RNA-based analysis of BRCA1 and BRCA2 gene alterations

Cancer Genetics and Cytogenetics 170 (2006) 93e101

RNA-based analysis of BRCA1 and BRCA2 gene alterations Fabrizia Bonattia, Chiara Pepea, Mariella Tancredia, Grazia Lombardia, Paolo Aretinib, Elisa Sensib, Elisabetta Falaschia, Giovanna Cipollinib, Generoso Bevilacquaa, Maria Adelaide Caligoa,* a

Section of Genetic Oncology, Division of Surgical, Molecular and Ultrastructural Pathology, University of Pisa and Pisa University Hospital, Via Roma, 57 Pisa, Italy b MGM, Institute of Molecular Genetics and Medicine, Via Volturno, 56/58 Pisa, Italy

Received 30 December 2005; received in revised form 12 May 2006; accepted 15 May 2006

Abstract

Alterations in BRCA1 and BRCA2 genes account for a large proportion of hereditary breast and ovarian cancers. Mutations and variants of unknown pathological significance have been identified in both genes; however, most of them have been studied only at the genomic level, and their effect on mRNA expression remains unknown. We identified two BRCA1 and six BRCA2 splice site variants, and one BRCA2 alteration at exon 14. Our aim was to ascertain the effect on RNA processing of the variants still unclassified. We found that BRCA1 c.IVS11 þ 1GOA, BRCA2 c.7252_7272delinsTG, BRCA2 c.IVS2 þ 1GOA, BRCA2 c.IVS13-2AOG, BRCA2 c.IVS21 þ 4AOG, and BRCA2 c.9345GOA lead to aberrant transcripts in lymphocytes. Five of these six splice site variants caused a complete inactivation of the mutant allele because they produced frameshift similar to previously described deleterious exonic variants. Therefore, we consider them to be true deleterious mutations, possibly associated with an increased lifetime risk of breast or ovarian cancer. BRCA1 c.IVS17 þ 6COG, BRCA2 c.IVS12-9del4, and BRCA2 IVS1-9del3 represent rare variants, not disrupting normal mRNA processing. The last two BRCA2 genetic variants had not been reported in the Breast Cancer Information Core BIC database. Ó 2006 Elsevier Inc. All rights reserved.

1. Introduction The identification of BRCA1 and BRCA2 genes has been a major advance in the understanding of familial forms of breast and ovarian cancer, because alterations of these two genes result in a strong predisposition to cancer [1,2]. The analysis of BRCA1 coding sequence by means of mutation screening methods based on polymerase chain reaction (PCR) sequencing protocols, such as direct sequencing, has allowed the identification of 800 different germline alterations mainly consisting of point mutations and small deletions or insertions. These alterations, found either within the coding sequence or at the introneexon junctions, introduce premature stop codons (BIC database, Breast Cancer Information Core: http://research.nhgri.nih. gov/bic/). Large genomic rearrangements have been identified in some families, using a variety of alternative technical approaches [3,4]; however, the frequency of

* Corresponding author. Tel.: þ39 050 992907; fax: þ39 050 992706. E-mail address: [email protected] (M.A. Caligo). 0165-4608/06/$ e see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.05.005

BRCA1 alterations described is lower than linkage analysis prediction. Missense mutations that affect amino acids critical for protein structure and function are generally assumed to be a likely cause of disease; however, a large number of missense mutations and intron variants routinely encountered in clinical and research laboratories cannot be readily distinguished as either disease-associated mutations or benign polymorphisms, and are classified in the BIC database as variants of uncertain pathological significance. This poses a major problem for genetic counseling. Furthermore, recent studies have shown that translationally silent variants, normally classified as allelic polymorphisms and considered to be neutral, can affect mRNA processing [5,6]. Accurate RNA splicing requires the absence of mutations in the cis-acting consensus elements known to be involved in RNA splicing: the conserved sequence motifs at the introneexon junctions, and at the branch point. Shapiro and Senapathy [7] showed a sequence of eight nucleotides to be highly conserved at the exoneintron boundary, the splice donor or 50 splice site [(A/C)AG|gt(a/g)agt]. The splice acceptor or 30 splice site also exhibits a highly

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F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101

conserved sequence of four nucleotides, preceded by a pyrimidine-rich region (tyttytytyyyyncag|G, where ‘y’ represents a pyrimidine and ‘n’ represents a nucleotide) [8,9]. Several groups are currently working on the molecular characterization of BRCA1 and BRCA2 splicing variants with relation to their role in breast cancer. Four major BRCA1 isoforms have been identified: a full-length splice variant; D(9,10) skipping exons 9 and 10; D11q, an inframe deletion of 3,309 nucleotides from exon 11 resulting from an alternative splice event; and D(9,10,11q) [10e12]. In BRCA2 isoforms identified so far, alternative splicing encompassed exons 20e23: the main isoform contains a 64-bp in-frame insertion derived from intron 20, and two additional isoforms were generated by using an alternative acceptor site at intron 22 [13]. Most important, a number of studies have been able to correlate the presence of specific mRNA variants to splice site mutations in BRCA1/2 genes [14e18]. For the present study, we had available 276 families in Italy (all with history of breast cancer, or ovarian cancer, or both) who had been tested for BRCA1/2 gene status. Of these, 46 families carried genomic mutations at BRCA1 and 24 at BRCA2. Eight genetic variants at the splice sites (2.2%) were also detected, two in BRCA1 and six in BRCA2; in one family, a complex BRCA2 genetic rearrangement was identified. Our objective was to clarify the effects of the detected variants on mRNA processing and to compare these results with those predicted by using a computer program.

astor.som.jhmi.edu/BayesMendel/brcapro.html) [20,21], and specifically implemented for penetrance estimates in the Italian population according to recent data [22]. 2.2. mRNA analysis RNA was isolated from peripheral blood lymphocytes (TriReagent; Molecular Research Center, http://www. mrcgene.com) and analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) using SuperScript ssp1 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocols. PCR amplification of splice site variants was extended to adjacent exons. Whenever genomic sequencing showed absence of variants, full-length cDNA from BRCA1 was subdivided into six overlapping amplicons (exon 1 to 7, exon 6 to beginning of exon 11, exon 10 to 12, end of exon 11 to exon 16, exon 15 to 19, and exon 18 to 24). Fulllength cDNA from BRCA2 was subdivided into eight overlapping amplicons (exon 1 to 5, exon 4 to 9, exon 8 to beginning of exon 10, exon 9 to beginning of exon 11, end of exon 10 to exon 12, end of exon 11 to exon 15, exon 14 to 18, exon 17 to 21, and exon 20 to 27). Amplified fragments were separated on a 2% agarose gel and visualized with ethidium bromide. RT-PCR products containing both the normal and the aberrant fragment were sequenced directly. In some experiments, individual bands were excised from the gel and sequenced using the same primer pairs as for PCR. The primer sequences are available from the corresponding author. 2.3. PCR direct sequencing

2. Materials and methods 2.1. Patients We selected eight probands showing variants localized around the splice sites (BRCA1 c.IVS11 þ 1GOA and c.IVS17 þ 6COG; BRCA2 c.IVS1-9del3, c.IVS2 þ 1GOA, c.IVS12-9del4, c.IVS13-2AOG, c.IVS21 þ 4AOG, and c.9345GOA), and one patient showing a genomic alteration of unknown pathological significance (BRCA2 c.7252_7272delinsTG). The DNA mutation numbering is based on the cDNA sequence for BRCA1 (GenBank: U14680) and BRCA2 (GenBank: NM_000059), where þ1 corresponds to the first base of exon 1. The nomenclature system for the description of changes (mutations) in DNA follows the recommendations of den Dunnen and Antonarakis [19]. In addition, 41 probands belonging to 41 unrelated Italian families with breast cancer, ovarian cancer, or both were selected from among 221 families negative for BRCA1/2 gene mutations after prediction analysis showed a high probability of carrier status. Prediction of germline mutations in BRCA1/2 genes based on family history was determined using CaGene software, derived from the BRCAPro model (http://

PCR direct sequencing (PCR-DS) was performed on an ABI model 3100 automated sequencer (Applied Biosystems, Foster City, CA), using a BigDye Terminator cycle sequencing reaction kit (Applied Biosystems), following the recommended protocol. Primer sequences, PCR conditions, and sequencing are available from the corresponding author. Sequencing analysis was performed by SeqScape version 1.1 software (Applied Biosystems). 2.4. Splicing prediction method To confirm aberrant splicing identified through mRNA analysis, we used BDGP software (Berkeley Drosophila Genome Project; available at http://www.fruitfly.org/ seq_tools/splice.html). as the splice site prediction algorithm to analyze the structure of donor and acceptor sites using two separate neural network recognizers developed as described [23]. We trained a back-propagation, feedforward neural network with one layer of hidden units to recognize donor and acceptor sites, using a novel optimized representative data set. We considered only genes having constraint consensus splice sites (i.e., ‘GT’ for the donor and ‘AG’ for the acceptor site). The output of the network is a score for potential splice sites.

F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101

3. Results 3.1. mRNA sequencing and splicing analysis Our 276 families (all with history of breast cancer, ovarian cancer, or both) had been tested for BRCA1/2 gene status. The PCR-DS analysis, encompassing both the complete coding sequence and the exoneintron junctions, allowed the identification of 46 families carrying genomic mutations at BRCA1 (16.7%) and 24 families carrying mutations at BRCA2 (8.7%). Eight genetic variants at the splice sites (2.2%), two of them in BRCA1 and six in BRCA2, were also detected, as well as a complex BRCA2 genetic rearrangement. The eight splice site alterations were tested for their possible effect on mRNA processing. Aberrant RNA splicing was detected in five cases (62.5%), four of them involving BRCA2 and one involving BRCA1 (Table 1). BRCA1 c.IVS11 þ 1GOA is located at the donor splice site of intron 11. cDNA amplification revealed two electrophoresis bands in both proband and control (Fig. 1A), one corresponding to the full-length transcript (5,600 bp) and the other to the BRCA1 isoform D11q (2,287 bp, retention of 123 bp from exon 11) [10,12]. A quantitative difference was observed between proband and control, with the patient showing a reduced amount of the higher molecular weight band. The patient is heterozygous for the polymorphism L771L, and the PCR-DS showed that one of two alleles is not transcribed in mRNA (Fig. 1B); we therefore hypothesized a monoallelic expression. BRCA2 c.IVS2 þ 1GOA, located at the donor splice site of intron 2, produced loss of exon 2, due to recruitment of an upstream acceptor site (Fig. 2A). The variant allele showed loss of the start translation point, normally located at exon 2. A frameshift was observed, generating a new start codon at position 551 and a stop codon at position 587. The PCR-DS of the region encompassing BRCA2 exon 1e2 revealed two overlapping sequences, one corresponding to the wild-type allele and the other to the variant allele D2 (Fig. 2B). BRCA2 c.IVS13-2AOG, located at the acceptor splice site of intron 13, produced the loss of exon 14, due to

95

recruitment of a downstream acceptor site. The alternative transcript generated a stop codon at position 2495 (Fig. 3A). The PCR-DS of the region encompassing BRCA2 exon 13e15 revealed two overlapping sequences, one corresponding to the wild-type allele and the other to the variant allele D14 (Fig. 3B). BRCA2 c.9345GOA, located at the donor splice site of exon 23, showed skipping of exon 23, generated by the use of an upstream splice site producing a stop codon at position 3042 (Fig. 4A). The PCR-DS of the variant allele D23 showed the sequence of the band lacking exon 23 (Fig. 4B). BRCA2 c.IVS21 þ 4AOG, causing loss of intron 21 donor site, led to consequent activation of a cryptic splice site at position IVS21 þ 46 (Fig. 5A). The resulting transcript retained a 46-bp fragment of intron 21 and generated a stop codon at position c.IVS21 þ 9 (Fig. 5B). BRCA1 c.IVS17 þ 6COG, BRCA2 c.IVS1-9del3, and BRCA2 c.IVS12-9del4 showed the same patterns of RTPCR products as in normal controls, when examined by electrophoresis on agarose gels. These results suggest that these three variants are normally expressed in mRNA and are likely to be translated into the wild-type proteins. One additional proband had shown a complex genomic rearrangement at the BRCA2 locus, consisting of a TG insertion and a 20-bp deletion of unknown origin. BRCA2 c.7252_7272delinsTG generated an anomalous stop codon in exon 14, at position 2341 of cDNA (Fig. 6A). mRNA processing remained unaltered, and cDNA presented the same alterations as genomic DNA: a total deletion of 6 amino acids. cDNA sequence analysis showed a wild-type transcript down to position 7252. Afterwards, two overlapping sequences became evident, one corresponding to the wild-type allele and the other to the variant (Fig. 6B). Sequence analysis of full-length BRCA1 and BRCA2 cDNAs of the 41 probands belonging to high-risk breast cancer families and negative for DNA alterations revealed functional BRCA1/2 mRNAs deriving from both alleles. We also detected the presence of the four major BRCA1 isoforms dfull-length, D(9,10), D(11q), and D(9,10,11q)d and the three BRCA2 isoforms due to alternative splicing in region encompassing exons 20e23.

Table 1 Effect on mRNA processing of eight BRCA variants analyzed Family

Gene

Varianta

Effect on mRNA processing

Effect on protein

PI PI PI PI PI PI PI PI PI

BRCA1 BRCA1 BRCA2 BRCA2 BRCA2 BRCA2 BRCA2 BRCA2 BRCA2

c.IVS11 þ 1GOA c.IVS17 þ 6COG c.IVS2 þ 1GOA c.IVS13-2AOG c.IVS21 þ 4AOG c.9345GOA c.7252_7272delinsTG c.IVS1-9del3 c.IVS12-9del4

Skipping of exon 11 None Skipping of exon 2 Skipping of exon 14 Retention of 47 bp of intron 21 Skipping of exon 23 Deletion of 18 bp (6aa) None None

None None Truncation Truncation Truncation Truncation Truncation None None

38 348 413 60 262 184 267 299 263 a

Mutation numbering is based on the cDNA sequence for BRCA1 (MIM no. 113705) and BRCA2 (MIM no. 600185) according to the recommendations of den Dunnen and Antonarakis [19] as described in section 2.1.

c.IVS11+1G>A

M

A

CTRL

F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101

96

B

cDNA sequence

6000bp 5000bp

Full length (5600bp) 11q (2287bp)

2500bp 2000bp

gDNA sequence

Fig. 1. BRCA1 c.IVS11 þ 1GOA. (A) The amplification of control (CTRL) and patient’s cDNA carrying the BRCA1 c.IVS11 þ 1GOA variant, showed two bands in agarose gel, one corresponding to the full-length transcript (5,600 bp) and the other to the BRCA1 isoform D11q (2,287 bp). BRCA1 c.IVS11 þ 1GOA is located at the donor splice site of intron 11. A quantitative difference was observed between proband and control, with the patient showing a reduced amount of the higher molecular weight band (M, molecular weight marker). (B) The polymerase chain reaction direct sequencing (PCR-DS) of BRCA1 c.IVS11 þ 1GOA: the patient is heterozygous for the polymorphism L771L (c.2431TOC; gDNA sequence), and one of the two alleles is not transcribed in mRNA (cDNA sequence).

3.2. Phenotypeegenotype correlations and cosegregation analysis

B

M

CTRL

A

c.IVS2+1G>A

BRCA1 c.IVS11 þ 1GOA was detected in a proband (II.5) affected by ovarian cancer (age at disease: 52 years), belonging to a hereditary breast and ovarian cancer (HBOC) family. Family history showed one first-degree relative (II.2) with breast cancer at 40 years. We established absence of the mutation in a healthy daughter of the proband (III.5) (Fig. 7A). BRCA2 c.IVS2 þ 1GOA was detected in a proband (III.1) affected by breast cancer (age at disease: 41 years), belonging to a hereditary breast cancer (HBC) family. Family history showed one second-degree relative (II.2) and one

third-degree relative (III.3) affected by breast cancer; median age at disease was 47.5 years. We could not perform a segregation analysis because all the patients had died (Fig. 7B). BRCA2 c.IVS13-2AOG was detected in a proband (III.5) affected by bilateral breast cancer (age at disease: 39 and 46 years), belonging to an early onset breast cancer (EOBC) family. Family history showed two firstdegree relatives (II.6 and III.6) and three second-degree relatives (II.2, II.5, and II.9) with breast cancer; median age at disease was 45.8 years. We could not perform a segregation analysis because all the patients had died (Fig. 7C).

Exon1

Exon2 Exon3 c.IVS2+1G>A

Full length (511bp) 2 (396bp)

500bp 400bp 300bp

Exon1

Exon2

CTRL

Fig. 2. BRCA2 c.IVS2 þ 1GOA. (A) The cDNA amplification showed a double band, one corresponding to wild-type transcript and the other corresponding to allele variant lacking exon 2 (CTRL, control; M, molecular weight marker). (B) The direct sequence of the cDNA sample showed two overlapping sequences at exon 2 level, one corresponding to the wild-type allele and the other to the variant allele D2.

CTRL

c.IVS13-2A>G

A

M

F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101

B Exon13

97

Exon14 Exon15

Full length (752bp)

700bp 600bp 500bp

c.IVS13-2A>G

400bp 14 (326bp)

300bp

Exon13

Exon14 CTRL

Fig. 3. BRCA2 c.IVS13-2AOG. The amplification of BRCA2 c.IVS13-2AOG variant D14 (A) showed a double band, one corresponding to wild-type transcript and the other corresponding to allele variant with the loss of exon 14 (CTRL, control; M, molecular weight marker). (B) The PCR-DS showed the overlapping of allele sequences, one corresponding to the wild-type allele and the other to the variant allele D14.

(II.4) with breast cancer; median age at breast disease was 54 years. We established absence of the mutation in three healthy members of the family: a sister of the proband (IV.3) and the sister’s two daughters (V.2 and V.3) (Fig. 7F). Using PCR-DS, we also analyzed proband BRCA status in DNA samples from tumor tissue and peripheral blood lymphocytes, to test for possible loss of heterozygosity (LOH). No LOH was detected at the BRCA2 locus; the BRCA1 locus showed the deletion of one allele at the tumor level. Note that median ages reported here were calculated for relatives only (i.e., excluding the proband), and for breast cancer only. If more than one cancer affected the same person, each tumor was considered to be independent and therefore each age at disease was entered separately in calculating median age at disease. 3.3. Splicing prediction analysis The BDGP computer program allows predicting the effects of a variant on splicing efficiency comparing the

B M

c.9345G>A

A

CTRL

BRCA2 c.9345GOA was detected in a proband (III.1) affected by breast cancer (age at disease: 40 years), belonging to an HBOC family. Family history showed one first-degree relative (II.3), three second-degree relatives (I.1 paternal, II.1, and II.8), and one third-degree relative (III.4) affected by breast cancer; median age at breast cancer was 46 years. We could not perform a segregation analysis because blood samples of other family members were not available (Fig. 7D). BRCA2 IVS21 þ 4aOg was detected in a proband (II.4) affected by ovarian cancer (age at disease: 31 years), belonging an HBOC family. Family history showed one first-degree relative (II.3) with breast cancer at 30 years. We established absence of the mutation in two healthy members of the family: a sister of the proband (II.6) and the sister’s daughter (III.6) (Fig. 7E). BRCA2 c.7252_7272delinsTG was detected in a proband (IV.1) affected by bilateral breast cancer (age at disease: 58 and 65 years), belonging to an HBC family. Family history showed two first-degree relatives (III.2 and IV.4), one second-degree relative (V.4), and one third-degree relative

Exon22

Exon24 c.9345G>A

Full length (455bp) 23 (290bp)

500bp 400bp 300bp 200bp

Exon22

Exon23 CTRL

Fig. 4. BRCA2 c.9345GOA. The amplification of BRCA2 c.9345GOA variant D23 (A) showed a double band, one corresponding to wild-type transcript and the other corresponding to allele variant with the loss of exon 23 (CTRL, control; M, molecular weight marker). (B) The PCR-DS of the variant allele D23 showed the sequence of the band lacking exon 23.

F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101

98

A EX 21

*

EX 22

“ins46” (352bp) Full length (306bp)

B M

CTRL

c.IVS21+4A>G

IVS21

400bp 300bp 200bp

Exon21

Intron21

Exon2

Exon22

c.IVS21+4A>G

CTRL

Fig. 5. BRCA2 c.IVS21 þ 4AOG. (A) The BRCA2 IVS21 þ 4AOG showed the loss of the donor site (indicated by asterisk *) and the consequent activation of a cryptic splice site in position c.IVS21 þ 46 (normal splicing is indicated in dashed line and aberrant splicing in continuous line). The variant allele gave rise to an anomalous mRNA with a 46-bp insertion (‘‘ins46’’), due to intron retention, leading to a truncated protein for the presence of a premature stop codon at position c.IVS21 þ 9 (CTRL, control; EX, exon; M, molecular weight marker). (B) The PCR-DS showed two overlapping sequences, one corresponding to the wild-type allele and the other to the variant allele.

4. Discussion Alternative splicing of pre-mRNA is a powerful regulatory mechanism that can affect quantitative and qualitative control of gene expression and functional diversification of proteins. Splice site variants detected by PCR-DS can be investigated by means of RNA-based sequencing. There are claims that such analysis can detect differential splicing, exon skipping, or nonsense-mediated mRNA decay that results in either the absence or low expression of mRNA harboring mutations. According to the December 2005 update

c.7252_7272delinsTG

M

A

CTRL

sequence containing the consensus splice site versus the sequence variant. It revealed loss of the corresponding consensus splice sites for BRCA1 c.IVS11 þ 1GOA, BRCA2 c.IVS2 þ 1GOA, BRCA2 c.IVS13-2AOG, and BRCA2 c.IVS21 þ 4AOG, with no change for BRCA1 c.IVS17 þ 6COG, BRCA2 c.IVS12-9del4. For BRCA2 c.IVS19del3 variants, the software failed to predict the presence of an acceptor splice site in intron 1 of BRCA2, probably because the start point of BRCA2 translation is normally located in exon 2 (Table 2).

B cDNA

gDNA 500bp 400bp

Wild type (470bp) c.7252_7272delinsTG (452bp)

CTRL cDNA/gDNA

Fig. 6. BRCA2 c.7252_7272delinsTG. (A) The cDNA amplification of exon 13e15 of BRCA2 showed two bands in agarose gel for the patient who carried the BRCA2 c.7252_7272delinsTG in exon 14 (CTRL, control; M, molecular weight marker). (B) The sequence analysis of the gDNA and cDNA showed two overlapping sequences, one corresponding to the wild-type allele and the other to the variant allele (CTRL: wild-type sequence of cDNA is the same as that of gDNA).

F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101 2

1 Unknown

82

1

I

A

I

1

99

2

80

80

2

3

1

2

67

5

4

B

33

6

7

9

8

II 1

3

2

II Breast

68

40

6

5

4

70 Breast 66 55

+

56

70

62

2

52

5

4

3

III 36

35

50

Ovary 1

III 1

81

+

7

6

-

41

IV

1

1

1

Colonrectum 89

2

1

2

D

Breast 30 Breast 30

66

2

55

17

2

IV 1

4

40

2

14

I

3

Breast Colonrectum

49

Breast

37

15

2

3

4

5

6

7

8

9

II 1

I

2

Breast 50 Lung Breast 48 Ovary 65 72

C

Unknown

78

92 Breast 57

66

43

2

1

4

6

5

III 1

II

2

4

3

Breast

6

5

7

84 Breast Breast Prostate

70

45 37

8

49

45

10

9

Lung Breast

45

+

11

Breast Colonrectum

71

Breast 40

61

1

I 1

3

2

5

4

58

63 68 2

F

6

III Prostate 55

49 53 +

Breast Breast

50

39 46

Breast 32

1 1

IV 19

2

84

1

2

I

E

70

1

2

5

4

3

II

1

30

3

4

38

36

43

60 Breast 51 60 Pancreas 72

7

1

+

42

40

6

32

-

37

-

33

5

Breast

59

2

1

V

4

63

72

Breast 58 Breast 65

3

2

IV

5

III

3

2

III

71

31

2

6

-

+

Lung Breast Ovary

50

4

Breast 60 Breast 60

Ovary

17

1

3

2

II

3

-

37

4

5

Breast

40

Fig. 7. Pedigrees of six families with the deleterious mutations identified. Filled symbols indicate individuals with invasive breast cancer (breast). Striped symbols indicate individuals with ovarian cancer (ovary). Grey symbols indicate individuals with cancer other than breast/ovary. The age at diagnosis of cancer is below each symbol. The age of death or the age registered at the time of pedigreee reconstruction is below symbols of healthy individuals if known. The arrow indicates the proband individual. Plus signs indicate individuals harboring the BRCA1/2 mutations. Minus signs indicate individuals without BRCA1/2 mutations. (A) PI 38 BRCA1 c.IVS11 þ 1GOA. (B) PI 413 BRCA2 c.IVS2 þ 1GOA. (C) PI 60 BRCA2 c.IVS13-2AOG. (D) PI 184 BRCA2 c.9345GOA. (E) PI 262 BRCA2 c.IVS21 þ 4AOG. (F) PI 267 BRCA2 c.7252_7272delinsTG.

of the BIC database, 6.2% of BRCA1 and 3.6% of BRCA2 mutations are splice site mutations. In the last few years, a number of studies have demonstrated the effect at the mRNA level of BRCA1 and BRCA2 splice site variants, such as BRCA2 c.8204GOA, which produces skipping of exon 17 [14], and BRCA1 c.IVS10-

2AOC, causing loss of exon 11 [15]. On the other hand, BRCA1 G1706A [16] and BRCA2 c.IVS25 þ 9AOC [17] represent rare variants, not disrupting normal splicing. For the present study, we selected 50 probands belonging to 50 unrelated Italian breast and ovarian cancer families, previously analyzed by PCR-DS for BRCA1/2 gene

F. Bonatti et al. / Cancer Genetics and Cytogenetics 170 (2006) 93e101

100

Table 2 BDGP splice prediction analysis: predicted variation in splicing efficiency in the normal sequence vs. variant sequences Predicted splicing efficiency, %

Sequencea Designation BRCA1, BRCA1, BRCA2, BRCA2, BRCA2, BRCA2, BRCA2, BRCA2,

c.IVS11 þ 1GOA c.IVS17 þ 6COG c.IVS2 þ 1GOA c.IVS13-2AOG c.IVS21 þ 4AOG c.9345GOA c.IVS1-9del3 c.IVS12-9del4

Normal

Variant

Normal

Variant

AACTTAGgtattgga AACAGgtataccaag AAAGCAGgtattgac ccattgcagCACAAC CCTTGAGgtgagaga ACTACCGgtacaaac ctgttttgcagACTTAT aattgtttcctagGCACA

AACTTAGatattgga AACAGgtatagcaag AAAGCAGatattgac ccattgcggCACAAC CCTTGAGgtgggaga ACTACCAgtacaaac ttttgcagACTTAT gtttcctagGCACA

45 92 81 56 98 57 0 64

0 92 0 0 0 0 0 64

Abbreviations: BDGP, Berkeley Drosophila Genome Project (software available at http://www.fruitfly.org/seq_tools/splice.html). a Notation: exons are indicated by capital letters; introns, lowercase. The canonic consensus is highlighted in sans serif bold face; the variants, sans serif italic.

status. Eight probands showed variants localized around the splice sites, one proband carried a genomic alteration of unknown pathological significance and the remaining 41 were negative for BRCA1/2 mutations. Aberrant RNA splicing was evidenced in five out of eight probands carrying genomic variants, and BDGP analysis was able to predict all as deleterious mutations for the loss of splice sites. Phenotypeegenotype correlation and cosegregation analysis established the absence of those mutations in healthy relatives (median age: 45 years). Our results show that BRCA2 c.9345 GOA, BRCA2 c.IVS2 þ 1GOA, BRCA2 c.IVS13-2AOG, BRCA2 c.IVS21 þ 4AOG, and BRCA2 c.7252_7272delinsTG variants produce frameshift similar to other previously described deleterious exonic variants. We therefore consider them to be true deleterious mutations, possibly associated with an increased lifetime risk of breast and ovarian cancer. BRCA1 c.IVS11 þ 1GOA leads only to the isoform D11q (2,287 bp, retention of 123 bp from exon 11). We note that the splicing efficiency predicted by BDGP for the donor site of intron 11 is low (45%). BRCA1 c.IVS11 þ 1GOA, BRCA2 c.IVS2 þ 1GOA, BRCA2 c.9345GOA, and BRCA2 c.IVS13-2AOG are reported in the BIC database as splice mutations [10e12]. BRCA2 c.IVS21 þ 4AOG is reported in the BIC database as a mutation of unknown pathological significance. In the present study, we found that the variant allele gave rise to an anomalous mRNA with a 46-bp insertion (due to intron retention) leading to a truncated protein for the presence of a premature stop codon at position c.IVS21 þ 9. BRCA2 c.7252_7272delinsTG in exon 14, not present in the BIC database, is a complex genomic rearrangement first detected by PCR-DS and then confirmed at the mRNA level; however, the presence of both BRCA2 alleles in tumors suggests the possibility of alternative mechanisms for disease progression. The remaining three variants were predicted by BDGP as likely to be neutral, and they showed no cDNA alterations or abnormal transcripts. BRCA1 c.IVS17 þ 6COG

had been reported on one occasion in BIC as variant of unknown pathological significance while BRCA2 c.IVS19del3 and BRCA2 c.IVS12-9del4 have not been reported in the BIC database. BRCA1/2 mRNA analysis revealed no cDNA alterations in any of the 41 negative probands, despite the high probability of their being carriers (predicted by the CaGene software). We also detected the presence of the major BRCA1 and BRCA2 isoforms previously described (Fig. 1A). In the present study, we characterized a group of variants identified by PCR-DS analysis, evidencing five novel mutations (1/276 5 0.36% in BRCA1 and 4/276 5 1.4% BRCA2) that represent 10% (5/50) of all mutations in our selected group of families. The mRNA analysis allowed to recognize families carrying BRCA1/2 alterations at a frequency higher than predicted by genomic analysis (17 and 9.8%, respectively). Notably, we observed a lower percentage of BRCA1 splicing mutations (1/47 5 2%) (6.2%) and a higher BRCA2 percentage (5/29 5 17.2%) in our cohort of samples, compared with 3.6% in the BIC database. References [1] Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Taytigian S, Liu O, Cochran C, Bennett LM, Ding W, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994;266:66e71. [2] Wooster R, Neuhausen SL, Mangion J, Quirk Y, Ford D, Collins N, Nguyen K, Seal S, Trant T, Averill D, et al. Localization of a breast cancer susceptibility gene, BRCA2, to chromosome 13q12-13. Science 1994;265:2088e90. [3] Tournier I, Bressac-de Paillerets B, Sobol H, Stoppa-Lyonnet D, Lidereau R, Barrois M, Mazoyer S, Coulet F, Hardouin A, Chompret A, Lortholary A, Chappuis P, Bourdon V, Bonadona V, Maugard C, Gilbert B, Nogues C, Frebourg T, Tosi M. Significant contribution of germline BRCA2 rearrangement in male breast cancer families. Cancer Res 2004;64:8143e7. [4] Hogervorst FBL, Nederlof PM, Gille JJP, McElgunn CJ, Grippeling M, Pruntel R, Regnerus R, Van Welsen T, Van Spaendonk R, Menko FH, Kluijt I, Dommering C, Verhoef S, Schouten JP, Van’t Veer LJ, Pals G. Large genomic deletions and

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