Human Molecular Genetics, 2002, Vol. 11, No. 23 2805-2814
© 2002 Oxford University Press
The nonsense-mediated mRNA decay pathway triggers degradation of most BRCA1 mRNAs bearing premature termination codons
1Laboratoire de Génétique UMR 5641, Faculté de Médecine Rockefeller, 69373 Lyon cedex 08, France, 2International Agency for Research on Cancer, 69372 Lyon cedex 03, France, 3Laboratoire de Génétique, Hôpital Edouard Herriot, 69437 Lyon cedex 03, France and 4Service de Génétique Oncologique, Institut Curie, 75248 Paris cedex 05, France
Received March 28, 2002; Accepted September 10, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Germline mutations in the BRCA1 gene are scattered over the 22 coding exons and most of them generate premature termination codons (PTCs). A mechanism called nonsense-mediated mRNA decay (NMD) is known to specifically degrade transcripts with PTCs; however, steady-state amounts of mutant BRCA1 mRNAs have very rarely been measured. Although growing evidence implicates downstream exon–exon junctions (EEJs) as critical determinants for discrimination between normal stop codons and PTCs, requirements concerning the minimal and maximal distance between PTCs and downstream EEJs are still debated. We assessed the relative amount of transcripts encoded by BRCA1 alleles harbouring 30 different truncating mutations in lymphoblastoid cell lines established from carriers from breast/ovarian cancer families. We found that NMD is triggered by 80% of PTC+ alleles and results in a 1.5- to 5-fold reduction in mRNA abundance. All truncating mutations located in the 3.4 kb long central exon are subject to NMD, irrespective of their distance to the downstream EEJ (305 to 3395 nt). PTCs not leading to NMD are either located in the last exon or very close to the translation initiation codon. We hypothesize that reinitiation could explain why transcripts carrying early PTCs escape NMD. This is the first study challenging the NMD rules, which have been established through the study of minigenes, by analysing a large series of mutant endogenous alleles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mutations in the BRCA1 gene, cloned in 1994 (1), are responsible for 80% of breast and ovarian cancer families and for 28% of breast cancer only families with at least four cancer cases (2). For women with a germ line BRCA1 mutation, the cumulative estimated risk by age 70 years for breast cancer is between 45 and 84% and the corresponding ovarian risk is between 27 and 66% (3,4). More than 90% of the numerous different germ line BRCA1 alterations identified (>600) truncate the coding sequence by introducing premature termination codons (PTCs) through nonsense, frameshift, splice site mutations or genomic rearrangements (Breast cancer Information Core—BIC at http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/). The relative steady-state amounts of mutant mRNAs bearing a PTC have rarely been measured. However, it has been noted that, for some truncating mutations, mutant transcripts were present at a much lower rate than their wild-type counterpart in lymphoblastoid cell lines (5–11).
More than 20 years ago, it was discovered in yeast that mutations truncating coding sequences reduce the messenger level without lowering its instantaneous rate of synthesis (12). This finding has since been extended successively to all eukaryotes examined, even in plants. The mechanism by which transcripts containing a PTC (PTC+ transcripts) are detected and degraded within cells has been called ‘nonsense-mediated mRNA decay’ (NMD) (reviewed in refs 13–16). Because of NMD, PTC+ transcripts do not generally lead to the synthesis of truncated proteins, which could have a dominant negative effect. Several trans-acting factors involved in NMD have now been identified in yeast, Caenorhabditis elegans and humans (reviewed in ref. 13). Some of them assemble on the ribosome when a stop codon is recognized to form the so-called ‘mRNA surveillance complex’. One of the main issues in understanding NMD resided in elucidating how cells discriminate between normal termination codons and PTCs. In vertebrates, it has been shown that the presence of an exon–exon junction (EEJ) located at least 50–55 nt downstream a stop codon distinguishes PTCs from normal stop codons (located within the last exon in 93% of all eukaryote genes examined). The mechanism by which EEJs are detected likely involves recognition by the surveillance machinery of a multi-protein complex (the ‘exon–exon junction complex’ or EJC) specifically deposited 20–24 nucleotides upstream of each junction during mRNA splicing (17–23). The encounter between the surveillance complex and an EJC triggers accelerated decay of the PTC+ mRNA and it has recently been suggested that this takes place after a pioneer round of mRNA translation (24). In yeast, where introns are present only within a few genes, stop codons are recognized as premature due to sequences defined as downstream sequence elements (DSEs) (25,26). These sequences promote interaction with at least one protein, marking the mRNA as aberrant when located 3' of a stop codon (27).
We aimed to determine to what extent BRCA1 truncating mutations are subject to NMD. The BRCA1 gene contains 22 coding exons, among which exon 11 encodes more than 60% of the 5.6 kb long ORF. As a result, 
60% of all truncating mutations are located within exon 11 and we thus wondered whether the absence of a close 3' EEJ would prevent NMD of the corresponding PTC+ mRNAs. This question seemed important to address not only to better understand how the NMD pathway functions but also to estimate how sensitive BRCA1 cDNA screening strategies are when NMD is not impaired, which is more than often the case. We also aimed to question the validity of the biological hypotheses underlying the genotype/phenotype correlations drawn for BRCA1 truncating mutations, mainly based on the analysis of mutation position and the assumption that truncated proteins could display differences in their residual function (28–30).
We have measured the amount of transcripts encoded by BRCA1 alleles harbouring 32 different mutations scattered all over the coding sequence in lymphoblastoid cell lines established from carriers from breast and/or ovarian cancer families. Thirty mutations truncate the coding sequence, one is a missense mutation and the last one leads to an in-frame exon skipping. We show here that most truncating mutations are associated with significantly reduced transcript amounts, while transcripts synthesized from alleles maintaining the reading frame are expressed at a steady level.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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PTCs corresponding to the 30 truncating mutations analysed in this study are scattered along the whole BRCA1 coding sequence (Table 1 and Fig. 1). Quantitation of both allele-specific transcripts was obtained by fluorescence measurement of their relative amount following electrophoretic separation of RT–PCR fragments based on size difference. This size difference was either generated directly by the mutation (insertion or deletion of at least 10 bp) (12 mutations, 16 families), or after digestion by a restriction enzyme whose cutting site is destroyed or created either by the mutation (5 mutations, 5 families) or by a sequence polymorphism (15 mutations, 19 families).
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Factors known to affect the reliability of PCR-based quantitation (quality and quantity of template, primer pair amplification efficiency) are not expected to influence our assay because we compare the amount of allele-specific transcripts within the same sample by using a single pair of primers. Whenever a polymorphism was used to discriminate between the two alleles, we tried to conduct a co-segregation study using several relatives to identify the mutation-carrying allele for each family. This was possible for only 8 families out of 19 because of limited access to family members and successful for 5 of them (4 mutations) because of lack of informativity in the remaining ones.
We first checked whether the two copies of the BRCA1 gene are expressed at the same level in the absence of mutations in lymphoblastoid cell lines. We measured the relative amount of each BRCA1 allele transcript for six non-carrier individuals heterozygous for the K1183R polymorphism. The ratio between the two alleles ranged from 48.5:51.5±2.3% to 53.1:46.9±3.8%, with a mean value of 51.4:48.6±3.5%, and is therefore very close to the expected ratio of 50:50.
The relative amounts of the two BRCA1 transcript species were measured in lymphoblastoid cell lines bearing one wild-type and one mutant allele (Fig. 2). When the mutation-carrying allele was known, the amount of the mutant transcript was expressed as a percentage, normalized to the wild-type one. Otherwise, we normalized the percentage of the less abundant transcript with the amount of the more abundant one. Among the 30 truncating mutations, 24 lead to a 30–82% decrease in the rate of transcripts synthesized from one of the two BRCA1 alleles. In 16 cases, we were able to identify which transcript bears the PTC: it was always the one present in reduced amount. Statistical analysis of these data showed that there is a significant difference between wild-type versus mutant BRCA1 mRNAs ratios (P=0.004) if we compare patients carrying PTC+ mutations to patients carrying PTC- mutations and controls.
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Remarkably, six truncating mutations had no significant consequence on transcript steady-state level (Fig. 2). Among these are the two most 5' (188del11–36ter; 185delAG–39ter) and the three most 3' (5382insC–1829ter; Arg1835ter; 5677insA–1853ter) truncating mutations tested. 188del11 and 185delAG change the frame in the same way and the resulting truncated open reading frame of 36 and 39 codons respectively use the same stop codon in exon 3. The PTCs generated by 5382insC, Arg1835ter and 5677insA are all located in the last exon (exon 24). The sixth truncating mutation not associated with reduced amount is the nonsense one located in the in-frame exon 18, Glu1694ter, which causes inappropriate skipping of the entire exon both in vivo (31) and in vitro (32). Therefore, the mutant mRNA does not contain the nonsense mutation.
Among the five truncating mutations for which transcript steady-state level could be assessed in more than one lymphoblastoid cell line established from independent individuals, results were homogeneous: 185delAG–39ter and 5382insC–1829ter never lead to reduced level, while 3599del11–1163ter and ins6kbEx13–1460ter always do. The missense mutation and the in-frame deletion do not mediate decay (Fig. 2), as expected and as described in the literature (33).
As NMD requires translation, translational inhibition within cells should specifically stabilize PTC+ transcripts subject to NMD without stabilizing wild-type, PTC- or PTC+ transcripts insensitive to NMD. In order to test this, lymphoblastoid cell lines established from 12 individuals bearing either a truncating mutation (185delAG–39ter; 795delT–233ter; 1623del5–503ter; Gln563ter; 3599del11–1163ter; ins6kbEx13–1460ter; del1kbEx17–1672ter; Glu1694ter; Arg1835ter) or a missense mutation (Cys64Gly) were grown in culture medium containing a translation inhibitor, puromycin, for 6 hours. The results are shown in Figure 3. As expected, addition of puromycin led to a marked rise in the steady-state level of transcript carrying the 233ter, 503ter, 563ter, 1163ter, 1460ter and 1672ter codons, demonstrating that degradation of these transcripts depends on translation. Conversely, we observed little variation in the amount of transcripts carrying Cys64Gly. These characteristics imply that NMD is likely to be responsible for the allele-specific reduction in levels of mRNA seen for most BRCA1 truncating mutations. Little variation was observed for mRNAs carrying 39ter, 1694ter or 1835ter, therefore confirming that these transcripts are not subject to NMD.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The NMD pathway has remained obscure for many years, with very little and often conflicting data generated. Nevertheless, two models have been proposed recently, which both acknowledge the implication of introns as critical determinants of PTC recognition, but differ in the way this recognition takes place. In the scanning model, after termination has occurred at a PTC, the surveillance complex encounters a mark, very likely an EJC (exon–exon junction complex), by scanning the mRNA in the 3' direction (34–36). In the termination/mRNP context model, the improper context of the termination event leads to inadequate remodelling of the mRNP and transcript destabilization (37).
A growing number of proteins marking downstream EEJs have been identified since 2000 (17–23), which strengthens the evidence for a link between splicing and NMD. However, discrepancies remain about the cellular localization of NMD and PTC position regarding EEJs. Noteworthy, most of the established NMD rules are based on studies of a limited number of PTCs in the ß-globin (35,38,39), the T-cell receptor-ß (TCR-ß) (34), the triosephosphate isomerase (TPI) (36,40,41) and the glutathione peroxidase 1 (GP1) (42) genes, which all contain a small number of average-sized exons. Therefore, these rules had to be challenged with further analyses of truncating mutations in other human genes with a more complex structure. Furthermore, it seemed important to analyse and compare mutant endogenous alleles to confirm or invalidate results obtained with transfected minigenes into which exons and introns were moved and PTCs were introduced by site-directed mutagenesis.
The BRCA1 gene was the ideal candidate, with 22 coding exons whose length extends from 41–3426 nt and more than 600 truncating mutations identified along the whole coding sequence. In this study, we have analysed BRCA1 transcript steady-state levels in 38 individuals bearing 30 different truncating mutations. We have shown that NMD is triggered by 24 PTC+ alleles (80%) and results in a 1.5- to 5-fold reduction in mRNA abundance. This level of reduction is within the range of what has been observed for the truncating mutations of APC in lymphoblastoid cell lines (2- to 3-fold) (43), of the LDL receptor gene in cultured fibroblast (1.5- to 3-fold) (44) or of the GPx1 mRNA in the absence of selenium in L and CHO cells (2- to 3-fold) (42). This level of reduction is also comparable to the reduction observed for the TPI (45) and the ß-globin (46) PTC+ mRNAs produced by transient transfection. On the other hand, down-regulation appeared much stronger for transfected TCR-ß transcripts (
30-fold), which commonly harbour PTCs as a result of programmed DNA rearrangement in T lymphocytes (34). It has been shown very recently that such a strong down-regulation was mediated by a down-regulating-promoting element that acts upstream of nonsense codons (47). Furthermore, the same region also confers a boundary-independent polar regulation to TCR-ß, such that a PTC located 196 nt 5' from the terminal downstream intron triggered robust NMD (50-fold), while intermediate position (nt -91 to -142) triggered less of an mRNA decrease (8- to 21-fold) and proximal PTCs (nt -16 to -31) caused modest NMD (2- to 4-fold) (48). This situation differs from what has been observed for other transcripts for which no polar effect has been reported and a minimal distance of 50–55 nt between the PTC and a downstream EEJ is required for NMD. The rationale for the need of a minimal distance could reside within the fact that the EJC has been shown to be deposited 20–24 nt upstream of EEJs and comprises to date at least 5 proteins in the cytoplasm (hUpf2, hUpf3, Y14, RNPS1, Magoh), which suggests that this complex is displaced by the translation machinery when it is too close to the PTC (13). It seems likely that TCR genes may have evolved a unique NMD pathway in response to frequent PTC acquisition during normal development (47,48).
In yeast, DSEs must be located within 150 nt downstream of the stop codon to be functional (49). In mammals, little is known about the existence of an upper limit for the distance between PTC and the closest 3' EEJ. By evaluating for the first time the steady-state level of a series of 14 transcripts carrying PTCs in a very large central exon, we were able to address this issue and found that all these transcripts are subject to NMD, irrespective of their distance from the exon 11–exon 12 junction (305–3395 nt). Previous data showed that a PTC located in the TPI gene more than 559 nt 5' of the following EEJ escaped NMD (36). One interpretation of this result is that the surveillance complex fails to scan more than 550 nt because it dissociates from the mRNA. However, our results are in agreement with those recently obtained for the ß-globin and the HSP70 genes, where PTCs located 654 nt upstream of an EEJ in the case of the former (39), 678 or 1203 nt in the case of the latter (50), lead to NMD. It implies that there may be no superior limit, which could be compatible with both models: the termination/mRNP context model does not include any upper limit restriction (37) and in the case of the scanning model, the surveillance complex may remain associated to the mRNA until encountering the first downstream EEJ or until the 3' end of the mRNA is reached. Alternatively, we can also imagine that some DSE-like regulatory sequences exist in human genes, as it has already been suggested (38,51) and could act as ‘failsafe marks’ when the distance between the PTC and the downstream EJC is too large. As such sequences have been excluded in a few cases (36,39,50,52), their presence might be restricted to a limited set of genes. We are conducting further studies in order to discriminate between these hypotheses.
Nonsense-mediated alterations in splicing (NAS) is another mechanism by which nonsense mutations can affect transcripts fate (53,54). One of the nonsense mutations included in this analysis, Glu1694ter, causes inappropriate skipping of the entire constitutive exon 18 into which it is located, both in vivo (31) and in vitro (32). It has been shown that skipping of exon 18 results from disruption of a splicing enhancer in the coding sequence (32). We show here that the mutant transcript, which retains the reading frame, is not subject to NMD, as expected.
Among the five other truncating mutations that do not lead to NMD, three introduce a PTC in the last exon, exon 24. These data therefore confirm the implication of downstream EEJs as critical determinants of PTC recognition. The other two mutations not leading to NMD are the two most 5' (188del11–36ter; 185delAG–39ter) truncating mutations tested that introduce the same PTC, producing ORFs of 36 and 39 codons, respectively. Studies have shown that, when the first AUG codon is shortly followed by an in-frame termination codon, post-termination ribosomes apparently resume scanning and may reinitiate translation at a downstream site (55). For these two BRCA1 mutations, an in-frame AUG that resides in a favourable context follows 50 nt after the PTC. As the analysis of the human TPI transcripts suggests that reinitiation downstream of a PTC will protect the transcript from NMD (56), we believe that translation reinitiation is likely to explain the failure of 185delAG and 188del11 to trigger NMD. If our hypothesis is correct, reinitiation could lead to the production of an N-terminally truncated BRCA1 protein missing the first 47 amino acids i.e. the RING-finger domain, providing that this truncated protein is stable. The closest downstream PTC analysed, 81ter, generated by the 332–11G>T mutation, is associated with reduced steady-state transcript level. Whether, in this case, the reading-frame is too long or the next in-frame ATG too far away (168 nt after the PTC) or inefficient to allow reinitiation is not known. It should be noted that in the case of the TPI transcript, translation reinitiation abrogates NMD when nonsense codons are located at position 1, 2 or 10 but not at position 23 (56).
Our results question the casual assumption that a PTC-containing allele encodes a truncated protein. Among the 30 truncating mutations tested, only six do not lead to reduced amount of transcript and could therefore potentially generate high level of truncated proteins: 185delAG, 188del11, Glu1694ter, 5382insC, Arg1835ter and 5677insA. Furthermore, the structure of the abnormal product could be inferred from the position of the PTC only in the case of these last three mutations. Current attempts to draw phenotype/genotype correlations to try to estimate whether the position of truncating mutations confers different risks of breast and ovarian cancers, on the basis of differential functional properties of protein domains (28–30). It has been found recently that the breast cancer risk associated with mutations in the central region (nucleotides 2401–4190) was significantly lower than for other mutations, whereas the ovarian cancer risk associated with mutations 3' to nucleotide 4191 was significantly reduced relative to the rest of the gene (28). Quantitation of mutant and wild-type proteins would be of importance in order to try to resolve the biological mechanism underlying this phenotype/genotype correlation. It should be noted that the two most common BRCA1 mutations, 185delAG and 5382insC, which are found in 0.8% and 0.4%, respectively, of the general population of Ashkenazi Jews (57), 5382insC also being the most frequently observed BRCA1 mutation in non-Jewish populations, are both among the very few mutations likely to give rise to high levels of truncated proteins.
In the same way, the functional activities of BRCA1 truncated proteins deduced from the experimental expression of PTC-containing cDNAs in transfected cells (58) should be considered with caution. Indeed, the use of constructs without introns sidesteps NMD and forces the cell to synthetize proteins that would not have been produced or only in limited amount otherwise.
We also show that success of RNA-based mutation detection methods applied to BRCA1 can be severely compromised by NMD when this mechanism is overlooked in mutation screening strategies. It therefore appears essential to inhibit NMD in samples by blocking protein synthesis before RNA extraction in order to stabilize PTC+ transcripts and to ensure efficient detection.
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MATERIALS AND METHODS |
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Families
The 40 families included in this study (Table 1) have been selected from different genetic counselling programs for breast and/or ovarian cancer out of an original set of 58 families: 29 families have been collected by Dr H.T. Lynch at the Creighton University School of Medicine (Omaha, NE), 8 by Dr D. Stoppa-Lyonnet at the Institut Curie (Paris, France), 2 by Dr C. Lasset at the Centre Léon Bérard (Lyon, France) and 1 by Dr C. Blanchet-Bardon at the Institut Gustave Roussy (Paris, France). They present an average of 5 breast cancer cases (4 diagnosed before 45 years old) and 2 ovarian cancer cases.
Thirty truncating mutations, 1 missense mutation and 1 in-frame exon deletion were identified in the BRCA1 gene in these families (Table 1) by direct sequencing, HDA, PTT, DGGE, cDNA analysis or Southern Blotting (8–11,31,59,60). Each mutation was further checked for this study by sequencing genomic or complementary DNA to avoid erroneous PTC location calculation as a result of errors in mutation nomenclature. The PTCs are scattered all over the BRCA1 coding sequence, as shown in Figure 1. One carrier per family was used for the analysis.
Six control individuals (i.e. non-carriers), belonging to two of the 40 families included in the study (F2770 and F2651a), have also been analysed in order to compare mRNA expression levels of the two wild-type BRCA1 alleles transcribed in lymphoblastoid cell lines.
Cell culture
Human lymphoblastoid cells lines were established by Epstein-Barr virus immortalization of patients' blood lymphocytes. Cells were maintained in RPMI 1640 medium (Sigma, Saint Quentin Fallavier, France) supplemented with 10% foetal calf serum and 1% penicillin-streptomycin (Sigma) in a 5% CO2 incubator at 37°C. For translation inhibition, lymphoblastoid cells were treated with a fresh puromycin solution (Sigma) at the final concentration of 100 µg per ml of culture for 6 hours (61) and harvested for genomic DNA (gDNA) or total mRNA isolation.
gDNA and mRNA extractions—RT–PCR amplification
gDNA and total mRNA were respectively isolated from 2x107 and 5x106 frozen cells using QIAamp DNA and RNeasy minikits (Qiagen, Courtaboeuf, France), according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized with 0.5–1 µg of total RNA and Moloney murine leukemia virus-reverse transcriptase (Roche, Meylan, France). cDNAs were amplified by PCR using the Platinium Taq DNA Polymerase (Gibco BRL, Cergy Pontoise, France) and specific BRCA1 primers (sequence available upon request), using a GenAmp PCR system 9600 thermal cycler (Perkin Elmer, Foster City, CA, USA) for 35 cycles for polymorphism screening on gDNA and 28 cycles for mutant versus wild-type transcript ratio measurements on cDNA.
Polymorphism screening
In order to be able to discriminate between the two allelic BRCA1 transcripts produced in every cell line we took advantage, when the mutation itself could not be used, of polymorphisms located in the coding sequence, which modify a restriction site ((62) and Table 2). The search for individuals heterozygous for such polymorphisms was done by endonuclease digestion of gDNA in 36 families carrying a BRCA1 mutation. Only nineteen families were found to have heterozygote members and the remaining 17 families were therefore not included in this study.
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Estimation of allelic transcripts level by a RT–PCR based method
PCR fragments generated from wild-type and mutant BRCA1 transcripts were discriminated directly by electrophoresis migration when the mutation induces a length difference superior to 10 bp between wild-type and mutant transcripts or using various restriction enzymes otherwise (Table 2), for mutations and polymorphisms modifying a restriction site. By scanning the different signals, the relative amount of allelic transcripts could be determined.
Prior to quantitation, cDNAs were first tested for the presence of contaminating gDNA, which could skew the ratio of mutated versus wild-type transcripts when using primer pairs located within a single exon (i.e. within exon 11). Contaminated cDNA were either discarded or purified from gDNA. After a 28 cycle cDNA amplification, PCR products were digested when needed with restriction enzymes (New England Biolabs, Beverly, MA, USA) directly or after purification with the Wizard PCR preps DNA purification (Promega, Charbonnières les Bains, France) or MiniElute (Qiagen) kits if strict buffer conditions were necessary. PCR (for direct visualization) or digestion products were electrophoresed in an 8 to 15% polyacrylamide gel (depending on the needed range of separation). Bands were visualized under a 520 nm UV light using the Fluor-S (Biorad) after DNA fixation by a 10% ethanol/0.5% acetic acid solution for 10 minutes and SYBR green staining (Sigma). Quantitative analysis was performed using the Quantity One software (Biorad, Marnes-la-Coquette, France). The intensity of each band being proportional to its size, intensity/size ratios were taken into account. Each experiment was repeated at least three times independently.
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ACKNOWLEDGEMENTS |
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We thank the family members who collaborated in this study. We also thank H.T. Lynch, for a long-term collaboration in the study of these families, C. Bonnardel and S. Pagès for their expert assistance and Marc Billaud, Monique Buisson, David E. Goldgar, Hervé Le Hir and Ivan Mikaélian for their support, their encouragement and their critical reading of the manuscript. This work was supported by program grants from le Comité Départemental du Rhône de la Ligue contre le Cancer and by the Institut Curie ‘Programme Incitatif et Coopératif: Génétique et Biologie des Cancers du Sein’. L.P.-V. is supported by a fellowship from the Ligue Nationale contre le Cancer.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +33 478777587; Fax: +33 478777220; Email: smazoyer{at}rockefeller.univ-lyon1.fr
Present address: Institut Gustave Roussy, Villejuif, France.
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