Estelle Lopez

Victoria Viart

Caroline Guittard

Carine Templin

Céline René

Déborah Méchin

Marie Des Georges

Mireille Claustres

Marie-Catherine Romey-Chatelain

Magali Taulan
email: magali.taulan@inserm.fr

Marie Des

Marie-Catherine Romey- Chatelain

Variants in CFTR untranslated regions are associated with Congenital Bilateral Absence of the Vas Deferens

Keywords: CBAVD, CFTR transcriptional regulation, microsatellite, unknown variant

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Congenital bilateral absence of the vas deferens (CBAVD), an autosomal recessive disorder, is a frequent cause of obstructive azoospermia and accounts for approximately 2-3% of male infertility in Caucasian populations. In 90% of cases, CBAVD is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. 1 2 To date, more than 1,600 genetic changes have been identified in this gene. A combination of severe mutations on both alleles of a patient usually leads to severe forms of classical Cystic Fibrosis (CF). 3 4 Other mutations that retain residual CFTR function (above 10%), result in mild CF or incomplete phenotypes. CBAVD patients carry two mild mutations or are compound heterozygous for a severe and a mild alterations of the CFTR gene. 1 5 Despite extensive testing for point mutations and large rearrangements [START_REF] Audrezet | Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms[END_REF][START_REF] Hantash | Characterization of a recurrent novel large duplication in the cystic fibrosis transmembrane conductance regulator gene[END_REF][START_REF] Taulan | Large genomic rearrangements in the CFTR gene contribute to CBAVD[END_REF][START_REF] Taulan | A novel double deletion underscores the importance of characterizing end points of the CFTR large rearrangements[END_REF] , a small proportion of alleles still remains unidentified in CBAVD patients. 1 10 Interestingly, we have detected two novel rare (TAAA) n repeats in the IVS9 of the CFTR gene in two CBAVD patients, heterozygous for a variation in the minimal promoter (-33G>A) [START_REF] Romey | First putative sequence alterations in the minimal CFTR promoter region[END_REF] or for the common CBAVD variant (TG12T5).

Microsatellites, also known as Short Tandem Repeats (STRs), are distributed throughout the genome [START_REF] Li | Microsatellites within genes: structure, function, and evolution[END_REF] . Since the last decade, scientists suggested that alterations in the length of simple DNA repeat sequences might be responsible for diseases, such as myotonic dystrophy type 2 [START_REF] Ranum | RNA-Mediated Neuromuscular Disorders[END_REF] and gynecomastia. [START_REF] Czajka-Oraniec | Association between gynecomastia and aromatase (CYP19) polymorphisms[END_REF] Accumulating data evidenced that microsatellite variations including tetranucleotides affect regulation of gene expression by inhibiting the transcription of these genes implicated in diseases 15 16 or by altering RNA processing [START_REF] Hedjran | Control of alternative pre-mRNA splicing by distributed pentameric repeats[END_REF] for STRs detected close to some intron/exon boundaries.

Several microsatellites have been identified in non-coding regions of the CFTR gene. [START_REF] Chehab | A dimorphic 4bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the delta F508 mutation: implications for prenatal diagnosis and mutation origin[END_REF][START_REF] Xu | Human CFTR gene sequences in regions flanking exon 10: a simple repeat sequence polymorphism in intron 9[END_REF][START_REF] Elce | Three novel CFTR polymorphic repeats improve segregation analysis for cystic fibrosis[END_REF] Whereas most are dinucleotides, two tetranucleotides (TAAA) n and (GATT) n repeats have been reported in intronic regions. [START_REF] Chehab | A dimorphic 4bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the delta F508 mutation: implications for prenatal diagnosis and mutation origin[END_REF] [START_REF] Xu | Human CFTR gene sequences in regions flanking exon 10: a simple repeat sequence polymorphism in intron 9[END_REF] Except for the (TG)mTn polymorphism tracts in the CFTR IVS8, known to dramatically alter the CFTR exon 9 skipping 18 21-25 , the role of repeats in the regulation of CFTR function and expression remains poorly defined.

The aim of this study was to evaluate whether the uncommon -33G>A promoter variant and the (TAAA) 6 and (TAAA) 8 tetranucleotide repeat alterations located deeply within intron 9 may contribute to the CBAVD phenotype. We were also interested in understanding the molecular mechanism by investigating the potential regulatory function of Forkhead box (FOX) transcription factors in the presence of these variants.

Material and methods

Haplotyping IVS9(TAAA)n repeats.

PCR reactions were carried out on 100 ng of human DNA (see primers in Supplementary Table 1). Aliquots of the amplicons [(TAAA)n units] were separated by multicapillary electrophoresis on an ABI 3130xl Genetic Analyzer running GeneMapper v4.0 for allele identification as previously described. [START_REF] Bareil | Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders[END_REF] Expression plasmids, gene reporter vectors and hybrid minigene constructs.

To perform gene reporter assays, the pGL3B-33A plasmid was generated from pGL3B-WT, pGL3-Basic reporter vector containing the wild-type human CFTR minimal promoter [START_REF] Romey | A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression[END_REF] For hybrid minigene assays, constructs containing the (TAAA) n repeats of the CFTR intron 9 were created as depicted in figure 3. The human CFTR genomic regions encompassing the exons 9 and 10 with their flanking intronic sequences were amplified in two separated PCRs.

The PCR products were subcloned into the XhoI/NheI digested pSPL3 plasmid, kindly gift by Dr. S. Tuffery-Giraud (Laboratory of Molecular Genetics, Montpellier, France). As positive controls, minigenes containing either a T5 or a T3 allele [START_REF] Disset | A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD)[END_REF] were generated by directed mutagenesis (Stratagene).

To prepare the gene reporter vectors pGL3C-(TAAA) n , a 1222-base pair fragment spanning the CFTR intron 9 region was generated by PCR from genomic DNA of individuals with (TAAA) 9 , (TAAA) 8 or (TAAA) 6 repeat. The PCR products were restricted with NheI and BglII endonucleases and then ligated into the pGL3-Control reporter vector (Promega) upstream the SV40 promoter.

To evaluate CFTR mRNA levels, expression plasmids containing either the wild-type or the mutated CFTR minimal promoter upstream the CFTR cDNA (pcDNA3.1-WT and pcDNA3.1-33A, respectively) were constructed. The full-length human CFTR cDNA was excised from the pTG5985 vector, generously given by Transgene SA, with ApaI/XhoI and ligated into the pcDNA3.1(-) expression vector. To drive the expression of the CFTR cDNA, the SV-40 promoter was replaced by the CFTR minimal promoter. For the (TAAA) n repeat, the (TAAA) n intron 9 region is inserted upstream the CFTR minimal promoter in the pcDNA3.1(-) expression vector containing the CFTR cDNA. All the primers are listed in Supplementary Table 1. All the constructs were verified by direct sequencing.

Cell culture and transient transfections.

HVP (SV40 ori-transformed epithelial vas deferens) cells, maintained in CMRL-1066 medium (Gibco, Invitrogen) and kindly provided by Dr. A. Harris (Weatherall Institute of Molecular Medecine, Oxford University, UK). HeLa (human cervical carcinoma) cells were cultured as previously reported. [START_REF] Taulan | First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding[END_REF] For transient transfections, cells, seeded at a density of about 10,000 cells/100 µl of medium in 96-well plates, were transfected with 60 ng of indicated reporter vector and 6 ng of internal control pRL-SV40 containing Renilla luciferase (Promega), to normalize for transfection efficiency, by using Fugene®6 transfection reagent (Roche). In transient co-transfection experiments, 4 ng of either pcDNA3(-) or pcDNA3-FOXI1 vector were also transfected. Similar transfections were performed in 6-well plates for protein extracts. For siRNA assays, transfections of cells with 40 nM FOXI1-targeting siRNAs (Santa Cruz, Clinisciences) were performed using Interferin reagent (PolyPlus, Ozyme). Non-targeting siRNA purchased from Santa Cruz (Clinisciences), served as a negative control. Dual Glo Luciferase Assay System (Promega) was used according to the manufacturer's recommendations.

To analyse the hybrid minigene expressions, 1-1.5x10 6 cells were platted into Nunclon® 35 x 10 mm cell culture dishes. After 24 hours, cells were transiently transfected with 6 μg of either pSPL3 empty vector or one minigene plasmid using Polyfect® transfection reagent (Qiagen). For the CFTR mRNA level analyses, 6 μg of the indicated pcDNA3.1 expression vectors alone or with 2.5 μg of FOXI1 expression vector or 40 nM of FOXI1-targeting siRNA were transfected. After 48 hours, the cells were lysed for RNA extraction. All hybrid minigene, expression vector and gene reporter data represented the result of at least three independent experiments performed in triplicates with independently purified plasmid DNA preparations.

Establishment of stable cell lines.

For chromatin immunoprecipitation assays, HVP cells were seeded at a density of about 300,000 cells in 35-mm culture dishes and were co-transfected with 80 ng of pCI-neo vector and 2 μg of indicated reporter vector by Polyfect® transfection reagent. Stably transfected clones were selected in medium containing G418 antibiotic (Promega). Clones of G418resistant cells were screened by direct sequencing.

Reverse-transcriptase PCR and RT-qPCR.

Total RNAs were extracted by using the RNeasy Plus Mini kit (Qiagen). RT were performed as previously described [START_REF] Rene | Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway[END_REF] , and cDNAs were amplified with either SD6/SA2/β-actin primers (hybrid minigene) or CFTR/GAPDH (expression vector), by using classical PCR or the LightCycler® 480 Real-Time PCR System (Roche), respectively. All PCR reactions were performed in triplicate in three independent experiments.

Chromatin Immunoprecipitation assays (ChIP)

ChIP assays were performed according to protocols previously described with minor modifications. [START_REF] Rene | Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway[END_REF] Prior to harvest, stably transfected HVP cells were cross-linked with formaldehyde. Purified crosslinked chromatin was immunoprecipitated using 3µg of either anti-FOXI1 antibody (AbCam) or anti-HA antibody (Roche), used to control for non-specific binding of DNA. Input and immunoprecipitated DNAs were analyzed using either primers specific of the CFTR minimal promoter or primers amplifying the CFTR intron 9 region containing the (TAAA) n repeat (Supplementary Table 1), with the LightCycler® 480 Real-Time PCR System (Roche). All PCR reactions were performed in triplicate. Experiments are repeated at least three times, averaged and expressed relative to the input signal and to negative control.

Immunoblot analyses

Total whole-cell proteins from transfected HVP cells were analyzed by western blot as previously described. [START_REF] Rene | Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway[END_REF] The membranes were incubated overnight with 1:1,000 diluted anti-FOXI1 (Ab-Cam) primary antibody in 5% skim milk. The protein levels of the β-actin housekeeping gene were assayed for internal control of protein loading.

Statistical analyses.

Data are expressed as the mean ± SE. Paired comparisons were made using student's t-test using InStat (GraphPad Software, version 3.0). Data were considered statistically significant at p<0.05. Relative intensities of equal areas were compared using Quantity One® quantification analysis software (Bio-Rad).

Results

The -33G>A transition in the CFTR minimal promoter leads to a decreased promoter activity

We first investigated the putative effect of a rare promoter substitution, -33G>A, on both the CFTR transcriptional activity and mRNA levels. As this sequence variation has been identified only in a patient with CBAVD, wild-type (pGL3B-WT) and mutated (pGL3B-33A) promoter constructs were tested in epithelial cell lines derived from tissues where spatial and temporal regulation of the CFTR expression is functional (HVP: vas deferens) or not (Hela: cervix). Compared to the wild-type, the promoter activity of the mutant construct was significantly reduced by approximately 54% (p<0.05) in HVP cell line whereas no significant variation was observed in Hela cells (Figure 1A). To ensure that the -33A variant is responsible for the observed inhibition, RT-quantitative PCR (qPCR) assays were performed from HVP and Hela cells. Since RNA transcripts of the patient were not accessible, we transfected these cell lines with expression vectors containing the CFTR cDNA under the control of the CFTR promoter (pcDNA3.1-WT or pcDNA3.1-33A). The results confirmed a diminution in the CFTR mRNA level in HVP cell line whereas no difference was observed in Hela cells (Figure 1B). Collectively, these findings established that the promoter -33A variant influences the CFTR transcription in a cell-specific manner.

The -33A variant affects promoter activity through allele-specific binding of the FOXI1 transcription factor

We then evaluated whether this single-nucleotide substitution perturbs the DNA binding affinity for transcription factors. In silico analyses, using TFsearch and Consite softwares, indicated that the CFTR minimal promoter region contains a binding site for FOX proteins overlapping the -33 position. Since FOXI1 (Forkhead Box I1) has been reported as a key regulator of genes involved in male fertility in epididymal epithelial cells 30 31 , we hypothesized that FOXI1 could participate to male infertility due to CBAVD by regulating the CFTR gene in HVP cell line. First, to test whether this factor is capable of binding in vivo to the endogenous CFTR promoter sequence, ChIP assays were carried out. The DNA immunoprecipitated with a non-specific antibody did not show any PCR signal (Figure 2A, lane 3). As negative control, we used the promoter of Caspase 8 gene, which lacks FOX binding sites. As observed in figure 2A (lane 2), FOXI1 binds to the CFTR minimal promoter.

We next employed quantitative ChIP analyses to obtain evidence that FOXI1 proteins bind differentially to the mutated CFTR promoter sequence. Because we could not have access to the patient's tissue, ChIP assays were performed using stable HVP cells expressing either the wild-type or the mutated CFTR promoter reporter construct (pGL3B-WT and pGL3B-33A).

The qPCR results reveal that FOXI1 binds to the minimal CFTR promoter in HVP cells with a higher avidity for the mutated promoter (Figure 2B).

To assess the functional activity of the FOXI1 factor, we transiently co-transfected the FOXI1 expression vector with either the wild-type (pGL3B-WT) or the mutated (pGL3B-33A) reporter plasmid in HVP cells. Our results show that the over-expression of FOXI1 proteins reduces the activity of the CFTR promoter, which is more significant in mutated context than in wild-type context (51% versus 37%) (Figure 2C, grey bars). To confirm the role of FOXI1 in the regulation of the CFTR transcription, we transiently co-transfected the reporter plasmids along with a FOXI1-specific small interfering RNA (siRNA). This resulted in a weak but significant activation of the luciferase activity in HVP cells (Figure 2C, white bars). The efficiency of the over-expression and the siRNA knockdown of the FOXI1 proteins were verified by immunoblotting experiments. Transient transfections of FOXI1-siRNA reduced FOXI1 protein levels by approximately 50% compared to a non-specific siRNA control (Figure 2D). RT-qPCR experiments were performed to analyse the CFTR mRNA levels in both wild-type and mutated contexts after FOXI1 over-expression (Figure 2E).

Although the effect of FOXI1 repression on the -33A allele versus the -33G allele is attenuated with RT-qPCR assays (Figure 2E), probably due to variation in constructs and approaches, both results suggested a markedly decrease of the CFTR expression for the mutated allele whereas only one nucleotide is changed. In addition, to confirm the negative role of FOXI1, we performed transient co-transfection experiments with either the AE4-promH1 or the AE4-promH3 reporter construct along with either the FOXI1 expression vector or FOXI1 specific siRNA in HVP cells. Our data demonstrated the strong activator role of FOXI1 on AE4-promH1 and AE4-promH3 transcription (Figure 2F) as previously reported. 30 32 Collectively, these data established that FOXI1 inhibits the CFTR promoter activity with a marked effect on the -33A allele.

Genetic analysis of previously undescribed (TAAA)n repeats sequence DNA motif in CFTR IVS9

By scanning microsatellite variability in the CFTR IVS9, in a sample of 254 control chromosomes and 86 chromosomes from CBAVD patients with 1 or 0 CFTR mutation, we identified five tetranucleotide (TAAA) n repeat tracts with number of motifs ranging from 9 to 13. This study showed that the 9-repeats allele is the most frequent (Table 1). In addition, analysis of DNAs, from CF patients homozygous for the p.Phe508del mutation and from CBAVD patients heterozygous for the p.Phe508del mutation and the [TG12T5] variant, revealed that the p.Phe508del mutations is mainly associated with the (TAAA) 11 tract (Table 1). Interestingly, we detected two previously undescribed variants, (TAAA) 8 and (TAAA) 6 , in two CBAVD patients carrying a (TAAA) 9 tract on the other chromosome. The patient with the 6-repeat allele is heterozygous for the -33G>A promoter variation [START_REF] Romey | First putative sequence alterations in the minimal CFTR promoter region[END_REF] ; the patient with the 8-repeat allele is heterozygous for the [TG12T5] variant. Accurate assignation of haplotypes was impossible (DNA from parents not available for segregation analyses). These two novel variants in IVS9 were not detected in a panel of 254 control chromosomes and 122 DNAs from the CBAVD subjects with the p.Phe508del and the TG12T5 alterations, and from the CF patients homozygous for the p.Phe508del mutation (Table 1). We hypothesized that these tetranucleotide repeats, located deeply within intron 9, could influence gene expression. 15 16 33 34 Also, we evaluated the functional importance of the new CBAVD (TAAA) 6 and (TAAA) 8 alleles compared to the most frequent (TAAA) 9 allele in general population.

The (TAAA) n repeat motif does not influence the splicing of CFTR exon 10

To investigate the putative impact of the IVS9 (TAAA) microsatellite on the RNA processing, CFTR exons 9-10 hybrid minigene constructs (Figure 3A) were transiently transfected into HVP and Hela cells, and the pattern of splicing was analyzed by RT-PCR with the SD6/SA2 primers. In the two tested cell lines, all PCR reactions produced one band of the expected size for wild-type mRNA transcript containing exons 9 and 10 (Figure 3B).

As positive controls, hybrid minigenes containing either a T5 or a T3 allele showed a significant decrease of correctly spliced transcript with the appearance of an aberrantlyspliced transcript containing only the exon 10. As previously reported [START_REF] Mastro | Mechanistic role of a disease-associated genetic variant within the ADAM33 asthma susceptibility gene[END_REF] , the same constructs with variable size containing (TAAA) n motif were also created to ensure that enclosed sequences to the motif repeats do not contain silencer or enhancer elements. No modulation of the spliced transcript has been observed (data not shown). Our findings indicated that the (TAAA) 6 and (TAAA) 8 repeats has no effect on exon 10 splicing.

The (TAAA) n repeat motif alters the CFTR transcription

In a second set of experiments, we speculated that the variation in the (TAAA) number might alter the activity of the CFTR promoter as previously reported for other genes. 16 34 To explore this possibility, we transfected in both HVP and Hela cells, Because tissues from patients are inaccessible, we used artificial vectors even if the constructions differ from the CFTR gene organization, in order to evaluate the impact of the (TAAA) n repeat on the CFTR transcription. Our results showed that the (TAAA) 8 and

(TAAA) 6 intronic variants significantly decrease the CFTR transcription suggesting the presence of a silencer element in the intron 9 (TAAA) n repeats of the CFTR gene. The decrease was not dependent on the location of the (TAAA) n repeat. We next hypothesized that this intronic transcriptional repression might reflect altered binding of one or more regulatory factors located within the (TAAA) microsatellite.

The (TAAA) n motif corresponds to DNA binding motif for FOX family factors

In silico analyses predicted binding sites for FOX proteins overlapping the (TAAA) n repeat. For the same reason that previously underlined in this paper, we assessed the possibility that FOXI1 factor could also regulate the CFTR transcription through these sequences in HVP cells. Transient co-transfection assays results indicated that enforced expression of FOXI1 protein induced a significant inhibition of the luciferase activity in the context of the CFTR intron 9 (Figure 5A). Results of siRNA assays confirmed the inhibitory role of the FOXI1 factor. We next confirmed the role repressor of FOXI1 at the CFTR mRNA level (Figure 5B).

To judge the FOXI1 binding ability on the (TAAA) n repeats, qChIP assays were performed using HVP cells stably transfected with (TAAA) 9 , (TAAA) 8 , or (TAAA) 6 allele.

Quantitative PCR realized with specific primers for a 350 bp-region encompassing the (TAAA) n repeat, revealed that the FOXI1 proteins bind to these intronic sequences (Figure 5C). Interestingly, we observed an increasing binding activity associated with the loss of (TAAA) repeats. Although our findings evidenced that the FOXI1 transcription factor negatively influenced the CFTR transcription, this attenuation seems to be independent of the loss of (TAAA) repeats suggesting that the CFTR repression is mediated by another unknown molecular mechanism.

Discussion

Because polymorphisms in the promoter region may modulate gene transcription by interacting with trans-acting elements, we set out functional analyses including in silico analyses, promoter reporter assays, and ChIP experiments, to explore the role of the -33G>A nucleotide change in promoter activity.

We demonstrated that the -33A allele inhibits significantly the CFTR promoter activity and alters the binding of the FOXI1 trans-acting factor and that the enforced FOXI1 protein expression negatively modulates the CFTR transcription. As FOXI1 proteins are here described as repressors for the first time, we confirmed our data by using reporter vectors containing promoter sequences of genes known to be positively transactivated by FOXI1. 30 32 Interestingly, the inhibitory effect of FOXI1 is stronger on the -33A allele than on the wildtype allele. Our findings suggest the contribution of FOXI1 in the physiopathology of CBAVD disorder through alteration of the CFTR promoter activity. The winged helix/forkhead box (FOX) gene family encodes a large class of nuclear proteins, characterized by a highly conserved forkhead DNA-binding domain. [START_REF] Katoh | Human FOX gene family (Review)[END_REF] The FOX proteins are involved in a wide range of developmental processes through regulation of tissue-specific expression of target genes during morphogenesis and differentiation. 38 39 Among the FOX transcription factors, recent attention have been drawn to FOXI1 (also known as HFH3, Fkh10 and FREAC6). Although FOXI1 was originally identified as a kidney-expressed forkhead gene 40- 42 , recent data indicated its pivotal regulatory role for normal sperm function. [START_REF] Blomqvist | Epididymal expression of the forkhead transcription factor Foxi1 is required for male fertility[END_REF] [START_REF] Cornwall | New insights into epididymal biology and function[END_REF] Blomqvist and collaborators showed that mice lacking FOXI1 fail to generate offspring. FOXI1 has been reported to function as a regulator of genes, important for the epididymal dependent maturation and storage of spermatozoa. [START_REF] Blomqvist | Epididymal expression of the forkhead transcription factor Foxi1 is required for male fertility[END_REF] In parallel, we provided experimental evidences showing that the (TAAA) n repeat sequence within the CFTR intron 9 has a functional role in the CFTR transcriptional regulation by using gene reporter assays, confirmed by CFTR mRNA expression.

Microsatellites have been described to alter the activity of genes when located in the promoter 15 34 or in introns. 16 33 In addition, Phylactides and coll. have previously reported that cis-acting elements located deep within CFTR introns promote the CFTR expression. [START_REF] Phylactides | Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene[END_REF] To determine the molecular mechanism that might explain the transcriptional impact of the STR, we conducted in silico analyses and functional assays. The results of in silico analyses revealed that the deletion of 1 or 3 (TAAA) repeats alter the putative binding for the tissuespecific FOX factors family. Using over-expression and RNA interference assays, we demonstrated that the FOXI1 factor inhibits the CFTR transcription, with similar extent in both wild-type and mutated context. However, ChIP analyses showed that the (TAAA) repeat shortening positively affects the affinity of FOXI1 transcription factor.

The role of the (TAAA) n alterations in regulating the CFTR transcription remains enigmatic. As the loss of (TAAA) repeat increased the ability of the FOXI1 binding and that enforced FOXI1 expression did not alter the CFTR promoter activity, a possible explanation is that microsatellite variation might form an alternative DNA structure that may affect gene transcription. FOX transcription factors regulate gene expression in virtue of their ability to bend or loop DNA, thereby facilitating communication between cis-element and trans-acting proteins. [START_REF] Cirillo | Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4[END_REF] Interestingly, FOXI1 is capable of remodelling chromatin structure and to maintain its DNA interactions in condensed chromatin. [START_REF] Yan | The forkhead transcription factor FoxI1 remains bound to condensed mitotic chromosomes and stably remodels chromatin structure[END_REF] In addition, the presence of the -33A nucleotide putatively creates another regulatory site through reinforcing the FOXI1 DNA binding that might affect the CFTR gene activity, with a markedly effect when both mutant alleles [-33A and (TAAA) 6 ] are considered. Recent investigations demonstrated that the CFTR gene regulation is modulated by interactions between promoter and intronic sequences. [START_REF] Ott | Novel regulatory mechanisms for the CFTR gene[END_REF] To conclude, this work suggested that the -33G>A promoter variant and (TAAA) with either the FOXI1 expression vector or the FOXI-specific siRNA are shown. Luciferase activities obtained from AE4-promH1 and AE4-prom H3 constructs was defined as 100%.

Other relative luciferase activities were expressed from this value. (* p < 0.05).

  , by site-directed mutagenesis (Stratagene). The pcDNA3-FOXI1 expression vector was generously gift by Dr. S. Enerbäck (Center of Medical Genetics, Institute of Biomedicine, Göterborg University, Sweden). The AE4-promH1 and AE4-promH3 reporter constructs were kindly provided by Dr. C.A. Hübner (Department of Human Genetics, UKE-Hamburg, Hamburg, Germany).

  Figure 4A. The results showed that in HVP cells, the pGL3C-(TAAA) 6 and pGL3C-(TAAA) 8

Figure 1 .

 1 Figure 1. Analysis of allele-specific transcriptional effects of the -33G>A CFTR promoter

Figure 3 . 9 and 10 .

 3910 Figure 3. Effect of deletion of (TAAA) motif repeats on the CFTR mRNA splicing.

Figure 4 .

 4 Figure 4. Analysis of allele-specific transcriptional effects of the (TAAA) n variants.

Table 1 : (TAAA) allele frequencies in control, CF and CBAVD subjects

 1

		% control	% CBAVD patients	% CF patients
				Mutations:	Mutations:
			0 or 1 identified	p.Phe508del/	p.Phe508del/
	Number of		mutation	TG12T5	p.Phe508del
	(TAAA)
	repeats	n=254	n=86	n=56	n=66
	13	2.75	1.20	0	0
	12	2.36	1.20	0	0
	11	8.66	10.46	50	94
	10	11.40	9.30	3.60	0
	9	74.80	75.60	46.40	6
	8	0	1.20	0	0
	6	0	1.20	0	0
	'n' the total number of chromosomes

Acknowledgments

This work was supported in part by the Centre Hospitalier Universitaire of Montpellier and the french association Vaincre La Mucoviscidose [grants TG0707 to E.L. and G0804 to V.V.].

could be associated with the CBAVD phenotype.

Conflict of Interest Statement

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D Gareth Evans

Michelle Harvie

Nigel Bundred

Anthony Howell

Evans Dg

Bundred N Howell

Uptake of breast cancer prevention and screening trials

published or not. The documents may come

Introduction

Recently breast cancer (BC) trials' uptake has improved. In 2008/2009 14,359 BC patients were recruited to UK trials representing 33% of incident cases, with 9.1% joining randomized trials [START_REF]NCRN/NIHR Breast trials recruitment[END_REF]. For the same year 93/428 (22%) BC patients at our institution entered randomized treatment trials and 70 of 248 (28%) over a more limited time span entered into a randomised study comparing three different diet/exercise programmes after surgery. This represents uptakes of 47%/45% of eligible patients. Prevention trials can improve the statistical power of their studies by recruiting individuals with a higher risk of the specific cancer. Indeed this might be expected to improve recruitment as people at higher risk of a cancer may be more motivated to join a trial. We previously reported a relatively low uptake of the double blind tamoxifen versus placebo trial IBIS1 amongst women very high risk of breast cancer (lifetime risk 40%+) [START_REF] Evans | Uptake of screening and prevention trials in women at very high risk of breast cancer[END_REF]. We have here enlarged this study to investigate uptake across a range of risks to a number of breast cancer prevention studies.

Patients and Methods

The Manchester Breast cancer Family History Clinic has now seen and assessed over 9000 women since 1987. Lifetime risks of breast cancer are calculated using a manual lifetables approach and using the Tyrer-Cuzick model [START_REF] Amir | Evaluation of Breast Cancer Risk Assessment Packages in the Family History Evaluation and Screening Programme[END_REF]. Records are kept of all women approached for prevention studies. We assessed the proportion of women who opted for three chemoprevention studies; two placebo controlled studies of tamoxifen (IBIS1) and anastrazole (IBIS2) and a randomized study using a combination of raloxifene and zoladex (RAZOR) vs. placebo as well as two dietary intervention studies; a non randomised trial to assess the effect of weight loss on markers of breast cancer risk over 12 months (Lifestyle Study) (n=40) [START_REF] Evans | Uptake of risk reducing surgery in unaffected women at high risk of breast and ovarian cancer is risk, age and time dependent[END_REF] and a randomised study comparing two different weight loss diets over 6 months (Intermittent diet study (n=54) [START_REF] Harvie | Adherence to a diet and exercise weight loss intervention amongst women at increased risk of breast cancer. The Open Obesity Journal[END_REF]. Uptake to these trials was compared across lifetime breast cancer risk categories to two screening trials (FH01/FH02) of mammography aged 40-45 and 35-39 years respectively (Table 1).

Lifetime risk categories were: very high risk women (lifetime risk 40%+); high risk (lifetime risk 26-39%) and moderate risk (lifetime risk 17-25%). Women were only counted as being approached if eligible. Women who had previously undertaken risk reducing mastectomy were excluded.

The drug and screening trials report the numbers of eligible subjects personally invited to the study by clinical staff within the Family History Clinic.

Recruitment to the weight loss studies involved a mail shot.

Results

In total 861 very high risk women (lifetime risk 40%+), 1713 high risk women (lifetime risk 26-39%) and 1785 women at moderate risk (lifetime risk 17-25%) were approached for at least one drug or diet trial. Uptake to drug trials was 5.3-13.6%. Uptake to the IBIS1 study was higher amongst high but not very high risk group. This was less evident for IBIS2, but the combined assessment showed a significant difference in uptake 12% vs 8.4% (p=0.004). RAZOR was initially targeted only at the very high risk group of 601 women. The recruitment rate was only 5.5% and therefore had to be opened out to the other risk categories. Complete records of high/moderate risk women approached were not available, but uptake was estimated to be higher: 11% high risk and 8% for moderate risk women. In contrast 699/737 (95%) and 372/401 (93%) women eligible for the screening trials (FHO1/FH02) were recruited with only 6 women returning their consent forms or stating in clinic saying they declined entry.

. The lower uptake rate for dietary studies reflects the general poor response to mail shots.

Only 12.5% (40/340) entered the lifestyle study and 6.2% (50/797) entered the intermittent study. There was no consistent pattern of uptake to these studies according to risk categories. This most likely reflects that motivation to join a weight loss study reflects a general desire to lose weight and is not motivated by risk reduction. Uptake to drug prevention studies thus remains low. In the highest risk group of unaffected BRCA1/2 mutations carriers only 22/256 (8%) have entered a prevention trial, whereas risk reducing surgery uptake was >50% after 7 years [START_REF] Harvie | The effect of intermittent versus chronic energy restriction on breast cancer risk biomarkers in premenopausal women[END_REF]. This also compared to 319/1713(18.6%) high risk (p=0.001) entering a prevention trial.

Discussion

We have shown that uptake to drug prevention trials remains disappointingly low at 5.3-13.6%. In contrast the great majority of women from all three risk categories have entered a non randomized screening trial. Uptake of screening is already known to be much higher than of prevention studies with the majority of highest risk women entering MRI screening [START_REF] Evans | Uptake of screening and prevention trials in women at very high risk of breast cancer[END_REF]. The reasons for the poor uptake to prevention trials need to be explored. Anecdotally our high risk women report not feeling comfortable with placebo studies as they will wish to be in an active treatment arm. It is likely that trials aimed at this very high risk group will need to consider dropping a placebo arm in favour of two proven preventive agents before recruitment is likely to rise. For entry into the STAR trial which involved randomization to either tamoxifen or raloxifene, but did not have a placebo, uptake was higher. A total of 184,460 women were screened using the modified Gail model to determine their breast cancer risk. Of these, 96,368 had a 5-year risk of at least 1.66% [START_REF] Vogel | Effects of tamoxifen vs raloxifene on the risk of developing invasive breast cancer and other disease outcomes: the NSABP Study of Tamoxifen and Raloxifene (STAR) P-2 trial[END_REF]. From this group, 20 616 (21.4%) agreed to be screened to determine full eligibility for the trial based on the medical criteria defined below; 20,168 were found to meet all eligibility criteria of the study. Of this latter group, 19,747 women expressed a desire to go forward with participation in the trial, signed a consent form, and were randomized to receive either tamoxifen or raloxifene. Participants were screened and enrolled through nearly 200 clinical centers throughout North America. This multicentre trial represents an adjusted uptake of 21%.

It is certainly possible that women at very high risk do not feel that the potential risk reductions of 35-70% are sufficient given their high absolute risk of breast cancer. Recent introduction of MRI screening in the UK might have improved uptake of prevention studies with the possibility of reliable early detection. However, if anything uptake of prevention trials appears to have reduced in more recent cohorts and uptake of risk reducing mastectomy has not dropped [START_REF] Evans | Uptake of risk reducing surgery in unaffected women at high risk of breast and ovarian cancer is risk, age and time dependent[END_REF]. Perhaps this is linked to widespread press coverage of adverse effects of certain cancer prevention drugs [START_REF] Senior | COX-2 inhibitors: cancer prevention or cardiovascular risk?[END_REF] and hormone treatments [START_REF] Beral | Breast cancer and hormone-replacement therapy in the Million Women Study[END_REF]. Improving risk communication may have some benefit in uptake according to research on uptake of screening [START_REF] Edwards | Personalised risk communication for informed decision making about taking screening tests[END_REF].

Recruitment to clinical trials is likely dependant on a number of factors such as having a dedicated trial manager, being a cancer or drug trial, and having interventions only available inside the trial [START_REF] Campbell | Recruitment to randomised trials: strategies for trial enrollment and participation study. The STEPS study[END_REF]. Training of study staff has also been highlighted as an important area [START_REF] Campbell | Recruitment to randomised trials: strategies for trial enrollment and participation study. The STEPS study[END_REF][START_REF] Ulrich | RTOG physician and research associate attitudes, beliefs and practices regarding clinical trials: implications for improving patient recruitment[END_REF][START_REF] Donovan | Development of a complex intervention improved randomization and informed consent in a randomized controlled trial[END_REF], and providing them with a best practices tool box that in the future would include successful mechanisms for eligibility screening and recruitment [START_REF] Ulrich | RTOG physician and research associate attitudes, beliefs and practices regarding clinical trials: implications for improving patient recruitment[END_REF]. Development of complex interventions to target poorly recruiting centres or individuals that include training has also shown some success [START_REF] Donovan | Development of a complex intervention improved randomization and informed consent in a randomized controlled trial[END_REF].

We are not aware of any other groups that have assessed uptake across a series of prevention trials, or to specify this by risk category. Uptake of tamoxifen in North America where it is licensed for prevention (this is not the case in Europe) has shown only 0.2% of the female population aged 40-79 taking the drug in the year 2000 [START_REF] Waters | Prevalence of tamoxifen use for breast cancer chemoprevention among U.S. women[END_REF].

However, this study was not able to assess uptake in those strictly eligible on risk grounds or after being approached with prevention as an option. Amongst BRCA1/2 mutation carriers a Canadian group reported 11% uptake (29/270) of either tamoxifen or raloxifene in women who had not undergone risk reducing mastectomy [START_REF] Metcalfe | Variation in rates of uptake of preventive options by Canadian women carrying the BRCA1 or BRCA2 genetic mutation[END_REF]. This figure is rather similar to our 8% uptake in mutation carriers for any drug prevention trial. It is likely that more must be done before we will get sufficient individuals into cancer prevention trials to move forward on an alternative to surgery in the highest risk groups.

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Table 1 :

 1 Uptake to prevention and screening trials in the Family History Clinic based on lifetime risk Patients invited to study by mail shot + The Lifestyle study only approached women at moderate and high risk $ -Estimated uptake: Complete records of high and moderate risk women approached were not available.

	Proportion of approached recruited
	by breast cancer risk categories

Acknowledgments:

We acknowledge the support of the Biomedical Research Centre at Central Manchester Foundation Trust, NIHR and the Genesis Breast Cancer Prevention Appeal.

Competing interest: None to declare