* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download OncJuly3 6..6
Epigenomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Metagenomics wikipedia , lookup
Genome evolution wikipedia , lookup
Non-coding DNA wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Pathogenomics wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
History of genetic engineering wikipedia , lookup
Genomic library wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Molecular Inversion Probe wikipedia , lookup
Genome (book) wikipedia , lookup
SNP genotyping wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Designer baby wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Genome editing wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Frameshift mutation wikipedia , lookup
Microevolution wikipedia , lookup
Microsatellite wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Helitron (biology) wikipedia , lookup
Point mutation wikipedia , lookup
ã Oncogene (1999) 18, 4160 ± 4165 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc SHORT REPORT Identi®cation of a 3 kb Alu-mediated BRCA1 gene rearrangement in two breast/ovarian cancer families Marco Montagna*,1, Maria Santacatterina1, Arianna Torri1, Chiara Menin1,2, Daniela Zullato1, Luigi Chieco-Bianchi1 and Emma D'Andrea1 1 Department of Oncology and Surgical Sciences, Oncology Section, Interuniversity Center for Research on Cancer, University of Padova, via Gattamelata, 64; I-35128 Padova, Italy; 2IST Biotechnology Section, Interuniversity Center for Research on Cancer, University of Padova, via Gattamelata, 64; I-35128 Padova, Italy Most of the hereditary breast cancers are attributed to constitutive alterations of either BRCA1 or BRCA2 genes; nonetheless, germline mutations of these genes in `high risk' families are found less frequently than expected from linkage data. Recent ®ndings suggest that major genomic rearrangements of the BRCA1 gene might account for at least some of the apparently mutation negative cases. We studied 60 aected probands belonging to families with a strong history of breast and/or ovarian cancer who scored negative for BRCA1 gene mutations by PTT and SSCP analysis. DNA was analysed by the Southern blotting procedure using three dierent restriction enzymes, and two probes obtained by RT ± PCR of the 5' and 3' BRCA1 coding sequence. A 3 kb deletion encompassing exon 17 and causing a frameshift mutation was identi®ed in two independently ascertained families. RT ± PCR and longrange DNA PCR were employed to characterize the rearrangement that was ®nally shown to be the result of a recombination between two very similar Alu repeats. This type of mutation is not identi®ed by the conventional methods of mutation detection which are based on PCR ampli®cation of single exons. Therefore, further search for gene rearrangements is needed to better de®ne the proportion of `high risk' families that might be explained by gross genomic alterations of the BRCA1 gene. Keywords: BRCA1; genomic rearrangement; breast cancer; ovarian cancer; Alu repeats About 5 ± 10% of all breast cancers are due to a strong genetic predisposition segregating as a highly penetrant autosomal dominant trait (Claus et al., 1991). Among the genes associated with the disease, BRCA1 and BRCA2 are thought to account for most of the families with multiple breast and/or ovarian cancers (Ford et al., 1998). Germline point mutations in these genes are scattered throughout their entire coding sequence, and mostly recur only in families sharing the same ancestry (BIC: The Breast Cancer Information Core); they *Correspondence: M Montagna Received 23 October 1998; revised 14 January 1999; accepted 16 February 1999 include nonsense mutations, small deletions and insertions causing frameshift mutations, missense mutations occurring at crucial aminoacid positions within well conserved domains, and mutations aecting the splice sites with loss of one or more exons in the transcript. The frequency of these types of mutations varies greatly depending on the racial or ethnic group, and, in general, is lower than expected from data based on linkage analysis (Szabo and King, 1997). In particular, in a recent report from the Breast Cancer Linkage Consortium it was estimated that only 63% of families linked to BRCA1 have a mutation detectable by the standard screening methods (Ford et al., 1998), thus suggesting that other types of gene inactivation might account for some of the predisposed kindreds. Based on the diminished or absent expression of one BRCA1 allele, an inhibition of BRCA1 transcription by `regulatory mutations' was advanced in some cases; to date, however, this kind of gene alteration has been rarely reported (Miki et al., 1994; Gayther et al., 1995; Serova et al., 1996; Xu et al, 1997). Major genomic rearrangements of the BRCA1 gene, which escape most of the current PCR-based mutation detection methods, are likely to account for at least some of the cases without apparent BRCA1 mutations (Petrij-Bosch et al., 1997; Swensen et al., 1997; Puget et al., 1997). This possibility is also supported by the high density of Alu repeats dispersed in the BRCA1 genomic sequence (Smith et al., 1996); indeed, Alu sequences can mediate major genomic rearrangements by promoting unequal crossing-over or other types of recombination. In this study we report the identi®cation of a 3 kb genomic deletion comprising exon 17 that caused a frameshift mutation leading to a premature stop codon in two independently ascertained breast/ovarian cancer families. Sixty Italian probands with breast and/or ovarian cancer and belonging to high risk families living in the Veneto Region were selected from a list of cases that scored negative for BRCA1 gene mutations as assessed by PTT of exon 11 and SSCP analysis of the remaining coding sequence (Montagna et al., 1996, and data not shown). Of the 60 families, 40 were site-speci®c breast cancer families, and 20 included both breast and ovarian cancer patients. Each family satis®ed at least one of the following selection criteria: (i) at least three breast and/or ovarian cancer cases with a ®rst-degree relationship; (ii) two cases of breast and/or ovarian BRCA1 gene rearrangements in Italian breast/ovarian cancer families M Montagna et al cancer with at least one of them aected before age 50; (iii) at least two ovarian cancer patients. A blood sample was obtained from each proband after written informed consent, and genomic DNA was analysed by Southern-blotting; each DNA sample was digested using HindIII, EcoRI and BamHI restriction enzymes, blotted and hybridized with probes pbr5' and pbr3' obtained by RT ± PCR of exons 2 ± 10 and 12 ± 24, respectively (both probes also include part of exon 11). The resulting restriction patterns were con®rmed on a restriction map of the BRCA1 genomic sequence derived from the L78833 GenBank sequence (Figure 1). Probe pbr5' was obtained by a nested RT ± PCR using previously reported primers (Friedman et al., 1994 and Hogervorst et al., 1995): c1F and 11AR for the ®rst PCR, BR2F2 and 11AiR for the second PCR. This probe disclosed an abnormal EcoRI restriction pattern in probands B96 and B37: however, a normal band pattern was obtained with both BamHI and HindIII. Further investigation of these two cases, using additional restriction enzymes as well as cDNA analysis, suggested the presence of an EcoRI restriction fragment length polymorphism (data not shown). On the contrary, probe pbr3' revealed aberrant bands in patient B74 after both HindIII and EcoRI DNA digestion (Figure 2); in particular, both restriction patterns were consistent with the loss of a *3 kb segment internal to the 19 kb HindIII fragment and removing an EcoRI site between exons 16 and 17 (Figure 1). The apparently normal restriction fragment pattern obtained with BamHI was likely due to the low resolution of the agarose matrix for high molecular weight fragments (*36 kb versus *33 kb). To investigate whether this putative rearrangement could aect the BRCA1 gene the BRCA1 transcript was analysed focusing on the region including exons 16 and 17, which are both located approximately within a 3 kb distance from the EcoRI site removed by the rearrangement. RT ± PCR of RNA from patient B74 was performed using primers located in exon 15 (forward primer) and exon 20 (reverse primer); in addition to the expected 737 bp band, a smaller and fainter band was observed in patient B74 and not in normal controls (Figure 3a). The size of the abnormal band was incompatible with the loss of the entire exon 16 which consists of 311 nucleotides, but was compatible with the loss of either exon 17, 18 or 19. A second PCR was therefore performed with a forward primer overlapping the junction between exons 16 and 17; in this case no abnormal bands were revealed, thus suggesting that the missing exon was number 17 (Figure 3b). The absence of exon 17 was ®nally demonstrated by sequence analysis of the cloned PCR-fragment corresponding to the mutant allele. This rearrangement caused a sequence frameshift in exon 18 leading to a premature stop codon at aminoacid position 1672, thus generating a truncated protein product. The intensity ratio between the wild type and the exon 17-deleted band in the RT ± PCR product of patient B74 clearly showed a lower expression of the Figure 1 Structure and restriction maps of the BRCA1 gene. Probes pbr5' and pbr3' and the corresponding exons are indicated at the top of the ®gure; HindIII, BamHI, and EcoRI restriction patterns are shown below the DNA exon-intron organization of the BRCA1 gene; numbers on the restriction maps refer to the size (in kb) of the restriction fragments involved in the rearrangement; arrows mark the location of the rearrangement breakpoints 4161 BRCA1 gene rearrangements in Italian breast/ovarian cancer families M Montagna et al 4162 Figure 2 Southern blot analysis of the DNA of six aected probands. Six mg of DNA derived from lymphoblastoid cells were digested overnight with EcoRI, BamHI, HindIII restriction enzymes, electrophoresed on 0.8% agarose gel, transferred onto Hybond-N+ nylon membranes (Amersham), and hybridized with the 32P-labeled pbr3' probe (exons 12 ± 24) obtained by a nested RT ± PCR using previously reported primers (Friedman et al., 1994; Hogervorst et al., 1995): primers 11PF and 24R for the ®rst PCR, and primers BR11F5 and BR24R2 for the nested PCR. Arrows indicate the aberrant bands caused by the rearrangement in patient B74. M, molecular weight markers mutant allele (Figure 3a). This allelic-imbalance is attributable to a mechanism known as nonsensemediated m-RNA decay, which drives the preferential degradation of transcripts in which premature translation termination has occurred (Jacobson and Pelts, 1996). To further characterize this rearrangement at the genomic level, we performed a long-range DNA PCR and ampli®ed the region from exons 16 ± 18 containing the breakpoint. A *7 kb fragment was obtained from both patient B74 and a normal control, while a *4 kb fragment, corresponding to the mutant allele, was ampli®ed selectively from patient B74, thus further con®rming the deletion of a *3 kb fragment (Figure 4a). The preferential ampli®cation of the 4 kb fragment in patient B74 is likely due to the dierent size of the mutant (4 kb) versus the wild type (7 kb) fragment. The breakpoint-containing region was then narrowed down by using dierent restriction enzymes; in this analysis, HincII was the most informative restriction enzyme, and mapped the deletion breakpoint to a 1000 bp HincII fragment (Figure 4b). Thus, a couple of PCR primers were designed to amplify this HincII fragment, which was then sequenced. A comparison with the L78833 GenBank sequence con®rmed a deletion of 3.2 kb, comprising exon 17. Moreover, the sequences adjacent to the breakpoint were disclosed to be part of two Alu repeats located in introns 16 (nt. 58 500 ± 58 798) and 17 (nt. 61 616 ± 61 918), and sharing an overall homology of 85% (Figure 5). This ®nding strongly suggested that the deletion was the result of a homologous recombination between two Alu sequences. Subsequent Southern blot analysis disclosed a restriction pattern in patient B58 that was identical to Figure 3 RT ± PCR analysis of patient B74 BRCA1 transcript. (a) RNA, isolated from lymphoblastoid cells using RNAzol (TelTest Inc.), was retrotranscribed with the avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) and PCR-ampli®ed with primers c8F and c10R (Friedman et al., 1994) located in exons 15 and 20, respectively; the arrow indicates the mutant allele in patient B74; (b), ampli®cation of the same samples using primer c10F, located over the junction between exons 16 and 17, and primer c10R BRCA1 gene rearrangements in Italian breast/ovarian cancer families M Montagna et al 4163 a b Figure 4 DNA ampli®cation and mapping of the breakpoint-containing region in patient B74. (a), long-range PCR was performed by amplifying the genomic sequence between exons 16 and 18 at the following conditions: after an initial denaturation (4 min at 958C) of the template DNA in PCR buer with primers 16F and 18R (Friedman et al., 1994), a combination of 5U of Taq DNA polymerase (Perkin Elmer) and 5U of Taq extender PCR additive (Stratagene) was added to the reaction; cycling conditions were as follows: 10 cycles at 948C ± 10 s, 648C ± 30 s, 688C ± 5 min followed by 22 additional cycles at 948C ± 10 s, 608C ± 30 s, 688C ± 5 min with an autoextension of 15 s/cycle, and a 688C ± 20 min ®nal extension. Arrows indicate the wild type (7 kb) and mutant (4 kb) alleles. C, normal control. M, molecular weight marker. (b) HincII restriction pattern of the mutant (B74) and wild type (C) alleles as shown in the scheme. The arrow indicates the HincII fragment containing the breakpoint. M, molecular weight marker BRCA1 gene rearrangements in Italian breast/ovarian cancer families M Montagna et al 4164 Figure 5 Comparison of the two Alu sequences involved in the rearrangement of patient B74. The HincII fragment containing the breakpoint was ampli®ed with primers 1/17F2 (5'-ggagaagccagaattgacag-3') and 1/17R2 (5'-ccagcaggtagataggagtt-3'), cloned with the `PCR TA cloning kit' (Invitrogene), and sequenced with the `PCR product sequencing kit' (Amersham). Sequence identity between the two Alu repeats is indicated by double dots; the rearranged sequence is shown in bold, the boxed sequence indicates the region where the breakpoint occurred a site-speci®c breast cancer family including seven breast cancers, whereas family B74 also presents two cases of ovarian cancer. This variability in phenotype suggests the presence of dierent modi®er factors in the two families. In this study we identi®ed a genomic rearrangement in two of 60 probands with undetectable BRCA1 mutations; this frequency might actually be higher as other rearrangements in genomic regions not well covered by the pbr5' and pbr3' probes and deletions of the entire BRCA1 locus might have been missed. The numerous Alu repeats present within the BRCA1 gene (Smith et al., 1996), and the recent observation of other genomic deletions (Petrij-Bosch et al., 1997; Swensen et al., 1997), one of which also includes exon 17 (Puget et al., 1997), suggest that (a) Alu-mediated recombination and recombination-like events constitute a further mechanism for the pathological inactivation of the BRCA1 gene, and (b) the genomic region surrounding exon 17 might be a site more prone than others to Alu-mediated rearrangements. The involvement of these types of mutations might have been underestimated to date, because most of the current mutation detection strategies are based on DNA ampli®cation of single exons. Our approach and/or long-range DNA PCR of the BRCA1 genomic sequence are apparently the best methods for identifying this type of mutation. This and further studies will help to complete the BRCA1 mutational spectrum, and explain at least some of the `high risk' families without apparent BRCA1 mutations. that obtained in patient B74, thus suggesting the occurrence of the same DNA rearrangement. PCR ampli®cation and sequence analysis of the HincII fragment from patient B58 con®rmed this hypothesis by showing an identical breakpoint. Furthermore, probands B74 and B58 were shown to share one allelic pattern over ®ve microsatellite markers (D17S1321, D17S855, D17S1322, D17S1323, D17S1327) spanning the BRCA1 gene (Neuhausen et al., 1996), thus suggesting a possible common ancestry for the two families (data not shown). It is noteworthy that the phenotypes in the two families are dierent despite the same mutation; B58 is Acknowledgements We thank the family members and the physicians (S Aversa, A Bononi, C Di Maggio, E Ferrazzi, A Fornasiero, V Fosser, O Nascimben, O Nicoletto, A Rosa-Bian, G Zavagno) who contributed to this study; M Quaggio for her expert technical assistance; P Gallo for artwork and P Segato for help in preparing the manuscript. M Montagna and M Santacatterina are fellows of the Associazione and Fondazione Italiana per la Ricerca sul Cancro (AIRC/FIRC), respectively. This study was supported by grants from Ministero della Ricerca Scienti®ca e Tecnologica (MURST), Italian Hereditary Breast/ Ovarian Cancer Consortium/AIRC-Coordinated Project, Regione Veneto/Ricerca Sanitaria Finalizzata, and Fondazione Cassa di Risparmio di Padova e Rovigo, Italy. References Claus EB, Risch N and Thompson WD. (1991). Am. J. Hum. Genet., 48, 232 ± 242. Ford D, Easton DF, Stratton M, Narod S, Goldgar D, Devilee P, Bishop DT, Weber B, Lenoir G, Chang-Claude J, Sobol H, Teare MD, Struewing J, Arason A, Scherneck S, Peto J, Rebbeck TR, Tonin P, Neuhausen S, Barkardottir R, Eyfjord J, Lynch H, Ponder BA, Gayther SA, Birth JM, Lindblom A, Stoppa-Lyonnet D, Bignon Y, Borg A, Hamann U, Haites N, Scott RJ, Maugard CM, Vasen H, Seitz S, Cannon-Albright LA, Scho®ld A, Zelada-Hedman M, and the Breast Cancer Linkage Consortium. (1998). Am. J. Hum. Genet., 62, 676 ± 689. Friedman LS, Ostermeyer EA, Szabo CI, Dowd P, Lynch ED, Rowel SE and King M-C. (1994). Nature Genet., 8, 399 ± 404. Gayther SA, Warren W, Mazoyer S, Russell PA, Harrington PA, Chiano M, Seal S, Hamoudi R, van Rensburg EJ, Dunning AM, Love R, Evans G, Easton D, Clayton D, Stratton MR and Ponder BAJ. (1995). Nature Genet., 11, 428 ± 433. Hogervorst FBL, Cornelis RS, Bout M, van Vliet M, Oosterwijk JC, Olmer R, Bakker B, Klijn JGM, Vasen HFA, Meijers-Heijboer H, Menko FH, Cornelisse CJ, den Dunnen JT, Devilee P and van Ommen G-JB. (1995). Nature Genet., 10, 208 ± 212. Jacobson A and Pelts SW. (1996). Annu. Rev. Biochem., 65, 693 ± 739. BRCA1 gene rearrangements in Italian breast/ovarian cancer families M Montagna et al Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, Bell R, Rosenthal J, Hussey C, Tran T, McClure M, Frye C, Hattier T, Phelps R, Haugen-Strano A, Katcher H, Yakumo K, Gholami Z, Shaer D, Stone S, Bayer S, Wray C, Bodgen R, Dayananth P, Ward J, Tonin P, Narod S, Bristow PK, Norris FH, Helvering L, Morrison P, Rosteck P, Lai M, Barrett JC, Lewis C, Neuhausen S, Cannon-Albright L, Goldgar D, Wiseman R, Kamb A and Skolnick MH. (1994). Science, 266, 66 ± 71. Montagna M, Santacatterina M, Corneo B, Menin C, Serova O, Lenoir GM, Chieco-Bianchi L and D'Andrea E. (1996). Cancer Res., 56, 5466 ± 5469. Neuhausen SL, Mazoyer S, Friedman L, Stratton M, Ot K, Caligo A, Tomlinson G, Cannon-Albright L, Bishop T, Kelsell D, Solomon E, Weber B, Couch F, Struewing J, Tonin P, Durocher F, Narod S, Skolnick MH, Lenoir G, Serova O, Ponder B, Stoppa-Lyonnet D, Easton D, King MC and Goldgar DE. (1996). Am. J. Hum. Genet., 58, 271 ± 280. Petrij-Bosch A, Peelen T, van Vliet M, van Eijk R, Olmer R, Drusedau M, Hogervorst FB, Hageman S, Arts PJ, Ligtenberg MJ, Meijers-Heijboer H, Klijn JG, Vasen HF, Cornelisse CJ, van't Veer LJ, Bakker E, van Ommen GJ and Devilee P. (1997). Nat. Genet., 17, 341 ± 345. Puget N, Torchard D, Serova-Sinilnikova OM, Lynch HT, Feuteun J, Lenoir GM and Mazoyer S. (1997). Cancer Res., 57, 828 ± 831. Serova O, Montagna M, Torchard D, Narod SA, Tonin P, Sylla B, Lynch HT, Feunteun J and Lenoir GM. (1996). Am. J. Hum. Genet., 58, 42 ± 51. Smith TM, Lee MK, Szabo CI, Jerome N, McEuen M, Taylor M, Hood L and King MC. (1996). Genomic Res., 6, 1029 ± 1049. Swensen J, Homan M, Skolnick MH and Neuhausen SL. (1997). Hum. Mol. Genet., 6, 1513 ± 1517. Szabo CI and King MC. (1997). Am. J. Hum. Genet., 60, 1013 ± 1020. The Breast Cancer Information Core on the internet (http : // www . nchgr.nih. gov / Intramural_research / Lab_ transfer/Bic/). Xu CF, Chambers JA, Nicolai H, Brown MA, Hujeirat Y, Mohammed S, Hodgson S, Kelsell DP, Spurr NK, Bishop DT and Solomon E. (1997). Genes Chromosomes Cancer, 18, 102 ± 110. 4165