Virginie Caux-Moncoutier

Laurent Castera

Carole Tirapo

Dorothée Michaux

Marie-Alice Remon

Anthony Laugé

Etienne Rouleau

Antoine De Pauw

Bruno Buecher

Marion Gauthier-Villars

Laurent Castéra

Marie- Alice Rémon

Jean-Louis Viovy

Dominique Stoppa-Lyonnet

Pharm.D Claude Houdayer
email: claude.houdayer@curie.net

EMMA, a

Keywords: BRCA1, BRCA2, mutation, screening, diagnosis, capillary electrophoresis BRCA1, BRCA2, mutation, screening, diagnosis, capillary electrophoresis, EMMA

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INTRODUCTION

The role of genetic screening is becoming increasingly important in cancer diagnosis, prognosis and treatment decisions. Developing fast, reliable and inexpensive methods to detect such mutations is therefore a major challenge for medicine.

Two main strategies are used to search for unknown mutations: direct sequencing and screening. The first strategy is currently considered to be the most reliable, although not totally flawless [START_REF] Eng | Interpreting epidemiological research: blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1[END_REF]. More specifically, reliable chemistry and software are prominent points to consider.

The second strategy is a two-step strategy involving preliminary screening for the presence of variants on amplicons, followed by sequencing of only those fragment(s) in which a variation was detected. This dramatically reduces the number of fragments that need to be sequenced. These screening strategies are mainly based on heteroduplex analysis (HDA), using either a dedicated liquid chromatographic system (Denaturing High Performance Liquid Chromatography, DHPLC) [START_REF] Spiegelman | High-accuracy DNA sequence variation screening by DHPLC[END_REF] or a real-time PCR machine (High Resolution Melting curve analysis, HRM) [START_REF] Wittwer | High-resolution DNA melting analysis: advancements and limitations[END_REF]. During a slow cool-down performed at the end of PCR amplification of a DNA fragment from a heterozygote sample, two homoduplex fragments (corresponding to the normal and mutated allele, respectively) and two heteroduplexes (with mismatched strands due to hybridization of a wild-type strand with a mutant strand) are obtained. The aim of HDA is to check for the presence of heteroduplexes. DHPLC and HRM are widely used in diagnostic laboratories. However, analysis conditions depend on melting domains, and optimization is required for each PCR fragment.

HDA can also be performed by electrophoresis: homoduplexes are separated from heteroduplexes due to differences in electrophoretic mobility. Separations are nowadays 4 mostly performed by multi-capillary electrophoresis, allowing for high automation, low cost and high throughput, as recently described in a diagnostic validation of Conformation-Sensitive Capillary Electrophoresis (Mattocks, et al.) . A novel HDA method has been recently developed (Houdayer, et al.2010). This method, called Enhanced Mismatch Mutation Analysis (EMMA) is based on the use of innovative matrices increasing the electrophoretic mobility differences between homoduplex and heteroduplex DNA [START_REF] Weber | A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis[END_REF].

Sensitivity is further improved by using nucleosides as additives to enhance single-base substitution detection. Nucleosides are expected to interact with mismatched bases of heteroduplexes, thereby increasing mobility differences with homoduplexes [START_REF] Weber | Improving sensitivity of electrophoretic heteroduplex analysis using nucleosides as additives: Application to the breast cancer predisposition gene BRCA2[END_REF]. Moreover, this method, in combination with adapted semiquantitative PCR conditions, can be used to simultaneously detect point mutations and large-scale rearrangement in a single run [START_REF] Weber | High-throughput simultaneous detection of point mutations and large-scale rearrangements by CE[END_REF]. This feature, combined with the use of a single set of separation conditions for all fragments and with the multiplexing capability of the method, leads to a considerable simplification and cost reduction compared to previous methods.

The use of EMMA for fast screening of BRCA1 and BRCA2 point mutations and large rearrangements is described below. Constitutional mutations of the BRCA1 [MIM 113705] and BRCA2 [MIM 600185] genes are associated with a risk of hereditary breast and ovarian cancer (HBOC). It is essential to identify BRCA1/2 mutations to provide appropriate counseling to patients and relatives, but this represents a challenging and time-consuming task, as the vast majority of mutations are unique and spread over the entire coding sequence.

In this paper, we report the results of BRCA1 and BRCA2 screening for point mutations and large rearrangements in 1,525 unrelated patients (372 for the validation step and 1,153 in routine diagnosis), and discuss the performance of this new strategy.

PATIENTS AND METHODS

Patients

Genetic testing for BRCA1 and BRCA2 was proposed to women based on individual and/or family history (herein defined as index cases i.e. the first family member in whom complete BRCA1/2 gene screening was performed). Individual inclusion criteria included: i) breast adenocarcinoma before the age of 36, ii) medullary adenocarcinoma without age limitation, iii) breast adenocarcinoma and ovarian cancer. Male breast cancer was also considered for genetic testing. Family history was defined as either i) 3 breast cancer cases in first-or second-degree relatives in the same lineage, ii) 2 breast cancer cases in first-or second-degree relatives (with a transmitting male), with one cancer before the age of 40 or one cancer before 50 and the other before 70 iii) 1 breast cancer case and one first-or second-degree relative (with a transmitting male) with ovarian cancer. During the course of this study, and in line with French recommendations, inclusion criteria were recently extended to women with isolated ovarian adenocarcinoma before the age of 70 (http://www.e-cancer.fr/lessoins/oncogenetique). A consecutive series of 1,525 ascertained cases, mostly of Caucasian descent, were studied. All patients attended a visit with a geneticist and a genetic counselor in a family cancer clinic, mostly at the Institut Curie, Paris, France. Patients gave their informed consent for BRCA1/2 gene analyses. .

Nucleic acid extraction

For mutation scanning purposes, DNA was extracted from 2 ml whole blood samples collected on EDTA using a modified perchlorate/chloroform procedure (first 465 samples), as [START_REF] Johns | Purification of human genomic DNA from whole blood using sodium perchlorate in place of phenol[END_REF], or the NucleoSpin blood L kit from Macherey Nagel (following 849 samples) or the Quickgene 610-L automated system from FujiFilm (last 211 samples) according to the manufacturer's instructions. Three different extraction procedures were used during this study in the context of a constant effort to reduce the time and labor involved, finally leading to the semi-automated solution from FujiFilm.

DNAs were calibrated to 50 ng/µl by UV spectrophotometric assay (Nanodrop). Absorbance ratios (260/280) and (260/230) had to be in the 1.8-2.0 and 2.0-2.2 ranges, respectively.

Enhanced Mismatch Mutation Analysis (EMMA)

Primer design. Primers were purchased from Fluigent, Paris, France. BRCA1 and BRCA2 coding sequences were divided into 81 amplicons analyzed in 24 multiplex PCRs including one internal control for large-scale rearrangement analysis (Primer sequences are available from Fluigent). Primers were designed to include flanking intronic sequences containing recognized splice sites and avoiding known polymorphism to prevent mispriming.

Due to the well-known limitations of capillary electrophoresis [START_REF] Rozycka | Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis[END_REF] and topological effects [START_REF] Weber | A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis[END_REF], false-negatives can occur in the 70bp from both extremities of the amplicon. As a result, larger and overlapping amplicons were designed so that the sequence of interest always fell outside these 70bp in at least one amplicon [START_REF] Weber | Improving sensitivity of electrophoretic heteroduplex analysis using nucleosides as additives: Application to the breast cancer predisposition gene BRCA2[END_REF]. One primer of each pair was labeled with 6-carboxyfluorescein (6-FAM). Amplicon synthesis. Primer mixes were prepared according to the manufacturer's instructions (Fluigent) and all multiplex PCRs were performed using a single condition with the Qiagen Multiplex PCR kit (Qiagen, Courtaboeuf, France). Briefly, samples were generated in a 10-µl reaction volume containing 100 ng of genomic DNA, 1µl i.e. 0.3 µM of supplied by the manufacturer (Qiagen). The PCR programs were run in a GeneAmp 9700 thermocycler (Applied Biosystems, Courtaboeuf, France) and consisted of a first denaturation step at 95°C for 15 min, followed by 23 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 90 s and extension at 72°C for 90 s, followed by a final extension at 72°C for 10 min. PCR products were denatured for 5 min at 96°C then gradually reannealed at -1°C/min to reach 25°C. On completion of PCR, PCR products were stored at -20°C until migration.

Analysis. PCR tube volume was completed with pure water up to 20µL. Wells in columns A and H were filled with 1 µL EMMA buffer 10x to compensate for the higher intensity of the capillaries at the extremities of the area. PCRs were electrophoresed with a single analytical condition (15kV at 30°C and a fifty cm length-to-detector array) on an ABI3100 (Applied Biosystems) using EMMA polymer and according to the manufacturer's instructions (Fluigent, Paris, France). Data were analyzed using dedicated software (Emmalys, Fluigent). Electrophoregrams were examined and scored by two operators.

Point mutations. PCR products showing abnormal EMMA profiles (e.g. multiple peaks, shouldering or peak widening) were re-amplified, purified and sequenced in both directions using the BigDye Terminator Cycle Sequencing V1.1 Ready Reaction kit (Applied Biosystems) with incorporation of the PCR oligonucleotides as extension primers, followed by electrophoresis in an ABI PRISM 3130XL Genetic Analyzer with analysis using the Collection and Sequence Analysis software package (Applied Biosystems). Nucleotide position was numbered on the basis of the coding sequence NM_007294.2 and NM_000059.3 for BRCA1 and BRCA2, respectively. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence according to recommended guidelines (available at http://www.emqn.org/emqn.php and www.hgvs.org/mutnomen). The initiation codon is codon 1. Electrophoregrams were examined and scored by two operators. All mutations were confirmed on a second blood or buccal swab sample.

Large rearrangements. EMMA and quantitative multiplex PCR of short fluorescent fragments (QMPSF) were both used to screen for BRCA1 and BRCA2 rearrangements because automated profile analysis was easier with QMPSF (see below). QMPSF was used as previously described [START_REF] Casilli | The contribution of germline rearrangements to the spectrum of BRCA2 mutations[END_REF] . All rearrangements were confirmed on a second blood sample and using another technique e.g. long range PCR, transcript analysis or a dedicated array-CGH [START_REF] Rouleau | High-resolution oligonucleotide array-CGH applied to the detection and characterization of large rearrangements in the hereditary breast cancer gene BRCA1[END_REF] . Spurious single-exon deletions due to mispriming were therefore unambiguously excluded.

Unknown Variants (UVs) interpretation

In silico analysis. Unfortunately, one half of the variations observed in the BRCA1/2 genes are UVs [START_REF] Hofstra | Tumor characteristics as an analytic tool for classifying genetic variants of uncertain clinical significance[END_REF], making biological and clinical interpretation a challenging task and consequently leading to clinically difficult situations. To facilitate subsequent genetic counseling, all identified UVs were submitted to in silico analysis using Alamut (Interactive Biosoftware), a decision-support system for mutation interpretation that integrates a splice prediction module. Apart from the impact on splicing, unknown variants were also analyzed for their putative "protein-based" impact by cross-species and Grantham score [START_REF] Grantham | Amino acid difference formula to help explain protein evolution[END_REF] comparisons, and were then scored using an in-house model (Supp. Based on previous knowledge [START_REF] Houdayer | Evaluation of in silico splice tools for decision-making in molecular diagnosis[END_REF], defects were defined as a minimum 10% decrease of the wild-type score. Emergence of a cryptic splice site was considered significant and worthy of RNA study when it scored at least 50% of the corresponding wildtype score. ESEs were not considered for routine purposes.

RNA was extracted from lymphoblastoid cell lines with and without puromycin treatment.

RNA was reverse-transcribed (Houdayer, et al., 2004) and the BRCA1/2 coding sequence surrounding the region of interest was amplified (primer sequences available on request).

Amplicons were purified and sequenced in both directions using the BigDye Terminator Cycle Sequencing V1.1 Ready Reaction kit (Applied Biosystems) with incorporation of the PCR oligonucleotides as extension primers, followed by electrophoresis in an ABI PRISM 3130XL Genetic Analyzer with analysis using the Collection and Sequence Analysis software package (Applied Biosystems). Normal controls were always included in these experiments.

Validation step

Point mutations. Before implementing EMMA as a routine diagnostic technique, a panel of known BRCA mutations was tested [START_REF] Weber | A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis[END_REF][START_REF] Weber | Improving sensitivity of electrophoretic heteroduplex analysis using nucleosides as additives: Application to the breast cancer predisposition gene BRCA2[END_REF] [and author's unpublished data]. A series of blind studies were then performed using DHPLC and QMPSF as reference techniques for point mutation and large rearrangement screening, respectively. DHPLC was chosen as a reference because it is considered to be the gold standard for mutation prescreening with more than 1,000 PubMed references [http://www.ncbi.nlm.nih.gov/sites/entrez]. Moreover, our laboratory has an extensive experience with this technique, with more than 4,000 patients screened for BRCA1/2 and RB1 point mutations (Houdayer, et al., 2004;[START_REF] Wagner | Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations[END_REF] and, more recently, large-scale rearrangements using the DHPLC-derived technique called MP/LC (Dehainault, et al., 2004).

The blind studies involved a total of 372 patients. Two hundred and seventy nine cases were first screened on BRCA1 by Fluigent in their mutation detection facility, and 93 cases were then screened on both BRCA1 and BRCA2 in our laboratory. PCR conditions and analyses were as described above. An equivalent sensitivity was demonstrated, as 127 variations (excluding polymorphisms) were found with both techniques. Actually, one variation was missed by EMMA (BRCA2, c.9364G>A/p.Ala3122Thr), but another variation missed by DHPLC was detected by EMMA (BRCA1, c.2311T>C/p.Leu771Leu). Large rearrangements. The principle of the method consists of quantitative evaluation of the relative proportion of a given fragment in the unknown sample with respect to a normal control. In order to compare two fragments amplified in two different tubes, peak intensities and areas are normalized to a non-mutated DNA fragment used as an internal control.

Fluorescence intensities were then normalized by adjusting the peaks and areas obtained for the control amplicons to the same level and the yield of each amplicon in the various samples was evaluated. As some profiles exhibit variations due to polymorphisms, rare variants and mutations, Emmalys analyzes variant and non-variant profiles separately and deletions are detected by a 50% decrease in peak intensity and area, while duplications are detected by a 1.5-increase in profile area. Firstly, a 30-sample dedicated large rearrangement panel was blindly tested using this procedure. This panel included 20 BRCA1 large rearrangements (deletions and duplications) and 10 normal controls. All samples were correctly scored. The second step of the validation consisted of identification of 3 large rearrangements present in the 372 patients. Two deletions encompassing exons 15 and 16 and 13 to 15, respectively, were correctly detected. The third deletion, a deletion of BRCA1 promoter, was not detected because the current EMMA primer panel does not explore this part of the gene (which is covered by our QMPSF assay).

Interpretation of polymorphisms according to their profiles.

One of the main challenges with prescreening methods is the correct identification of polymorphisms. This does not constitute an issue for genes with a very low polymorphic content such as RB1 but is a major hurdle for BRCA genes, which exhibit a large number of polymorphisms throughout their sequence. In this case, the advantages of prescreening methods are lost if each variant peak has to be sequenced [START_REF] Mattocks | Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning[END_REF]). The situation becomes much simpler if the method allows correct recognition of polymorphisms according to their profile. This requires a high profile specificity and reproducibility. Profiles Reproducibility was evaluated within series and between series, by superimposition of profiles from the same variant. Profiles matched very accurately (Figures 1A,B). A high profile specificity was also observed, as illustrated e.g. Figure 2 illustrating 2 polymorphic profiles and the co-occurrence of these two polymorphisms in the same patient.

Polymorphisms were consequently expected to be identified from their variant profile. To validate this hypothesis, all variant profiles interpreted as polymorphisms in this series of 93 patients were sequenced (for a total of 1,200 sequences), and sequencing results were compared to EMMA interpretation. In every case, the expected polymorphism was found and no extra polymorphism was found in the remaining variant profiles, corresponding to a sensitivity and specificity of 100% in this series. To ensure correct interpretation, polymorphisms should be interpreted together and compared between each other instead of being compared to a normal control.

Mutations Identified in Routine Screening

DNAs from 1,153 hereditary breast/ovarian cancer patients were subjected to routine EMMA screening followed by sequencing of variant fragments. An average of 10 to 15% of PCRs had to be re-amplified due to low signal-to-noise ratio (see discussion section) and 3% of variant amplicons were sequenced. The 137 mutations found are reported in Supp. Tables S1A-B. The overall mutational detection rate according to our inclusion criteria was 11.9% i.e. 6% and 5.9% on BRCA1 and BRCA2, respectively. The unknown variant detection rate (Supp. Table S2) was 33.5% i.e. higher than previously reported because of the length of the intronic sequences analyzed that includes a 70bp "safe zone" (see "primer design"). This increased number of intronic UVs may represent a drawback of the design which is why 13 subsequent in silico splice analysis is recommended to confidently predict their putative impact on splicing. UV detection rate was approximately twofold higher in BRCA2 (23.8%) than in BRCA1 (9.7%). This figure is explained by the respective length of their coding sequences, which in turn means that fewer mutations are found in BRCA2 as compared to BRCA1.Mutations were similar in type and relative proportion in BRCA1 and BRCA2 (Table 1). They mainly resulted in truncated proteins or RNA decay. BRCA2 yielded a slightly higher rate of frameshift mutations, due to a greater number of frameshift deletions (51.5% vs.

33.3% of the mutational spectrum for BRCA2 and BRCA1, respectively). Frameshift mutations are by far the largest class of mutations, followed by nonsense and splice mutations (average 20% and 9% for both genes). Other mutations comprised rare mutations, i.e. missense, in-frame and large rearrangements. Four large rearrangements were found on BRCA1 and 2 on BRCA2. In other words, point mutations and large rearrangements accounted for 94.2% and 5.8% of the mutational spectrum of BRCA1, respectively. For BRCA2, point mutations accounted for 97.1% of the mutational spectrum whereas large rearrangements represented 2.9% of the mutational spectrum

The most frequent mutations found were c.5266dupC on BRCA1 and c.2808_2811del on BRCA2 (each found 6 times) followed by BRCA2 c.5946delT (4 times). Four other mutations were each found 3 times, i.e. c.1A>G, c.68_69del and c.4986+6T>C on BRCA1 and c.1773_1776del on BRCA2. This lack of frequent mutations probably reflects the mixed ethnic origin of the study population derived from Paris and suburbs.

According to our criteria, 18 UVs putatively leaded to a splice anomaly. Lymphoblastoid cell lines were available in 10 cases and another blood sample has yet to be obtained from the remaining 8 cases. Two UVs on both BRCA1 and BRCA2 actually lead to a splice defect. The Apart from splicing, 3 other UVs were prominent protein-damaging candidates with scores greater than 16 i.e. c.92T>A/p.Ile31Asn and c.122C>T/p.Pro41Leu, c.280C>T/p.Pro94Ser on BRCA1 and BRCA2, respectively. They are included in class 4 of the recently proposed classification system [START_REF] Plon | Sequence variant classification and reporting: recommendations for improving the interpretation of cancer susceptibility genetic test results[END_REF].

Throughput

Patients were initially analyzed by series of 96, according to the same organization as previously used with DHPLC. Following a learning curve period, series of 30 patients were subsequently analyzed. It now takes seven working days for complete BRCA1/2 screening in 30 patients by one technician (excluding DNA extraction and sequencing).

EMMA

This is the first report of a large-scale study using EMMA, some aspects of this methodology therefore need to be discussed. Capillary electrophoresis was developed as an attractive strategy for mutation detection in the early 1990's [START_REF] Khrapko | Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis[END_REF]. However, its broad diffusion was hampered by design constraints and lack of sensitivity [START_REF] Rozycka | Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis[END_REF]. The recently described Heteroduplex Analysis by Capillary Array Electrophoresis [START_REF] Perez-Cabornero | A new strategy to screen MMR genes in Lynch Syndrome: HA-CAE, MLPA and RT-PCR[END_REF], also called Conformation-Sensitive Capillary Electrophoresis (CSCE) [START_REF] Mattocks | Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning[END_REF]) represents a real improvement, but it also presents certain disadvantages. It uses a home-made polymer recipe that does not allow batch-to-batch reproducibility. Consequently, all fragments with altered peak patterns need to be sequenced, dramatically increasing the amount of sequencing needed for genes such as BRCA1/2, which involve frequent polymorphisms; large rearrangements cannot be detected and mutational screening must be completed by another method.. EMMA retains the simplicity and throughput of CSCE, but as a result of a series of additional developmentscomprising industrialized ready-to-use separation matrix and specific software, it overcomes the above limitations. It allows easy and reliable recognition of polymorphisms according to their 16 profile, and it is the first technique able to detect large rearrangements and point mutations in a single run [START_REF] Weber | High-throughput simultaneous detection of point mutations and large-scale rearrangements by CE[END_REF].

. Some technical remarks and guidelines for implementation and use, based on our experience, are listed below. Regarding DNA extraction, 3 different procedures were used during the course of the study (see Materials and Methods" section) without any incidence on data quality. Due to its high sensitivity, in order to take full advantage of the potential of the technique for direct polymorphism identification, specific PCR amplifications must be used since nonspecific products could generate shouldering for a wild-type sample, making the profile difficult to interpret. Nonspecific products may also be generated by primer degradation with time, but this is easily detected because profiles lose their reproducibility.

Capillary electrophoresis always involves slight run-to-run and capillary-to-capillary mobility shifts, so it is therefore important to compare peaks after correct peak alignment, which can be tricky with conventional software in the case of complex profiles. The Emmalys software appeared to effectively deal with this difficulty, as suggested by the robustness of polymorphism analysis.

Strikingly, heteroduplexes could be either slower or faster than homoduplexes and no rule was found that could link one type of nucleotide substitution to one type of mobility difference or profile shape. Electrophoretic mobility does not only depend on the mismatch bases, but also on the local sequence environment of this mismatch, since distortions of the DNA helix at one base pair are propagated to the neighboring bases [START_REF] Weber | A high-throughput mutation detection method based on heteroduplex analysis using graft copolymer matrixes: application to Brca1 and Brca2 analysis[END_REF]. As a consequence of the above, a heteroduplex can exceptionally comigrate with the peak corresponding to the following/preceding peak in the multiplex electrophoregram. Therefore, if an amplicon from a patient shows an abnormal profile, but does not show any variation on sequencing, it could indicate that the altered amplicon is actually a neighboring amplicon.

This can be easily confirmed by observing the result of large rearrangement analysis, which should in turn mimic a deletion on the neighboring peak. Similarly, the quantitative analysis used to detect large rearrangements can also be helpful to detect allele drop-out due to mispriming. This information is important in order to detect situations in which one allele would not be amplified and therefore not analyzed. Moreover, the nucleotide variation inducing the mispriming can also be deleterious. Obviously, in such a case another primer pair must be used to distinguish this allelic drop-out from a bona fide deletion.

Finally, to ensure reliable and easy interpretation of polymorphisms, it is recommended to work on series of 30 samples or more. Below this range, it may prove difficult to correctly interpret polymorphism profiles because of an insufficient number of reference profiles.

Distinct injection protocols are available to ensure optimum results (EMMA high-low). In a first, "low" injection mode, samples are injected for 10s -20s at 1kV -4kV then electrophoresed at 15kV for 45 min. This injection mode has a sufficiently low consumption to allow a second run to be performed from the same sample using a "higher injection" protocol, in the event of occasional unexpected problems (missed injection, capillary failure, microbubble, etc). All of these "tricks", recommendations and checkpoints are summarized in the Analysis Pipeline, Figure 3.

The viscosity of EMMA separation medium is higher than that of other solutions conventionally used in ABI instruments. This requires certain modifications of the capillary filling and apparatus operation sequences (provided by Fluigent), compared to standard procedures. For instance, this involves longer array filling time, capillary rinsing with buffer every 100 runs, and capillary array exchange every 400 runs. Replacing EMMA buffer 1x daily is also recommended to avoid loss of resolution. System performance and integrity should be checked once a week using a control sample (a 4-plex PCR with variant profiles for each peak, available from Fluigent). As for any heteroduplex analysis, homozygous mutations cannot be detected by EMMA on the direct sample, but this can be circumvented by adding a wild-type DNA in the sample to force heteroduplex formation, as previously demonstrated with DHPLC [START_REF] Ferec | Analysis of genomic CFTR DNA[END_REF]. The cost of consumable items used to screen BRCA1 and BRCA2 by EMMA is estimated to be less than 100 euros per patient (based on price list). This estimation excluded the costs of DNA extraction and sequencing, purchase of the sequencer, instrument depreciation and labor. The markedly reduced need for downstream sequencing (typically 3%), as a result of polymorphism identification, is also a factor for cost reduction [START_REF] Sevilla | Testing for BRCA1 mutations: a cost-effectiveness analysis[END_REF].

This technology allows a high throughput and our laboratory now generates close to 9000 amplicons per month for diagnostic purposes; it should be stressed that the management of such a large volume of data requires a robust data management system. We have therefore constructed a FileMakerPro® database which integrates information from sampling to the final report, but which also interacts with the Institute's databases. This allows careful followup of the entire analysis [Laugé A et al., poster 664 Assises de Génétique 2010].

The advantages of EMMA compared to our previous DHPLC-based strategy are obvious: they comprise a single condition for amplification and run, a higher throughput and lower costs (lab technician time decreased to one quarter and overall cost decreased to one third, compared to DHPLC in our hands). The simultaneous detection of point mutations and large rearrangements is another advantage. However promoter regions are not covered and this must be taken into account, since deletions of the promoter regions are a probably rare but significant cause of HBOC [START_REF] Brown | Germline BRCA1 promoter deletions in UK and Australian familial breast cancer patients: Identification of a novel deletion consistent with BRCA1:psiBRCA1 recombination[END_REF][START_REF] Caux-Moncoutier | Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study[END_REF]. Although technical performance was acceptable, the complexity of the currently available Emmalys interface prevents routine use of large rearrangement detection. On the other hand, Emmalys was found to be robust and user-friendly for point mutation detection. More generally, we believe EMMA should be considered as an alternative to the High Resolution Melting curve analysis (HRM) technique. HRM is a very fast and elegant tubemethod also allowing simultaneous detection of point mutations and large rearrangements in one experiment [START_REF] Coulet | A One-Step Prescreening for Point Mutations and Large Rearrangement in BRCA1 and BRCA2 Genes Using Quantitative Polymerase Chain Reaction and High-Resolution Melting Curve Analysis[END_REF][START_REF] Rouleau | Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome[END_REF]. Its main advantage over EMMA is the absence of post-PCR manipulation. However, based on the recent literature, HRM appears to have a number of drawbacks (which are overcome by EMMA), notably: i) its sensitivity depends on the nucleotide composition of the amplicon (melting domains).

HRM diagnostic guidelines for BRCA1 screening recommend extreme caution when analyzing high-GC, low-GC contents and fragments containing many different variants [START_REF] Van Der Stoep | Diagnostic guidelines for high-resolution melting curve (HRM) analysis: an interlaboratory validation of BRCA1 mutation scanning using the 96-well LightScanner[END_REF][START_REF] Wittwer | High-resolution DNA melting analysis: advancements and limitations[END_REF] ii) interpretation of polymorphisms according to their profile would need probes or sequence analysis to exclude the presence of a mutation with an identical melt profile [START_REF] Nguyen-Dumont | Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis[END_REF].

Spectrum of Mutations

The mutation detection rate in BRCA genes largely depends on the patient inclusion criteria and selecting high-risk patients from breast and ovarian families would provide the geneticist with a higher mutation detection rate than including patients based exclusively on their personal history. That being said, the sensitivity of EMMA was 11.9% in our series of 1,153 patients, which is in line with expectations with regards to patient inclusions and compared to previous results using DHPLC as well as data from French laboratories (http://www.e-cancer.fr).

Considering the mutational spectrum, the recurrent so-called Ashkenazim mutations (BRCA1 68_69del, 5266dupC and BRCA2 5946delT) represented 9.5% of our identified mutations, which is why prescreening for these 3 mutations is proposed during genetic counseling to patients reporting an Eastern European origin. Apart from these mutations, we did not observe a larger panel of recurrent mutations which would justify a first-line screening procedure, as described in other populations [START_REF] Revillion | Multiplex single-nucleotide primer extension analysis to simultaneously detect eleven BRCA1 mutations in breast cancer families[END_REF]. Few deleterious inframe/missenses mutations were found but this could actually reflect an interpretation bias in view of the fact that 386 unknown variants were detected (detection rate 33.5%). In other words, these 386 variants no doubt included deleterious neutral/missense/intronic mutations, which is why scoring variants to highlight prominent candidates for future collaborative studies is of special relevance for optimization of molecular diagnosis. There is considerable debate about the usefulness of large rearrangement screening in BRCA genes. The main argument against such screening, is that the time and efforts needed for such screening might be used more efficiently by screening more patients but for point mutations only. Previous reports [START_REF] Engert | MLPA screening in the BRCA1 gene from 1,506 German hereditary breast cancer cases: novel deletions, frequent involvement of exon 17, and occurrence in single early-onset cases[END_REF][START_REF] Mazoyer | Genomic rearrangements in the BRCA1 and BRCA2 genes[END_REF]suggested that large rearrangements accounted for an average of 10% of the BRCA1 mutational spectrum. Although our present results are lower (5.8%), we can confidently confirm that it is worth searching for BRCA1 large rearrangements in genetic diagnosis, regardless of the patient's family history. The situation is less clear for BRCA2 because previous studies, including those from our team [START_REF] Casilli | The contribution of germline rearrangements to the spectrum of BRCA2 mutations[END_REF][START_REF] Tournier | Significant contribution of germline BRCA2 rearrangements in male breast cancer families[END_REF], were based on smaller series and selected high-risk families [START_REF] Agata | Large genomic deletions inactivate the BRCA2 gene in breast cancer families[END_REF][START_REF] Engert | MLPA screening in the BRCA1 gene from 1,506 German hereditary breast cancer cases: novel deletions, frequent involvement of exon 17, and occurrence in single early-onset cases[END_REF][START_REF] Woodward | Large genomic rearrangements of both BRCA2 and BRCA1 are a feature of the inherited breast/ovarian cancer phenotype in selected families[END_REF]. These studies led to the overall conclusion that BRCA2 genomic rearrangements are worth investigating in high-risk families. This does not mean that these mutations are associated with a higher risk, but that due to the very low contribution of large rearrangements in the BRCA2 mutational spectrum, the search for these mutations should be limited to patients with a severe family history associated with a high BRCA2 mutation detection rate. Two BRCA2 large rearrangements, one complete deletion and one duplication of exons 19, 20 (Figure 4) were found in the present series of 1,153 consecutive patients. The deletion was found in a young breast cancer patient with no family history i.e. a low-risk case. Conversely, the duplication was found in a high-risk family: the and 61 years of age, respectively. The overall probability of identifying a BRCA mutation in our index case was 59.4% (24.6% for BRCA1 and 34.9% for BRCA2) according to the BRCAPRO model [START_REF] Parmigiani | Determining carrier probabilities for breast cancersusceptibility genes BRCA1 and BRCA2[END_REF] and 57.9%(12.1% for BRCA1 and 45.8% for BRCA2) according to the BOADICEA model [START_REF] Antoniou | The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions[END_REF]. Interestingly, her older affected sister was found to be negative using a combination of direct sequencing for BRCA1/2 and a limited 5-site rearrangement panel for BRCA1(Comprehensive BRACAnalysis®, Myriad® Genetics laboratories Inc. Salt Lake City, USA). Following identification of the BRCA2 duplication in our index case, genetic testing and appropriate genetic counseling were made available to the relatives e.g. 6 unaffected females (at least) and the affected sister. As expected, the affected sister carried the BRCA2 duplication. More generally, and as this kind of mutation might also be found in low-risk situations, this study argues in favor of methods allowing simultaneous and convenient detection of point mutations and large rearrangements to enable clinical geneticists to meet the constraints of routine genetic testing.

General conclusions and perspectives

The main advantages of EMMA are that it provides a marked increase of throughput (or cost reduction at a given throughput) compared to DHPLC and is based on standard sequencers routinely used in laboratories. It can therefore be easily implemented in laboratories familiar with capillary electrophoresis, and allows screening and sequencing on the same platform. Due to its flexibility, EMMA could also be used for rapid BRCA screening of a subset of patients e.g. before poly(ADP-ribose) polymerase inhibitor treatment (Audeh, et al., 2010 ;[START_REF] Tutt | Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial[END_REF]. SOLiD NGS platforms (ABI-Life technologies) are available in our Institute, we tested this technology for BRCA diagnostic applications [Houdayer C et al., European Society of Human Genetics meeting 2010, P11.089]. The SOLiD chemistry appears to be reliable for diagnostic purposes, but implementation of a SOLiD diagnostic pipeline remains complex at the present time for reasons of sample preparation, bioinformatics, fast-evolving protocols and costs.

EMMA obviously cannot compete with SOLiD in terms of throughput, but indeed only a small number of diagnostic laboratories actually need such amazing sequencing capacities.

The throughput and therefore data handling constraints of the 454 NGS machine (Roche) are still enormous, although lower than those of SOLiD, but well known sensitivity/specificity problems in homopolymer runs have to be taken into account [START_REF] Emrich | Gene discovery and annotation using LCM-454 transcriptome sequencing[END_REF][START_REF] Wicker | 454 sequencing put to the test using the complex genome of barley[END_REF]. The present study therefore suggests that EMMA represents a valuable short-term and medium-term option for many diagnostic laboratories looking for an easy, reliable and affordable strategy, enabling fast and sensitive analysis for a large number of genes. The electrophoregrams in grey and black represent a normal control and the duplicated patient, respectively. Profiles are superimposed then normalized using the internal control.Two ratios are used to determine the presence or absence of a large-scale rearrangement: R1 is the ratio between peak intensity of the fragment of interest over peak intensity of the internal control. R2 (indicated for each peak) is the ratio of R1 of the sample of interest over control R1 defined as a mean R1 value based on several control samples. Duplicated exons are indicated by an arrow. Exon 19 is investigated in 2 overlapping amplicons (upper panels A, B), while exon 20 is investigated with a single amplicon (bottom panel C).

F

. In-house algorithm for UV scoring A restrictive definition of functional domains was used to avoid redundancy between "conservation across species" and "known functional domain" items. The amino-acid positions 24-64 (RING finger), 503-508 (SLN1), 607-615 (SLN2), 1189, 1457, 1524, 1542 (serine phosphorylation sites), 1649-1736 (BRCT1) and 1756-1855 (BRCT2) were therefore considered as functional domains for BRCA1. Functional domains for BRCA2 were as follows: amino-acid positions 18-105 (transactivation), 987-1069, 1198-1293;1407-1498 ;1501-1589;1649-1735;1822-1914;1955-2035;2036-2112 (BRC repeats) (BRC repeats), 3263-3269 (SLN1) and 3381-3385 (SLN2). Scores from 12 and above are considered "likely deleterious".

F

  Fluigent), 2µl of water and 5µl i.e. 1X Qiagen Multiplex PCR Master mix

Figure S1 )

 S1 FigureS1) validated on a series of 378 hereditary breast/ovarian cancer patients and previous

  Patients harboring unknown variants putatively leading to splice defects following Alamut in silico analyses were further investigated at the cDNA level.

  based strategy was designed for fast screening of BRCA1 and BRCA2 point mutations and large rearrangements in 1,525 unrelated hereditary breast and/or ovarian cancer (HBOC) patients: 1,153 patients were screened in a routine setting following a validation step enrolling 372 patients.

  from the same fragment must therefore be distinguished, and for the same variant, the same profile should be observed in independent experiments.

  c.547+3A>T / IVS08+3A>T and c.4484G>T / p.Arg1495Met on BRCA1 showed decrease of the donor site with subsequent exon 8 and exon 14 skipping, respectively. The c.316+5G>C / c.7975A>G / p.Arg2659Gly on BRCA2 also showed decrease of the donor site with subsequent exon 3 and exon 17 skipping.

  hundred and twenty five unrelated breast and/or ovarian cancer patients were studied, as part of the routine clinical management provided by the Institut Curie, Paris, France. To our knowledge, this is the largest complete mutational screening of BRCA1/2 genes reported to date in Europe.

  the above precautions and issues, which require the level of care typically required by molecular diagnosis, EMMA uses standard and well-known sequencers and a set of ready-to-use reagents, thereby facilitating upgrading to this new technology.

  her older sister developed bilateral breast carcinoma at 48, 64 and 57, 63 years of age, respectively. Their paternal aunt and grandmother also developed breast cancer at 45

  future of EMMA should be considered in the context of growing sequencing capacities provided by the next-generation-sequencing (NGS) platforms. Since 2

  FIGURE LEGENDS

Figure 1B .

 1B Figure 1B. Reproducibility within series. Screenshot from Emmalys showing superimposition of 33 different samples harboring the same polymorphism (BRCA1 exon 13), from a series of 93 patients.

Figure 2 .

 2 Figure 2. Profile specificity Screenshots from Emmalys with 3 polymorphic profiles from 2 adjacent codons (BRCA1 exon 11). In grey: normal monomorphic profile, in black: polymorphic profiles. From left to right: c.2077G>A/p.Asp693Asn, c.2082C>T/p.Ser694Ser and the combination of both (c.2077G>A/p.Asp693Asn plus c.2082C>T/p.Ser694Ser). All polymorphic profiles are clearly distinct.

Figure 3 .

 3 Figure 3. EMMA : analysis pipeline Schematic representation of a routine EMMA analysis.

Figure 4 .

 4 Figure 4. Duplication of BRCA2 exons 19 and 20. Three screenshots from Emmalys showing the 3 multiplex PCRs incorporating BRCA2 exons 19 and 20 (panels A, B and C). Exons under study and the amplicon number (when the exon is investigated in overlapping amplicons) are indicated at the top of the corresponding peaks.The electrophoregrams in grey and black represent a normal control and the duplicated patient, respectively. Profiles are superimposed then normalized using the internal control.Two ratios are used to determine the presence or absence of a large-scale rearrangement: R1 is the ratio between peak intensity of the fragment of interest over peak intensity of the internal control. R2 (indicated for each peak) is the ratio of R1 of the sample of interest over control R1 defined as a mean R1 value based on several control samples. Duplicated exons are indicated by an arrow. Exon 19 is investigated in 2 overlapping amplicons (upper panels A, B), while exon 20 is investigated with a single amplicon (bottom panel C).

29 Page 29 of 47 John Wiley & Sons, Inc.

	Human Mutation
	Tables
	o r F o r F o r
	P P P
	e e e
	e r e r e r
	R R R
	e e e
	v i e v i e v i e
	w w w

Table 1 .

 1 Deleterious mutations found according to their type.

		BRCA1 (number and %)	BRCA2 (number and %)
	Frameshift	37 (53.6%)	47 (69.1%)
	Nonsense	16 (23.2%)	13 (19.1%)
	Splice	8 (11.6%)	4 (5.9%)
	Large rearrangements	4 (5.8%)	2 (2.9%)
	Missense	4 (5.8%)	1 (1.5%)
	In frame	0 (0%)	1 (1.5%)
	Total	69 (100%)	68 (100%)
	Mutation detection rate	6%	5.9%
	(1153 patients)

Supplementary Table S1B. Deleterious mutations found on BRCA2

  Recurrence numbers are in brackets. Nucleotide position was numbered on the basis of the coding sequence NM_000059.3. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

	Intron 23	c.3305A>G c.5467+148delT			p.Asn1102Ser
	Site Site	Description Description c.3418A>G (4) c.5468-10C>A (2)			Expected Consequence Expected Consequence p.Ser1140Gly
	Exon 12 Exon 14	c.4165_4166delAG c.7069_7070delCT c.3600G>T		p.Ser1389X p.Leu2357ValfsX2 p.Gln1200His
	c.4201C>T c.7480C>T Description (*) these UVs were detected following allele drop-out (see text) Exon 13 Exon 15 Site Expected Consequence p.Gln1401X p.Arg2494X
	Exon 11	c.4258C>T c.7612A>T c.3608G>A			p.Gln1420X p.Lys2538X p.Arg1203Gln
	o r P e e r F o r P e e r c.4327C>T c.4372C>T c.4484G>T (2) c.4810C>T c.4945_4947delAGAinsTTTT (2) c.4986+6T>C (3) c.5071dupA c.5266dupC (6) c.5278-?_*1381+?del (deletion of exons 21 to 24) c.5307T>A c.5503C>T c.5541C>A F o r P e e r c.7617+1G>T c.7680dupT c.7795_7797delGAA c.7805G>C c.8021delA c.8029_8030delGA c.8032-8033dupAG c.8207delT c.3640G>A c.3804T>C c.3823A>G (4) c.4113G>A (2) c.4185+3A>T c.4185+21delTG c.4185+21_22dupTG c.4255G>C Site F Exon 14 Exon 16 Intron 16 Exon 17 Exon 20 Exons 21 to 24 Exon 21 Exon 24 Intron 15 Exon 16 Exon 18 Exon 12 Intron 12 Exon 13 o r P e e r Exons 19 to 20 c.8332-?_8632+?dup (duplication of exons 19 and 20) Exon 20 c.8548_8551del4 c.9026_9030del5 Exon 23 c.9097delA F o r F o r Intron 13 c.4358-37G>A Exon 14 c.4445A>G Exon 15 c.4636G>A c.4485-32C>T Intron 14 F o r F o r c.9154C>T Exon 24 c.9253dupA P P c.4485-57T>C c.4675+31C>T Intron 15 P P Intron 24 c.9257-1G>A e e r e e r c.4675+81T>C c.4691T>C c.4812A>G e e Exon 16 c.4840C>T e r e r	p.Arg1443X p.Gln1458X p.Arg1495Met p.Gln1604X p.Arg1649PhefsX30 p.Thr1691AsnfsX4 p.Gln1756ProfsX74 p.Tyr1769X p.Arg1835X p.Cys1847X p.Gln2561SerfsX5 p.Glu2599del p.Arg2602Thr p.Lys2674ArgfsX2 p.Glu2677LysfsX3 p.Asp2679GlyfsX16 p.Leu2736ProfsX2 p.Glu1214Lys p.Asn1268Asn p.Ile1275Val p.Gly1371Gly p.Glu1419Gln p.Glu2850GlnfsX12 p.Tyr3009SerfsX7 p.Asp1482Gly p.Asp1546Asn p.Thr3033LeufsX29 p.Arg3052Trp p.Thr3085AsnfsX26 p.Leu1564Pro p.Gln1604Gln p.Pro1614Ser
	Exon 17 Intron 16	c.4993G>C c.4987-20A>G	R R R R R R R R	p.Val1665Leu
	Intron 17 Exon 19 Intron 19	c.4987-25A>G c.5074+107C>T c.5074+108G>A (3) c.5075-74C>T c.5177G>T c.5193+64T>G c.5194-14T>A	e v i e w e v i e w e v i e w e v i e w e v i e w e v i e w e e v i e v i e p.Arg1726Ile w w
		c.5194-165_162dup
	Exon 20	c.5203G>A			p.Glu1735Lys
		c.5213G>A			p.Gly1738Glu
	Intron 20	c.5277+55A>T
	Intron 21	c.5332+182dupA (*)
		c.5332+78C>T
		c.5333-130T>C (2)
		c.5333-44A>T
		c.5333-61G>C
		c.5333-134C>A (4)
	Intron 22	c.5406+63G>T
	Exon 23	c.5412C>T (2)			p.Val1804Val

Supplementary Table S2B. Unknown variants found on BRCA2

  Recurrence numbers are in brackets. Nucleotide position was numbered on the basis of the coding sequence NM_000059.3. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

	Site	Description		Expected Consequence
	Exon 2	c.-11C>T (5)
		c.-12T>C
		c.64G>A		p.Ala22Thr
	Intron 2	c.67+62T>G (12)
		c.67+82C>G (14)
		c.68-7T>A (7)
	Exon 3	c.122C>T		p.Pro41Leu
	Intron 3	c.198A>G c.223G>C (3) c.280C>T c.316+5G>C F o r F o r F o r		p.Gln66Gln p.Ala75Pro p.Pro94Ser
		c.316+6T>C c.316+12A>G P P P
		c.316+13A>G e e e c.316+57C>T c.317-12G>A c.317-92delA e r e r e r
	Exon 4	c.324T>C c.400C>A	R R R	p.Asn108Asn p.Leu134Ile
	Intron 4 Intron 5 Intron 6	c.425+52A>G c.475+3A>T c.476-5C>T c.476-24A>G c.516+21A>T (4) c.516+54T>C c.517-19C>T (4)	e e e v i e v i e v i e w w w
		c.517-23_22delTA
		c.517-74G>C
		c.517-89G>A
	Exon 7	c.532A>C		p.Lys178Gln
	Intron 7	c.631+18G>A
		c.631+25C>T
		c.631+66C>T
		c.632-16A>C
		c.632-75T>C
	Intron 8	c.682-30A>C
		c.682-32A>G
	Exon 10	c.943T>A		p.Cys315Ser
		c.978C>A		p.Ser326Arg

Page 28 of 47 John Wiley & Sons, Inc.Human Mutation

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

The authors would like to thank J. Weber, J. Champ, C. Bureau, P. Michel, I. Eugène, C. Gilbon, S. Miglierina and C. Rougeron for helpful support during the course of this study, as well as O. Béra, F. Cornelis, P. Laurent-Puig and I. Mortemousque for referring patients. This paper describes work conducted in the context of the PREDICAN and Polygen projects. PREDICAN is a collaborative project supported by Medicen and la Région Ile de France.

Polygen is supported by ANR (Agence Nationale pour la Recherche).

Commercial interest:

The EMMA technology is dependent on Curie Institute/CNRS/University Pierre et Marie Curie joint patents DSL and JLV are shareholders of Fluigent

Description Expected Consequence Exons 1 to 27 c.( ?_-227)_(*902_ ?)del (complete deletion) c.145G>T (2) p.Glu49X Exon 3 c.289G>T p.Glu97X Intron 6 c.516+1G>T c.1238delT p.Leu413HisfsX17 c.1310_1313del4 p.Lys437IlefsX22 c.1511_1512delCT p.Ser504TyrfsX9 c.1593dupA p.Glu532ArgfsX3 c.1773_1776del4 (3) p.Ile591MetfsX22 Exon 10 c.1813dupA p.Ile605AsnfsX11 c.1929delG p.Arg645GlufsX15 c.2588dupA p.Asn863LysfsX18 c.2612C>A p.Ser871X c.2808_2811del4 (6) p.Ala938ProfsX21 c.2899_2900delCT p.Leu967ArgfsX14 c.3195delT p.Asn1066IlefsX11 c.3645_3646del2ins7 p.Phe1216LysfsX14 c.3744_3747del4 (2) p.Ser1248ArgfsX10 c.4136dupA p.Ile1380AspfsX2 c.4284dupT p.Gln1429SerfsX9 c.4965C>A p.Tyr1655X c.5197dupT p.Ser1733PhefsX10 c.5576_5579del4 p.Ile1859LysfsX3 c.5616_5620del5 p.Lys1872AsnfsX2 c.5645C>A p.Ser1882X c.5682C>G p.Tyr1894X c.5909C>A (2) p.Ser1970X c.5946delT (4) p.Ser1982ArgfsX22 c.6079dupA p.Arg2027LysfsX22 c.6082_6086del5 p.Glu2028LysfsX19 c.6275_6276delTT (2) p.Leu2092ProfsX7 c.6359C>G p.Ser2120X c.6373dupA p.Thr2125AsnfsX4 c.6405_6409del5

Supplementary Table

 S1A. Deleterious mutations found on BRCA1 Recurrence numbers are in brackets. Nucleotide position was numbered on the basis of the coding sequence NM_007294.2. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

Site


Eugénie Mutez

Frederic Lepretre

Emilie Le Rhun

Lydie Larvor

Aurélie Duflot

Vincent Mouroux

Jean-Pierre Kerckaert

Martin Figeac

Kathy Dujardin

Alain Destée

Frédéric Leprêtre

‡ Aurélie Duflot

Marie-Christine Chartier-Harlin
email: marie-christine.chartier-harlin@inserm.fr

SNCA locus duplication carriers: from genetics to Parkinson's disease phenotypes

Keywords: Parkinson, alpha-synuclein, SNCA, duplication, overexpression Parkinson, alpha-synuclein, SNCA, duplication, overexpression

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Genetic factors have been shown to contribute to the etiology of Parkinson's disease (PD). Missense mutations of the SNCA locus (MIM# 163890) were the first to be identified as a cause of familial PD [START_REF] Polymeropoulos | Mutation in the alpha-synuclein gene identified in families with Parkinson's disease[END_REF]. More recently, multiplications of the SNCA locus were reported in 11 families (and 4 isolated patients) with PD (Lesage & Brice, 2009). This new type of mutation has shed light on the importance of the level of expression of the wild-type gene and suggests that overexpression of alpha-synuclein is enough to cause PD. Our comparison of the SNCA-duplicated patients in family P59 with the Iowa kindred carrying a SNCA triplication prompted us to suggest that the severity of PD correlates with the gene dosage [START_REF] Singleton | Alpha-synuclein locus triplication causes Parkinson's disease[END_REF][START_REF] Chartier-Harlin | Alpha-synuclein locus duplication causes familial Parkinson's disease[END_REF][START_REF] Ross | Genomic investigation of α-synuclein multiplication and Parkinsonism[END_REF][START_REF] Ibáñez | Alpha-synuclein gene rearrangements in dominantly inherited parkinsonism: frequency, phenotype, and mechanisms[END_REF]. However, the rarity of this multiplication phenomenon makes it difficult to know whether the phenotype is due to SNCA alone or to other genes involved in the same genomic rearrangement. For example, the cognitive dysfunction seen in some subjects might be due in part to the presence of MMRN1 (MIM# 601456) in the multiplicated locus [START_REF] Nishioka | Clinical heterogeneity of α-synuclein gene duplication in Parkinson's disease[END_REF]. This gene is also present in the duplicated region of the P59 kindred also reported as FPD-131. This duplication was evaluated as the longest described to date and was expected to encompass 33 to 34 genes [START_REF] Ibáñez | Alpha-synuclein gene rearrangements in dominantly inherited parkinsonism: frequency, phenotype, and mechanisms[END_REF]. Moreover, it has been suggested that non-allelic, homologous recombination (NAHR) causes the multiplication of this locus (with mediation by low copy repeats (LCRs)) [START_REF] Ross | Genomic investigation of α-synuclein multiplication and Parkinsonism[END_REF]. However, characterization of the exact breakpoint sequence will be required to confirm or refute this hypothesis.

Hence, in the present study, we sought to (i) expand and deepen our description of the clinical phenotype of all the members of the P59 kindred, (ii) determine the level of expression of SNCA in both lymphoblastoid cell lines and peripheral blood mononuclear cells (PBMCs), (iii) identify the exact number of genes present at the genomic SNCA locus, (iv) explore the variations of expression of the duplicated genes in PBMCs and (v) define the exact breakpoint sequence and the mechanism underlying this locus multiplication.

MATERIALS AND METHODS

Patients

P59 was the first SNCA duplication kindred to be identified within a set of 9 families in the Parkfanord study [START_REF] Chartier-Harlin | Alpha-synuclein locus duplication causes familial Parkinson's disease[END_REF]. Further screening of 149 families did not reveal any other SNCA duplication carriers and confirmed the rarity of this type of event. Moreover, no deleterious punctual mutation in SNCA, LRRK2 (MIM# 609007) or GBA (MIM# 606463) genes was found in the affected members of this parkinsonian kindred with autosomal dominant inheritance (data not shown). In order to describe the clinical phenotype in detail, after obtaining IRB approval and informed consent, the ten members of family P59 were examined in a movement disorder clinic by physicians trained in PD and blinded to the genetic status of the subjects. The diagnosis of PD was based on the United Kingdom Parkinson's Disease Society Brain Bank criteria. Extensive neuropsychological investigations (including tests known to be particularly sensitive to cognitive dysfunction in PD) were performed on all participants. Global cognitive efficiency was assessed using the Mattis dementia rating scale (MDRS). Test procedures evaluating working and episodic memory and executive function have been described elsewhere [START_REF] Dujardin | A case of severe dysexecutive syndrome consecutive to chronic bilateral pallidal stimulation[END_REF].

DaTSCAN® single-photon emission computed tomography

Single-photon emission computed tomography (SPECT) with the radioligand ( 123 I) FP-CIT (DaTSCAN) was performed in all subjects. Interpretation was based on semiquantitative analysis of uptake in the caudate nucleus and the putamen (Binding Index calculated by using the occipital cortex as the radioactivity uptake reference).

SNCA gene dosage

The SNCA gene dosage was determined in a semi-quantitative PCR with genomic DNA (ABI Prism 7700 sequence detector, Applied Biosystems, Foster City, USA). Exons 1 and 7 of SNCA were amplified for each member of the P59 kindred. The ribosomal protein S18 (RPS18) gene (MIM# 180473) was amplified as an endogenous reference. DNA sample ND00139 from the Iowa kindred (http://ccr.coriell.org/) was used as a positive control of triplication of the SNCA locus [START_REF] Singleton | Alpha-synuclein locus triplication causes Parkinson's disease[END_REF]. All the primer sequences used for this study are given in Supp. Table S1.

Fluorescent in situ hybridization analysis

We derived Epstein-Barr-virus (EBV)-transformed lymphoblastoid cell lines for each subject and we resampled PBMCs from alive subjects. Fluorescent in situ hybridization (FISH) was performed using bacterial artificial chromosome clones from the RP11 library according to standard procedures. These clones include RP11-115D19 (encompassing a part of the SNCA gene), RP11-77E12 (encompassing parts of the SNCA and MMRN1 genes) and RP11-347P22 (encompassing the FAM190A gene).

Determination of the size of the duplication using comparative genomic hybridization (CGH) arrays

Genomic DNA from 2 subjects was labeled with Cy3 and Cy5-dCTP, respectively, then 244k CGH arrays (Agilent, Santa Clara, CA, USA) were scanned according to the manufacturer's instructions. The acquired images were analyzed using Agilent's CGH Analytics software. Spot intensities were corrected for the background and only spots with both Cy5 and Cy3 signal intensities at least 1.7 times greater than the local background were selected. Data were normalized against the median of Cy5/Cy3 ratios from all spots. At least 3 consecutive probes with an abnormal ratio (arbitrarily set between -0.3 and 0.3) indicated a genomic multiplication or deletion event.

Sequence analysis of the duplication junction

We used a PCR walking strategy to pinpoint the exact breakpoints of the duplicated segment. As reported previously [START_REF] Ross | Genomic investigation of α-synuclein multiplication and Parkinsonism[END_REF], rearrangements may occur within DNA repeat structures such as Alu repeat subtypes or long interspersed nuclear elements (LINEs). Sequence and genomic information on the locus was obtained from the UCSC Human March 2006 Assembly database (www.genome.ucsc.edu). The centromeric and telomeric flanking limits observed in our CGH analysis included two LINE-1 elements. These two LINEs (L1PA2 at 5' and L1PA4 at 3') were selected for further analysis. Primers were designed to amplify the theoretical breakpoint in these regions. The experiment was repeated until a PCR fragment of about 500bp was obtained for sequencing. Both strands were sequenced using the ABI BigDye Terminator Kit v. 3.1 (Applied Biosystems).

SNCA relative expression analysis

RNA from immortalized leukocytes was extracted with the RNeasy® mini kit and was reverse-transcribed using the QuantiTect® Kit (Qiagen, Courtaboeuf, France). Next, the SNCA cDNA was PCR-amplified. The method enables the estimation of the relative expression of SNCA compared to three reference genes (RPS18, GAPDH MIM# 138400 and β-actin ACTB MIM# 182790) using SYBR green PCR master mix on an 7500 Real-Time PCR System (Applied Biosystems) following the manufacturer' instructions. Standard curves of each amplified gene were created to calculate the PCR efficiency. The relative expression levels were calculated method using the comparative C T method [START_REF] Schmittgen | Analyzing real-time PCR data by the comparative C(T) method[END_REF]. All PCR reactions were performed at least in duplicate. We compared SNCA gene expression in immortalized cell lines derived from (i) Iowa kindred member carrying a SNCA triplication, (ii) two P59 subjects with SNCA duplication and (iii) seven controls (three P59 subjects without SNCA duplication and four unrelated controls).

Next, we conducted a second SNCA expression analysis in PBMCs from subject III4 in duplicate and 15 healthy unrelated controls with the same procedure using RPS18 as reference gene.

Microarray procedure

To define the expression of the genes present within the duplication, we performed Agilent one-color whole human genome 44K microarrays according to manufacturer's instructions. The experimental design included the subject III4 in triplicate and 20 unrelated controls free of any personal or familial neurological disorders (mean age 58.9 ± 13.4) and the microarray procedure and normalization was described previously (Mutez et al., 2010). Data analyses were performed with GeneSpring GX 7.3.1 software from Agilent. A Volcano plot was used to identify differentially expressed genes by comparing the SNCA duplication carrier in triplicate to the controls, using a ≥1.2fold change and p<0.05 as the significance threshold (Welch's t-test). Furthermore, we compared our results with the significantly deregulated genes (p<0.05 t-test) from (i) an Affymetrix transcriptomic analysis of PBMCs between 50 PD patients and 22 controls from the GEO dataset GDS2519 (www.ncbi.nlm.nih.gov/sites/gds) [START_REF] Scherzer | Molecular markers of early Parkinson's disease based on gene expression in blood[END_REF] and (ii) an Affymetrix transcriptomic analysis of substantia nigra between 16 PD patients and 9 controls from the GEO dataset GDS2821 [START_REF] Lesnick | A genomic pathway approach to a complex disease: axon guidance and Parkinson disease[END_REF]. Family P59's pedigree is presented in Fig. 1A. The study subjects included 4 PD patients (II1, II2, III1 and III2) and 6 asymptomatic or paucisymptomatic individuals (III3, III4, III5, III6, III7 and III8). Subject II1 had deceased before the study and no DNA sample was available -only historical data were noted. His age at PD onset was 39 and the disease duration was 23 years. Subject II2 presented a classical PD phenotype with rest tremor, bradykinesia, rigidity and asymmetrical onset at the age of 65 and a disease duration of 19 years. Subject III1 began his disease at 48 years old with bradykinesia. He developed a classical PD with Levodopa response. At death, the disease duration was 9 years. Subject III2 was diagnosed with PD at age of 40 and she did not present any sign of dementia. At time of the last evaluation, she was bedridden and met the criteria for PD dementia [START_REF] Emre | Clinical diagnostic criteria for dementia associated with Parkinson's disease[END_REF] with global efficiency degradation, working memory impairment, episodic memory disorder and dysexecutive syndrome; she deceased a few months later. Her disease duration was 18 years. Subject III3 presented a slight postural tremor and a dysexecutive syndrome with a MDRS score of 131/144. Subject III4 was 52 at evaluation and presented a rigidity of the left arm and also a global cognitive efficiency degradation (MDRS score 130/144), a working memory impairment and a dysexecutive syndrome. Subject III5 was 58 and presented a slight bradykinesia without cognitive complaint and a MDRS score of 136/144. Subject III6 had presented mystic and megalomaniac delirium at the age of 37. He had been treated with neuroleptics for 10 years and the symptoms had never recurred. He was 47 at time of evaluation and showed a postural tremor and bradykinesia related to the neuroleptics' use. There was a cognitive complaint with working memory impairment, episodic memory disorder and dysexecutive syndrome. The MDRS score was 136/144. No parkinsonian clinical or neuropsychological abnormality was observed for subjects III7 and III8 (MDRS scores of 141 and 144/144, respectively). Apart from osteoarthritis, all these subjects did not present another debilitating condition, for example cardiovascular, hematologic disorders or cancer.

RESULTS

Subjects (clinical evaluation and DaTSCAN results)

DaTSCAN SPECTs were normal for subjects III3, III5, III6, III7 and III8. Conversely, there were a severe defect in putamen and pallidum with right predominance for subject III2 and a hypo-uptake in right putamen for subject III4. DaTSCAN images and semi-quantitative analyses for subjects III2, III3 and III4 are presented in Fig. 1B and1C.

SNCA gene dosage and FISH

By using semiquantitative PCR to determine gene dosage, we found 4 subjects (II2, III1, III2 and III4) with a heterozygous SNCA duplication of exons 1 and 7 (Fig. 2A). To provide independent confirmation, FISH was performed in lymphoblastoid cell lines from the different family members. Again, a SNCA locus duplication was found in subjects II2, III1, III2 and III4 with 3 FISH probe signals (Fig. 2B). Moreover to evaluate if mosaicism could be present, we scored the FISH probe signals in a minimum of 100 interphase nuclei from non immortalized lymphocytes from subject III4 and controls (III3, III5, III6, III7 and III8). The duplication signals were present only in subject III4 in more than 80% of the interphase cells.

Identification of the duplicated genomic segment

High-density CGH array analysis brought a better definition than the microsatellite markers previously used [START_REF] Ibáñez | Alpha-synuclein gene rearrangements in dominantly inherited parkinsonism: frequency, phenotype, and mechanisms[END_REF] and showed that the duplication spanned 4.928 Mb and encompassed 31 known and putative genes (Fig. 3A, Table 1) instead of the expected 33 to 34 genes. The location of the probes at the extremity of the duplication indicated that the centromeric end of the duplication mapped to a 9555 bp region between exons 10 and 11 of the MAPK10 gene (MIM# 602897), whereas the telomeric end occurred within an 11188 kb region into the putative FAM190A gene (KIAA1680). The genes were annotated with their symbol, their description and RefSeq accession number. FC1 corresponds to the fold change (expression ratio) between subject III4 and the 20 controls. The fold change value was indicated for the significantly deregulated genes with the corresponding Agilent probe. FC2 corresponds to the fold change of the significantly deregulated genes between PD patients' PBMCs and controls obtained from the GEO dataset GDS2519; FC3 corresponds to the fold change of the significantly deregulated genes between PD patients' substantia nigra and controls obtained from the GEO dataset GDS2821. NS = no significant difference between the PD patients and controls and A = absent flag in the microarray procedure. The last column corresponds to the functions and associated diseases linked to the genes (http://www.ncbi.nlm.nih.gov/omim or http://www.ncbi.nlm.nih.gov/gene).

Sequence analysis of the duplication junction

By using a PCR walking strategy, we sequenced PCR amplicons to produce the breakpoint junction between the L1PA4 and L1PA2 elements (Fig. 3B, 3C and3D). The junction of these two elements results in the deletion of 349 bases (301 from L1PA4 and 48 from L1PA2). The analyses of the L1PA4 and L1PA2 sequences with the L1Base (Penzoker et al., 2005), indicated that these 2 LINEs sequences had inactive ORF. We identified 95% sequence similarities for 751 pb between L1PA4 and L1PA2 using BLAST program (http://blast.ncbi.nlm.nih.gov/), suggesting that the duplication event was generated by NAHR between LCRs [START_REF] Inoue | Molecular mechanisms for genomic disorders[END_REF].

Level of expression of the duplicated genes

SNCA level expression in lymphoblastoid cell lines from controls (2 copies), duplication carriers (3 copies) and triplication carrier (4 copies) revealed an expression gradient which correlated with the number of SNCA copies using 3 reference genes (Fig. 4A). Since the expression phenotype of lymphoblastoid cells in culture may change with the EBV transformation [START_REF] Lee | Expression phenotype changes of EBV-transformed lymphoblastoid cell lines during long-term subculture and its clinical significance[END_REF], we decided to confirm these results using fresh leukocytes. We were able to obtain PBMCs from subject III4 since subject III2 had deceased meanwhile. Again, we observed an overexpression of SNCA of 2.38 fold (± 0.41) in the III4 PBMCs compared to 15 healthy controls (Fig. 4B). To extend these results to the other duplicated genes, we used a transcriptomic approach with Agilent microarrays. Among the duplicated interval, 14 genes were significantly deregulated between the 3 RNA samples from subject III4 and 20 unrelated controls (Table 1). Again, we observed a SNCA overexpression in PBMCs from subject III4. Of interest, all the significantly deregulated genes present in the duplication showed an overexpression. Moreover, we compared our results with the literature data from GEO datasets. We observed a significantly deregulation for 3 genes belonging to the duplicated segment in PBMCs from PD patients (GDS2519) and for 10 genes in the substantia nigra from sporadic PD patients (GDS2821) (Table 1).

DISCUSSION

The French P59 kindred provided us with the opportunity to describe the relationship between the genotype and the clinical phenotype in parkinsonian conditions caused by largest known duplication of the SNCA locus. The clinical features were similar to those of classic PD, with mid-life disease onset and late-onset dementia. In addition, we identified a subject (III4) at a paucisymptomatic stage of the disease; the subject carried the SNCA locus duplication and displayed an asymmetric dopaminergic defect in DaTSCAN SPECT images. Hypo-uptake has not been observed in the other asymptomatic SNCA duplication carriers [START_REF] Ahn | alpha-Synuclein gene duplication is present in sporadic Parkinson disease[END_REF]. The disease being age-dependant, this difference might be explained by their younger age (from 44 to 51). Our data confirm that DaTSCAN can demonstrate a striatal dopaminergic deficiency even in a presymptomatic stage of the disease and suggests that subject III4 is at a more advanced stage of the disease and might soon develop the entire PD phenotype. However, the interval between SPECT abnormalities and PD development remain unknown.

In the present study, we showed that the expression levels in immortalized cells from two affected members were significantly elevated. The overexpression correlated with the number of multiplicated SNCA alleles and the clinical phenotype, since SNCA expression was greater in the triplicated cell line than in duplicated cell lines. Furthermore, we confirmed its overexpression with a fold change of 2.38 in fresh leukocytes from the P59 carrier compared to controls. Given that the functional effect of duplication cannot often be confirmed by examination of the substantia nigra from deceased patients, we believe that analysis of SNCA expression levels of lymphoblastoid cell lines or PBMCs is a valuable investigational technique. These findings demonstrate a biological dosage effect of SNCA locus multiplication in human cells. Our data further emphasize the importance of SNCA expression levels on the development of PD. SNCA overexpression was also recently implicated in the PD pathogenesis associated with the SNCA A53T substitution with an allelic imbalance and an epigenetic regulation (Voutsinas et al., 2010). Such overexpression may also contribute to sporadic cases since an association exists between the PD risk and the REP1 polymorphism within the SNCA promoter, which controls the level of alpha-synuclein expression in PD patients [START_REF] Maraganore | Collaborative analysis of alpha-synuclein gene promoter variability and Parkinson disease[END_REF].

In addition to SNCA, we confirmed that thirty other genes or putative transcripts are present within this 4.928 Mb, which is thus the largest identified SNCA duplicated locus leading to PD. To investigate their potential involvement in the PD phenotype, we analyzed their expression in the PBMCs from the P59 carrier by a transcriptomic approach. Nine of them were not expressed at a sufficient level in these cells and seven of them are not dosage-sensitive in those cells (Table 1). Two probes confirmed the overexpression of SNCA in the duplication carrier PBMCs. Moreover, we demonstrated that 13 other genes present in the duplicated locus were significantly overexpressed. Especially, FAM13A (MIM# 613299), MMRN1, NAP1L5 (MIM# 612203) together with SNCA presented a two-fold overexpression in these cells (Table 1). To determine if these genes were overexpressed in other PD conditions, we compared these results with published data. Interestingly, these genes are not overexpressed in PBMCs from other PD patients compared to controls. Thus, their overexpression seems directly linked to the presence of the duplication and may impact the phenotype of this patient. We cannot preclude that RNA overexpression will not lead to a protein dysfunction. Nevertheless, 10 out of the 31 duplicated genes were deregulated in substantia nigra from sporadic PD subjects (Table 1) suggesting that these genes might contribute to the disease development. Among them, expression of FAM13A gene was increased both in PBMCs duplication carrier and brain tissue of PD patients compared to controls, but was slightly decreased in other PD PBMCs. Although little is known on its role, this may suggest a particular role of FAM13A in the substantia nigra of duplicated patients that has not been found yet. MMRN1 is also overexpressed in the duplicated carrier PBMCs and since, when multiplicated, it was suggested to play a role in the clinical features notably on the cognitive impairment [START_REF] Nishioka | Clinical heterogeneity of α-synuclein gene duplication in Parkinson's disease[END_REF]; we also investigated cognitive functions. In our kindred, subject III2 met the criteria for dementia 17 years after PD onset [START_REF] Emre | Clinical diagnostic criteria for dementia associated with Parkinson's disease[END_REF]. Our tests revealed that two subjects (III2 and III6) had both cognitive complaints and cognitive dysfunction. However, only III2 carried the SNCA duplication. Likewise, we observed poor cognitive test results for subjects III3, III4 and III5 but only III4 carried the pathogenic mutation. These data support the hypothesis whereby the 30 other genes present within the duplication do not significantly contribute to the cognitive impairment. However, we cannot rule out the possibility that one or more of these genes may exacerbate susceptibility to cognitive dysfunction already present in the kindred members or contribute to another clinical condition. Indeed, osteoarthritis was present in duplication carriers and recent analyses reported a slightly increased risk for PD among patients with osteoarthritis [START_REF] Rugbjerg | Risk for Parkinson's disease among patients with osteoarthritis: a Danish cohort study[END_REF]. Nevertheless, since arthritis is one of the most common diseases affecting almost every person over age 60, to date it is difficult to draw further conclusion, on the presence of duplicated genes involved in osteoblast maturation or mineralization and the arthritis reported in the patients carrying the duplication (Table 1, last column). This table also indicates that several variants in the vicinity of SNCA, GPRIN3 (MIM# 611241) and MMRN1 were associated with PD risk by genome wide association studies (GWAS) [START_REF] Satake | Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease[END_REF][START_REF] Simón-Sánchez | Genome-wide association study reveals genetic risk underlying Parkinson's disease[END_REF]. However, GWAS data alone are not able to define these genes as a molecular cause in the disease. These single nucleotide polymorphisms are indeed localized in the intergenic regions and without further analyses showing the functionality of these variants; they could either reflect the association with other causative polymorphisms affecting the SNCA gene or the involvement of another sequence within this locus. Today, the most plausible explanation is the overexpression of SNCA in the disease, but other mechanisms such as mRNA stability, preferential splicing or miRNA might also exist. Indeed, SNCA gene showed a reduction of expression of 0.6 and 0.7-fold in idiopathic PD brain tissue and PBMCs, respectively; while it was overexpressed in P59 SNCA duplication carrier (Table 1). The significance of this decrease of total SNCA expression in idiopathic PD brain and PBMCs indicates that even if the clinical phenotype between sporadic PD and duplication carrier was similar, the underlying primary mechanisms can differ at least in a number of patients.

In the last part of the present study, we sought to define the duplication's boundaries and thus decipher the multiplication mechanism. The genome rearrangement was confirmed by several methods. We provided a better definition of the duplication's boundaries and demonstrated that 31 genes were located within the duplicated region. Next, by using a PCR walking strategy to sequence the breakpoint junction, we discovered that a rearrangement had occurred between two LINE-1 retrotransposons (L1PA2 and L1PA4) and not between Alu repeats, as it could have been expected [START_REF] Ross | Genomic investigation of α-synuclein multiplication and Parkinsonism[END_REF]. A 349 bp deletion was observed at the junction between the LINEs. Of the many transposable elements in the human genome (and which account for almost half of the latter), LINE-1 (L1) sequences are thought to be the major driving force behind genome expansion [START_REF] Babashok | Progress in understanding the biology of the human mutagen LINE-1[END_REF]. A transpositionally active L1 element comprises a 5'-untranslated region (UTR), two open reading frames (ORFs) (which notably code for proteins involved in RNA binding and genome integration), a 3'-UTR. However, most copies of L1 are 5'-truncated and often only consist of part of the 3'-UTR. The two L1 elements (L1PA2 and L1PA4) in family P59 had inactive ORFs. Even when these transposable elements are not active, they can nevertheless cause human disease by creating insertional mutations in genes and by contributing to genetic instability through NAHR (leading to rearrangements, such as duplications) [START_REF] Belancio | LINE dancing in the human genome: transposable elements and disease[END_REF]. In the duplicated locus, we identified a >750 pb length homology sequence between L1PA2 and L1PA4 with several perfect sequence matches; which is a required condition to generate NAHR [START_REF] Inoue | Molecular mechanisms for genomic disorders[END_REF]Conrad et al., 2009). Moreover, this region of chromosome 4q21 has a high DNA flexibility and A/T-content probably which is suspected to promote the occurrence of fragile sites [START_REF] Rozier | Characterization of a conserved aphidicolin-sensitive common fragile site at human 4q22 and mouse 6C1: possible association with an inherited disease and cancer[END_REF]. Taken as a whole, these data strengthen the idea that homologous recombination has preferred positions and architectural DNA characteristics, since rearrangement breakpoints are associated with LCRs more frequently that would be expected by chance alone [START_REF] Shaw | Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease[END_REF].

In conclusion, for the first time, we identified the exact sequence of the breakpoints from SNCA locus multiplication carrier and demonstrated the involvement of LINEs in SNCA locus duplication which led to SNCA overexpression and abnormal dopamine metabolism in the carriers. We showed that DaTSCAN SPECT, immortalized cell lines and PBMCs are effective tools for demonstrating the preclinical and biological features of PD. While our data highlight the importance of the SNCA overexpression as the major cause of the PD phenotype in our kindred, we also demonstrate that the duplication affects the expression of other genes. Therefore their potential role in PD phenotype should deserve further attention. Concerning the PCR walking strategy, the numbers correspond to the primers as indicated in Figure 3C.

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Figure 1 .

 1 Figure 1. Presentation of the kindred's pedigree, with the DaTSCAN results. (A) Pedigree of the P59 family. Subject III1 was the probant (arrow). Subject III4 was colored in gray, since she carried the SNCA duplication at a prodromal stage of the disease. (B) DaTSCAN images for subjects III2, III3 and III4. (C) Semi-quantitative analysis of radioligand uptake in the brain, with details of the Binding Index (left/right values). Abnormal values are given in red type.

Figure 2 .

 2 Figure 2. Identification of SNCA duplication by gene dosage and FISH (A) SNCA gene dosage assay for exons 1 and 7. The dosage was normalized to the RPS18 gene and compared with a triplication carrier from the Iowa kindred. (B) Identification of 3 FISH signals (represented by arrows) in interphase nucleus showing the duplication.

Figure 3 .

 3 Figure 3. Identification of the duplicated segment (CGH) and the breakpoint junction. (A) CGH analysis between subjects III4 and III5, showing the duplication highlighted in pink (at locus 4q21.3-q22.1) encompassing 31 genes. (B) Agilent CGH probes enclosing L1PA2 and L1PA4. These probes are also represented in A. (C) Graphical representation of the hypothetical rearrangement and duplication, showing the location of each of the 12 probes used in PCR and the sequence analysis. The deleted segment is represented in red. (D) Breakpoint junction sequence for the L1PA4 and L1PA2 elements (in red, the 349 deleted bases). The bases involved at the breakpoint junction are capitalized.

Figure 4 .

 4 Figure 4. Expression of SNCA in lymphoblastoid cell lines and PBMCs. (A) SNCA mRNA expression levels in immortalized leukocytes from subjects with SNCA locus multiplications. The duplicated group corresponds to lymphoblastoid cell lines from subjects III2 and III4. The triplicated cell line was obtained from the Iowa kindred. Expression levels were normalized against healthy controls. The 3 reference genes were GAPDH, RPS18 and ACTB. (B) SNCA mRNA expression levels in PBMCs of 15 healthy controls and the III4 subject in duplicate. The bars indicated the average values, arbitrarily set at 1 for the controls. The reference gene was RPS18.

  Stavrou EF, Karousos G, Dasoula A, Papachatzopoulou A, Syrrou M, Verkerk AJ, van der Spek P, Patrinos GP, Stöger R, Athanassiadou A. 2010. Allelic imbalance of expression and epigenetic regulation within the alpha-synuclein wild-type and p.Ala53Thr alleles in Parkinson disease. Hum Mutat 31:685-691.

Table 1 : List of the 31 known and putative genes present in the duplicated locus.

 1

	Symbol	Gene Name	RefSeq	Agilent Probe	FC1	FC2	FC3	Function/Associated disease
	ABCG2	ATP-binding cassette, sub-family G, member 2	NM_004827	A_23_P18713	NS	NS	NS	Chemoresistance Colon, breast cancer
	AFF1	AF4/FMR2 family, member 1	NM_005935	A_24_P414332	1.44	NS	NS	Acute leukemia
	C4ORF36	chromosome 4 open reading frame 36	NM_144645	A_23_P377339	A		A
	DMP1	dentin matrix acidic phosphoprotein 1	NM_004407	A_23_P133153	NS	A	A	Dentinogenesis Ricket, osteomalacia
	DSPP	dentin sialophosphoprotein	NM_014208	A_23_P373541	A	NS	NS	Dentinogenesis
	FAM13A	family with sequence similarity 13, member A	NM_014883	A_23_P370651 A_23_P7132	2.24 A	0.85	1.51	Chronic obstructive pulmonary disease
	FAM13AOS	FAM13A opposite strand	NR_002806	A_24_P614362	1.63		A
	FAM190A	family with sequence similarity 190, member A	NM_001145065	A_32_P306678	A		A	Attention deficit hyperactivity disorder
	GPRIN3	GPRIN family member 3	NM_198281	A_24_P76649	1.36		0.65	Parkinson's disease GWAS
	HERC3	hect domain and RLD 3	NM_014606	A_23_P121586	A	NS	1.41	Ubiquitin-ligase
	HERC5	hect domain and RLD 5	NM_016323	A_23_P110196	NS	NS	1.40	Ubiquitin-ligase Cell cycle
	HERC6	hect domain and RLD 6	NM_017912	A_23_P250353	1.38	NS	0.68	Ubiquitin-ligase
	HSD17B11	hydroxysteroid (17-beta) dehydrogenase 11	NM_016245	A_23_P21644 A_23_P408271	1.65 1.58	NS	NS	Short-chain alcohol dehydrogenases
	HSD17B13	hydroxysteroid (17-beta) dehydrogenase 13	NM_178135	A_24_P362969	A			Short-chain alcohol dehydrogenases
	IBSP	integrin-binding sialoprotein	NM_004967	A_23_P144549	A	NS	A	Skeletal mineralization
	KLHL8	kelch-like 8	NM_020803	A_23_P328729 A_24_P54808	1.53 1.44		1.38 1.25	Ubiquitin-ligase
	MAPK10	mitogen-activated protein	NM_002753	A_23_P45025	A	NS	NS	Epilepsia (Lennox-Gastaut)

Table S1 : Primer sequences used for gene dosage, SNCA expression, PCR walking strategy and breakpoint junction sequencing.

 S1

	PCR Use	Primer sequences (5'-3')
	SNCA exon 1 gene dosage	CCTTCAAGCCTTCTGCCTTTC CGAATGGCCACTCCCAGTT
	SNCA exon 7 gene dosage	TCTTTGCTCCCAGTTTCTTGAGA TGGAACTGAGCACTTGTACAGGAT
	SNCA expression	ATGTAGGCTCCAAAACCAAG TACCCTTCCTCAGAAGGCA
	RPS18 expression	CCTGGATACCGCAGCTAGGA GCGGCGCAATACGAATGCCCC
	ACTB expression	CACGGCATCGTCACCAACT AGCCACACGCAGCTCATTG
	GAPDH expression	TACATGTTCCAATATGATTCCACC ACCTTCTTGATGTCATCATATTTG
		1 TCAGAAGAGGCATATTACAT
		2 GTGACAACATTCTAAAAGGG
		3 TTCCTTATGATTCCACTTAG
		4 GCACAGACAAACAAAAAGAC
		5 AAGACCATCGAGACTAGGAA
	PCR Walking Strategy and	6 ACACCTCTACGCAAATAAAC
	Break junction sequencing	7 CCAAGTCAATCCTAAGCC
		8 AATCATGCTGCTATAAAGAC
		9 AAAAGTGGGTGAAGGATATG
		10 CCCTTTTAGAATGTTGTCAC
		11 CAAAACAGAGAAAACAGAAG
		12 ATGTGGGTCTGTAGCAAC

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ACKNOWLEDGMENTS

The authors thank the subjects for their participation in this research study. We wish to thank Professor Marc Steinling (Nuclear Medicine Department, Lille University Hospital, Lille, France) for the DaTSCAN analyses, Dr Joris Andrieux (Laboratory of Medical Genetics, Lille University Hospital, France) for his help in the FISH experiment. We are particularly grateful to the Institut de Recherches sur le Cancer de Lille for its assistance.

Contract grant number: PHRC PARKFANORD (2005/1914).