutation detection in relation to the inclusion criteria is summarized in Table 2. The highest detection rate (75%) of disease-causing mutations was identified in the group of families with HOC and HBOC syndrome with at least 3 cases of cancer (inclusion criteria I.). The majority (67.5%) of deleterious mutations in this group was attributable to BRCA1. The detection rate of BRCA1 and BRCA2 mutations in HOC and HBOC families with only two cancer cases was also high, 69.2% (inclusion criteria II.). Mutations in BRCA2 gene were most frequently found in HBC families with at least 3 cases diagnosed (inclusion criteria I.).

4 Foretova et al.

Table 1a. Spectrum of BRCA1 Germline Mutations Detected in Czech High-risk Breast/ovarian Cancer Patients

BIC traditional nomenclature

Approved systematic nomenclature

Effect on aminoacids

Numb. of families

World-wide appearance

Mutation published by

c.172T>A

c.53T>A Missense p.Met18Lys

2 Recently in Czech Republic and Australia

Machackova et al., 2001 Southey et al., 1999

c.187_188delAG (known as 185delAG)

c.68_69del2 Frame-shift p.Glu23fsX13

2 Very frequent in Europe and North America, founder in Ashkenazi Jews

BIC database

c.234T>C

c.115T>C Missense p.Cys39Arg

3 Very rare, only 2 cases recently reported in USA

BIC database

c.300T>G c.181T>G Missense p.Cys61Gly

6 Very frequent in Europe (founder in Middle Europe), and North America

BIC database Friedman et al., 1994

c.1135dupA c.1016dupA Frame-shift p.Lys339fsX6

1 Frequent in Norway and USA BIC database

c.1806C>T c.1687C>T Nonsense p.Gln563X

2 Frequent in North-Western and Middle Europe and USA

BIC database

c.2382G>T c.2263G>T Nonsense p.Glu755X

2 Very rare, reported in Hungary and Czech Republic

Ramus et al., 1997; Machackova et al., 2000

c.2607_2616dup AAGTATCCAT

c.2488_2497dup10 Frame-shift p.Lys833fsX

3 Recently only in Czech Republic

Machackova et al., 2001

c.2881delA c.2762delA Frame-shift p.Gln921fsX48

1 Novel mutation unpublished

c.3761_3762delGA c.3642_3643del2 Frame-shift p.Glu1214fsX3

1 Recently only in Czech Republic Machackova et al., 2001

c.3819_3823del GTAAA

c.3700_3704del5 Frame-shift p.Val1234fsX7

8 Frequent mainly in Middle and Eastern Europe and Canada

BIC database

c.3875_3878del GTCT

c.3756_3759del4 Frame-shift p.Leu1252fsX10

3 Very frequent, mainly in Western Europe and North America

BIC database

c.4284_4285delAG c.4165_4166del2 Frame-shift p.Ser1389fsX

1 Rare, detected in Germany, Netherlands, USA

BIC database

c.4304G>A c.4185G>A exon skipping p.Ser1363fsX10

1 Very rare, detected in Germany, USA

BIC database Claes et al., 2003

c.5385dupC (known as 5382insC)

c.5266dupC Frame-shift p.Gln1756fsX73

22 Most frequent mutation in Europe and North America, founder mutation in many Eastern European countries and in Ashkenazi Jewish population

BIC database

c.5629G>A c.5510G>A Nonsense p.Trp1837X

1 Rare in France and USA BIC database

Based on mRNA BRCA1 RefSeq: U14680 – numbering explained in the section ”Mutation Screening” Novel mutations - in bold.


Table 1b. Spectrum of BRCA2 Germline Mutations Detected in Czech High-risk Breast/ovarian Cancer Patients

BIC traditional nomenclature

Approved systematic nomenclature

Effect on aminoacids

Numb. of fam.

World-wide appearance

Mutation published by

c.690_691delAA c.462_463del2 Frame-shift p.Gln154fsX3

1 Very rare, recently only in Netherlands

BIC database

c.1617_1618delAG c.1389_1390del2 Frame-shift p.Thr463fsX3

1 Very rare, recently only in Belgium and USA

BIC database

c.2041dupA c.1813dupA Frame-shift p.Ile605fsX10

1 Frequent in Western Europe and North America

BIC database

c.3034_3037delAAAC c.2806_2809del4 Frame-shift p.Lys936fsX22

1 Very frequent in Spain and USA

BIC database

c.3304A>T c.3076A>T Nonsense p.Lys1026X

1 Very rare, recently only in USA

BIC database

c.3972_3975delTGAG c.3744_3747del4 Frame-shift p.Ser1248fsX9

1 Very rare, recently only in USA and Italy

BIC database

c.5073_5074delCT c.4845_4846del2 Frame-shift p.Leu1615fsX3



c.5873C>A c.5645C>A Nonsense p.Ser1882X

1 Frequent in Netherlands, Canada, USA, Italy

BIC database

c.6677_6678delAA c.6449_6450del2 Frame-shift p.Lys2150fsX24


c.6696_6697delTC c.6468_6469del2 Frame-shift p.Gln2157fsX17

1 Frequent in USA, Italy BIC database

c.6866delC c.6638delC Frame-shift p.Ser2213fsX16



c.6982dupT c.6754dupT Frame-shift p.Ser2252fsX8


c.8138_8142delCCTTT c.7910_7914del5 Frame-shift p.Phe2638fsX

7 Very rare in Germany, Denmark, USA

BIC database

c.8397_8400dupTGGG c.8169_8172dup4 Frame-shift p.Trp2725fsX5


c.8765_8766delAG c.8537_8538del2 Frame-shift p.Glu2846fsX21

7

Very frequent in Canada and USA

BIC database

c.9346-2A>G c.9118-2A>G cryptic splice p.Val3040fsX19

1 Frequent only in Finland

Huusko et al. 1998; Machackova et al., 2000

c.9631delC c.9403delC Frame-shift p.Leu3135fsX27

1 Frequent only in Poland BIC database

Based on mRNA BRCA2 RefSeq: U43746 – numbering explained in the section ”Mutation Screening” Novel mutations – in bold

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Table 2 Summary of BRCA1 and BRCA2 Mutations Identified in Relation to Inclusion Criteria and Phenotype

Inclusion criteria and phenotype

Number of families /patients

BRCA1 mutation (%)

BRCA2 mutation (%)

Overall mutation

(%) I. HOC + HBOC 40 27 (67.5%) 3 (7.5%) 30 (75%) I. HBC 63 10 (15.9%) 13 (20.5%) 23 (36.5%) I. Overall 103 37 (35.9%) 16 (15.5%) 53 (51.5%) II. HOC + HBOC

13

8 (61.5%)

1 (7.7%)

9 (69.2%)

II. HBC 81 9 (11.1%) 9 (11.1%) 18 (22.2%) II. Overall 94 17 (18.1%) 10 (10.6%) 27 (28.7%) I.+II. Familial cases HOC + HBOC

53

35 (66%)

4 (7.5%)

39 (73.6%)

I.+II. Familial cases HBC 144 19 (13.2%) 22 (15.3%) 41 (28.5%) I.+II. Familial cases - overall 197 54 (27.4%) 26 (13.2%) 80 (40.6%) III.A Bilateral breast cancer patient

9

1 (11.1%)

0

1 (11.1%)

III.B Bilateral ovarian cancer patient 3 1 (33.3%) 0 1 (33.3%) III.C Patient with breast and ovarian cancer 4 2 (50%) 2 (50%) 4 (100%) III. Sporadic bilateral or both cancers - overall 16 4 (25%) 2 (12.5%) 6 (37.5%) IV. Early onset unilateral ovarian cancer patient

5

0

0

0

IV. Early onset unilateral breast cancer patient 32 1 (3.1%) 1 (3.1%) 2 (6.2%) IV. Sporadic early onset -overall 37 1 (2.7%) 1 (2.7%) 2 (5.4%)

Abbreviations: inclusion criteria - see section of Material and methods; mutation - only deleterious mutations summarized in Table 1 are considered; HOC – hereditary ovarian cancer syndrome; HBOC – hereditary breast and ovarian cancer syndrome; HBC – hereditary breast cancer syndrome. We analyzed 19 families with at least one woman diagnosed with breast and ovarian cancer (the patient with

duplicity was not always available for mutation analysis in familial cases), and 4 sporadic breast and ovarian cancer cases. In 19 out of 23 unrelated cases (82.6%) a disease-causing mutation was identified (16 cases with BRCA1 mutation, 69.6%; 3 cases with BRCA2 mutation, 13%).

Screening of 32 patients with sporadic early-onset unilateral breast cancer resulted in the identification of two germline mutations (6.2%), one in BRCA1 (c.5266dupC) and one in BRCA2 (c.7910_7914del5). Both women were diagnosed at the age of 32. They were from families with a low number of relatives where genetic predisposition for breast cancer was carried through the paternal line. No germline mutation was found among 5 women with sporadic early-onset unilateral ovarian cancer.

DISCUSSION

We studied the spectrum of the BRCA1 and BRCA2 mutations in 197 breast/ovarian cancer families, and 53 sporadic breast/ovarian cancer cases. A majority of disease causing mutations detected was small deletions and insertions resulting in frame-shift or nonsense substitutions causing premature stop codons. Four novel pathogenic mutations (1 in BRCA1 and 3 in BRCA2) are reported, each of them was found only once. The mutation c.2762delA in BRCA1 was discovered in a family with HOC syndrome where ovarian cancer occurred in index patient at the age of 38 and in her mother at the age of 55. All three novel BRCA2 mutations caused HBC syndrome. In the family with c.6449_6450del2 the index case was diagnosed with breast cancer at the age of 41, her mother at the age of 29. In the family with c.6754dupT the index patient had breast cancer at the age of 47, her mother had bilateral breast cancer at 51and 52. The mutation c.8169_8172dup4 was found in a family where the daughter was diagnosed with breast cancer at 57, her mother had breast cancer at 49, her aunt at 68.

Two splice site mutations were detected: c.4185G>A in BRCA1 causing an exon 12 skipping, and c.9118-2A>G in BRCA2 gene causing an activation of a cryptic acceptor splice site 8 bp downstream to the wild type acceptor resulting in deletion of the first 7 bp of exon 24. Both splice site mutations lead to a frame-shift and premature termination of translation. The effect of these splice site alterations was shown at mRNA level (Claes et al., 2003).

Three different missense mutations detected in BRCA1 in 11 high-risk breast and/or ovarian cancer families (Table 1a), c.53T>A, c.115T>C and c.181T>G, are localized in a highly conserved structure of the C3HC4 RING domain. The RING domain of BRCA1 has been described as a binding site for proteins which might be important


for correct BRCA1 function (Jensen et al., 1998; Ruffner et al., 2001). Frequently detected missense substitution within the co-ordinating cysteine residues in the RING finger domain of BRCA1 (c.181T>G, founder mutation in Central European countries) was shown by many authors to segregate with the disease in several breast cancer families in Caucasians. With high probability, the same effect might be associated with the rare missense mutation c.115T>C, because the 100% conserved residues C39, H41, C61 and C64 represent the second Zn2+-binding site of the RING domain. A c.115T>C missense mutation has been detected in three Czech families with hereditary breast and ovarian cancer syndrome but was not present in our panel of 100 control chromosomes. The c.53T>A alteration has been reported in two Czech families and one Australian family with hereditary breast and breast-ovarian cancer syndrome but was not present in the control groups of both populations (Machackova et al., 2001; Southey et al., 1999). The M18 is a highly conserved residue localized 6 amino acids upstream of the core C3HC4

RING finger motif and was described to be an important part of the hydrophobic core of the N-terminal helix important for heterodimerisation of BRCA1 with other proteins (Brzovic et al., 2001). The c.53T>A results in the replacement of a nonpolar, hydrophobic methionine to a polar, hydrophilic lysine. This replacement may affect N-terminal helix folding, stability of the protein and may result in the disruption of the tertiary structure of the complex RING motif. We assume that c.53T>A is very likely to be a disease-causing mutation (Machackova et al., 2001).

The variety of mutations found in our patients reflects the spectrum of mutations found in other European countries. The BRCA1 mutations c.5266dupC, c.3700_3704del5 and c.181T>G were found in 37 %, 13.5 % and 11 % of 59 BRCA1 mutation positive patients, respectively. Two mutations in the BRCA2, c.7910_7914del5 and c.8537_8538del2, were each seen in 24% of 29 BRCA2 mutation carriers. All those five mutations account for 56.8 % of detected mutations.

The Czech population is mainly of Slavic origin. Due to its location (central Europe) and historical influences during the past centuries (part of the Austro-Hungarian Empire) an influx of other nations was also significant. Although there were over 118 000 Jews living in the Czech and Moravian part of the Czechoslovakia before the World War II, the current Jewish population was diminished to 12000 during the holocaust and consecutive emigration waves (www.kehilaprag.cz). Thus the BRCA1 mutation c.68_69del2 (known as 185delAG), predominant in Jewish population, was seen only twice and the BRCA2 mutation c. 5946delT (known as 6174delT) was not detected at all in this study. The BRCA1 mutation c.5266dupC (known as 5382insC), frequently found in Ashkenazi Jews, European countries, and North America, represents also the most frequent mutation in the Czech population.

In conclusion, we found disease-causing mutation in BRCA1 or BRCA2 in 40.6 % of 197 women with a positive family history of breast/ovarian cancer. Mutation detection rate was 73.5 % in families with HOC and HBOC syndrome, 28.5 % in families with HBC syndrome, 37.5% in patients with sporadic bilateral breast, ovarian or both cancers and 6.2 % in women with sporadic unilateral early-onset breast cancer. No mutation was found in 5 early-onset ovarian cancer cases. Four novel mutations are reported, one in BRCA1 and three in BRCA2 gene. Two splice site mutations were proven to lead to the premature termination of translation. Although the 5 most frequent mutations in BRCA1 and BRCA2 account for 56.8 % of all detected mutations, screening of the whole coding region of both BRCA1 and BRCA2 is necessary for the molecular genetic testing in the Czech high-risk breast/ovarian cancer patients.

ACKNOWLEDGMENTS

We would like to acknowledge all the medical geneticists and clinicians for the recruitment of patients for genetic analysis, especially Krutilkova V, MD; Plevova P, MD; Silhanova E, MD; Fiedler Z, MD; Gregor V, MD; Subrt I, MD; Cwiertka K, MD; Hejtmankova M, MD. We thank Mr. Patrick Broeker who worked on the preparation of the article and the revision of the English text.

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Documents

BRCA1 and BRCA2 mutations in women with familial or early-onset breast/ovarian cancer in the Czech Republic