Probe-based quantitative PCR assay for detecting constitutional and somatic deletions in the NF1 gene: application to genetic testing and tumor analysis.
Deletions of the NF1 gene also occur somatically, e.g., in tumors such as dNFs arising in patients with NF1. A key event in the initiation of neurofibroma development is biallelic inactivation of the NF1 gene (13-15). dNFs are composed of different cell types, but only Schwann cells (SCs) bear a double inactivation of the NF1 gene (16-18). Approximately 75% of the somatic mutational spectrum of the NF1 gene in NF1associated dNFs is accounted for by point mutations (i.e., nonsense, missense, small insertion/deletion, and splicing mutations) and intragenic deletions. The remainder (approximately 25%) present as a loss of heterozygosity in large genomic regions that include the NF1 gene (19, 20). The mechanistic causes of loss of heterozygosity are mitotic recombination in 62% of cases and genomic deletions of 80 kb to 8 Mb in the remaining 38% (20).
To date, NF1 constitutional deletions have been identified with multiple techniques, such as microsatellite analysis with intragenic markers (21-23), interphase fluorescence in situ hybridization (FISH) analysis via the use of probes within and flanking the NF1 gene (11,22,24,25), multiplex ligation-dependent probe amplification (MLPA) with commerciallyavailable kits (23, 26), and arraycomparative genomic hybridization (27). Microsatellite analysis (20, 28, 29), FISH (30), MLPA (19, 20, 29, 31), and array comparative genomic hybridization (32) have also been used to characterize somatic deletions encompassing the NF1 gene, together with other techniques, such as single-nucleotide polymorphism (SNP) analysis (32), paralog ratio analysis (20, 32), and SNP array (20).
Quantitative real-time PCR (qPCR) has been used to confirm intragenic constitutional deletions in NF1 (26); however, qPCR has not been used routinely to detect constitutional NF1 microdeletions. qPCR represents an alternative methodology because of its high analytical sensitivity and low imprecision, its relatively low screening cost, and its fast assay-development time. We describe a probe-based qPCR assay for detecting all 4 types of NF1 constitutional microdeletions and somatic deletions that affect the NF1 region. We also compare our qPCR results with those obtained via other techniques.
Materials and Methods
PATIENTS AND SAMPLES
This study included 59 samples: 14 venous blood samples previously tested by FISH and microsatellite markers and found to carry a constitutional deletion of NF1 in each case (21, 33); 16 samples from surgically excised dNFs; and 5 samples from selective SC cultures ([SC.sup.NF1-/-]) derived from dNFs, previously analyzed by MLPA, paralog ratio analysis, and SNP array and found to bear a somatic NF1 deletion in each case [(20) and data not shown]. In addition, we used a set of 24 control samples, each of which presents 2 copies of the NF1 gene: 10 dNF samples, 5 blood samples, 4 skin samples, 3 fibroblast samples, and 2 [SC.sup.NF1] samples. All patients gave written informed consent for the molecular studies performed.
Total DNA was extracted from blood samples with different methodologies: the salting-out procedure, the Wizard Genomic DNA Purification Kit (Promega), and the FlexiGene DNA Kit (Qiagen). DNA was extracted from dNFs and skin with the Gentra Puregene Kit (Qiagen). The QIAamp DNA Mini Kit (Qiagen) was used to extract DNA from cells (SCs and fibroblasts). All extractions were performed according to the manufacturer's instructions. A NanoDrop[R] spectrophotometer was used to quantify DNA and to measure purity and quality. DNA integrity was assessed by gel electrophoresis. All DNA samples included in this study presented with high purity and integrity.
qPCR EXPERIMENTAL SETUP
Primers and probes for the qPCR assay were developed with Roche Universal ProbeLibrary (UPL) technology. UPLs are hydrolysis probes of 8- to 9-nucleotide locked nucleic acid that are labeled at the 5' end with the fluorescent dye 6-carboxyfluorescein and at the 3' end with a quencher dye. The combination of the hydrolysis probe and the primer pair provided the specificity required for each particular genomic locus of interest. The design of each of the primers (desalted and purified; Sigma Life Science) was subjected to an in silico PCR and BLAT search analysis to evaluate their specificity, which was later assessed experimentally by PCR and agarose gel electrophoresis before qPCR experiments were conducted. The sequences of the primers and probes used in this study are listed in Table 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/ vol59/issue6.
qPCR experiments were performed in a LightCycler[R] 480 Real-Time PCR System with white Multiwell Plate 384 plates (Roche Diagnostics). Conditions for amplification were as follows: 95[degrees]C for 10 min; 45 cycles of 95[degrees]C for 10 s, 60[degrees]C for 30 s, and 72[degrees]C for 1 s; and 40[degrees]C for 30 s. The linear dynamic range (LDR) and efficiency (E) of the primers were evaluated (see Table 1 in the online Data Supplement). Then, to minimize technical variation between runs, we divided all samples under study into 3 panels of 20 samples each according to the sample-maximization method (34, 35). Each reaction in all experiments included 5 ng DNA template, 4 [micro]L of 2X LightCycler 480 Probes Master Mix (Roche Diagnostics), 0.1 [micro]mol/L UPL probe, and 0.75 [micro]mol/L of each primer, in a total volume of 8 [micro]L [except for reactions for the L1PA locus--a long interspersed repeat element (LINE) in the L1 family--which included 0.2 [micro]mol/L UPL probe and 1.2 [micro]mol/L of each primer]. PCRs for each primer set and sample were performed in triplicate. Each set of PCR assays included both negative controls without template and a dilution series of a particular template for calculating the E value of the primer pair in each run. In addition, a calibrator sample of known copy number was included in triplicate in every assay.
qPCR DATA ANALYSIS
We designed a Microsoft Excel spreadsheet to analyze qPCR data for copy number calculations. We used formulas from the qBase relative quantification framework (34), which are based on the Pfaffl method (36). In brief, we averaged the 3 quantification cycle (Cq) numbers obtained from each triplicate with the second-derivative maximum method in the Light-Cycler[R] 480 software [as long as the difference in Cq between the replicate with the highest value and the replicate with lowest value was <0.3 for all genes and <0.2 for the L1PA locus (35)]. We then calculated the [DELTA]Cq value for the difference between the unknown sample and the calibrator sample ([DELTA]Cq = [Cq.sub.unknown] " [Cq.sub.calibrator]). The relative quantity (RQ) was later calculated as: RQ = [E.sup.-[DELTA]Cq]. We calculated the normalized relative quantity (NRQ) as: NRQ = RQ/NF, where NF is the normalization factor. The NF in turn is the geometric mean of the RQ values for the 2 selected reference loci--the ADARB1 (adenosine deaminase, RNA-specific, B1) gene and the L1PA locus--for the particular sample. The value for the stability parameter, M (37), for this particular NF was previously calculated for the entire set of samples and was <0.2 (m = 0.168) (35). Finally, we calculated relative copy number (R N) as: R N = NRQ/RF, where RF is a rescaling factor. The RF is the geometric mean of the NRQ values of a set of 24 control samples bearing 2 NF1 copies. R N values close to 1 indicate the presence of 2 NF1 copies, and R N values close to 0.5 indicate an NF1 deletion. A 99% I for R N values indicating 2 NF1 copies was calculated for each interrogated locus (see Table 1 in the online Data Supplement).
We also used the MLPA technique to assess the NF1 copy number status of both samples with constitutional and somatic deletions. We performed MLPA reactions in duplicate with the SALSA MLPA Kit P122-C1 NF1 Area (MRC-Holland) and 40 ng DNA, in accordance with the manufacturer's instructions. Once ligated and amplified, PCR fragments were separated by capillary electrophoresis (ABI 3130xl Genetic Analyzer; Applied Biosystems). Peak intensities were analyzed with Peak Scanner Software (Applied Biosystems) and normalized for peak heights, as described elsewhere (20). In brief, peak height values were exported to an Excel spreadsheet. Relative probe signals were calculated for a particular sample by dividing the peak height of each of the 28 pairs of probes encompassing the NF1 region by the sum of the peak heights of the 11 pairs of reference probes (not located in the NF1 region). The ratio obtained for each individual relative probe height was then normalized for that specific probe to the mean obtained with 3 control samples (with 2 copies of the NF1 region). For genomic regions present in 2 copies in a sample, these calculations were expected to yield an RCN value of approximately 1.0. A value <0.8 was considered to indicate a deletion.
MICROSATELLITE MULTIPLEX PCR ANALYSIS
In parallel, we used microsatellite multiplex PCR analysis (MMPA) as previously developed in our laboratory (28) to assess the NF1 copy number status of tumor samples with NF1 somatic deletions and to calculate the percentage of cells within the tumors with 2 NF1 copies. This technique allows the simultaneous amplification of 16 microsatellite markers. MMPA reactions were performed in duplicate with the Multiplex PCR Kit (Qiagen) and 50 ng DNA. Information regarding the amplification protocol, data analysis, and calculations is described elsewhere (28).
[FIGURE 1 OMITTED]
DEVELOPMENT AND VALIDATION OF THE qPCR ASSAY
The qPCR assay was set up with UPL probes and their corresponding specific primer pairs. For copy number assessment, we selected 11 genomic loci distributed along a 2.8-Mb region encompassing the NF1 gene (Fig. 1). To distinguish Type-1, Type-2, Type-3, and atypical NF1 microdeletions specifically, we decided to interrogate 3 regions within the NF1 gene (NF1-5', NF1 -C, and NF1-3'), 5 loci that closely flank REP-A, REP-B, REP-C, SUZ12, and its pseudogene (SUZ12P1) [TBC1D29, TBC1 domain family, member 29; CRLF3, cytokine receptor-like factor 3; RNF135, ring finger protein 135; UTP6, UTP6, small subunit (SSU) processome component, homolog (yeast); RHOT1, ras homolog family member T1], and 1 locus within SUZ12P1 (SUZ12P1). Two other loci located distal to the NF1 gene [SSH2, slingshot homolog2 (Drosophila); PSMD11, proteasome (prosome, macropain) 26S subunit, non-ATPase, 11] were also included. In addition, the MAP2K4 (mitogen-activated protein kinase kinase 4) gene, located in 17p, was selected as a control (i.e., a locus with 2 copies). Moreover, we selected 2 reference genes, ADARB1 (21q22.3) and the LINE repetitive sequence L1PA (which is interspersed through the genome) to further normalize copy number data, as has been suggested (35).We chose UPL probes for our study because of their advantages of higher specificity, higher PCR E values, and avoidance of primer-dimer signal that hydrolysis probes show, in contrast to fluorescent dyes such as SYBR Green. We also chose UPL probes for their flexibility of use and their reduced cost compared with DNA-sequenced hydrolysis probes.
Some experiments were performed to validate the qPCR assay. First, a set of 2-fold dilutions of pooled DNA samples (80 to 0.156 ngper reaction in triplicate) was used to determine the LDR and the E value of the primers used (34). All designed primers showed a large LDR (at least 9 orders of magnitude) and high E values, which ranged from 1.88 to 2.11 for the loci analyzed (see Table 1 in the online Data Supplement).
Then, we determined the range of RCN values for a 2-copy status of the NF1 gene by analyzing 24 control samples. The mean (SD) NRQ for the 12 loci and 24 samples interrogated was 0.99 (0.08) (Fig. 2). The mean calculated 99% CI of RCN for the 12 loci was 0.81-1.23. An RCN value below the lower limit of the CI for a locus was considered to indicate deletion of that particular locus.
The qPCR assay also showed low intraassay and interassay imprecision (see Table 1 in the online Data Supplement).
[FIGURE 2 OMITTED]
DETECTION OF NF1 CONSTITUTIONAL DELETIONS
After validating the qPCR assay with a panel of 24 control samples, we tested the performance of the qPCR assay by using a set of 14 DNA samples previously characterized and bearing a constitutional deletion of the NF1 gene: 5 Type-1 deletions, 6 Type-2 deletions, and 3 atypical deletions. No Type-3 deletion was tested. All deletions were detected with the qPCR assay (Fig. 3; see Fig. 1 in the online Data Supplement). The mean (SD) RCN value within the deleted loci was 0.53 (0.07). Patient NF-01 bears a type 2 microdeletion. The DNA sample from this patient showed a mean RCN value of 0.68 (0.05) within the deleted loci. This result could reflect the presence of mosaicism for this microdeletion. Thus, when we considered all of the loci known to be deleted in our sample set, the qPCR assay showed 100% diagnostic sensitivityand 99.2% diagnostic specificity (see Table 2 in the online Data Supplement). The SUZ12 locus, which is essential for distinguishing a Type-1 deletion from a Type-2 deletion, showed 100% diagnostic sensitivity and 96.9% diagnostic specificity. These results were confirmed in a parallel analysis of these samples with the MLPA technique (see Fig. 1 in the online Data Supplement).
DETECTION OF NF1 SOMATIC DELETIONS
We also tested the performance of the qPCR assay for detecting somatic copy number losses by using samples from tumors and cells bearing different known somatic NF1 deletions. Because the [NF1.sup.-/-] cellular component is not total, not only within a dNF (and other NF1-associated traits) (17) but also in tissues showing mosaicism for a constitutional NF1 deletion, we first checked the reliability of the qPCR assay for detecting deletions in the context of mosaicism.
[FIGURE 3 OMITTED]
To assess the performance of the qPCR assay in admixtures of NF1-deleted and-nondeleted DNA samples, we set a cutoff value for the maximum percentage of NF1-nondeleted cells present in a tumor (or tissue) that the qPCR assay could tolerate and still detect the presence of a somatic NF1 deletion. To choose this cutoff, we checked the performance of the qPCR assay with several admixtures of 2 DNA samples, one with 2 NF1 copies and the other with a single NF1 copy. We prepared 11 serial dilutions of NF-59 (NF1 constitutional deletion) and NF-60 (sample with 2 NF1 copies) with different DNA percentages of the 2 samples in quintuplicate (i.e., 0% to 100% of the sample with 2 NF1 copies). For each admixture, the mean (SD) of the calculated RCN for the 6 loci (NF1-5', NF1 -C, NF1-3', CRLF3, RNF135, and UTP6) was plotted against the percentage of DNA with 2 NF1 copies present in the DNA admixture (Fig. 4A). To calculate the cutoff value for the percentage of cells with 2 NF1 copies that the qPCR assay could tolerate and still detect the presence of an NF1 deletion, we used the mean lower 99% confidence limit for all loci (0.81) (Fig. 4A). Hence, the qPCR assay detected NF1 somatic deletions in dNFs containing less than 56% of NF1-nondeleted DNA.
We also set a cutoffvalue for the MLPA technique by using the same serial DNA admixtures used for the qPCR. These samples were analyzed in duplicate, and for each admixture we plotted the mean (SD) of the calculated copy number of the 11 loci (from the CRLF3 3780 probe to the SUZ12 3786 probe) against the percentage of DNA with 2 NF1 copies present in the DNA admixture (Fig. 4B). We used an RCN value of 0.8 to obtain the cutoff value for the highest percentage of NF1 -nondeleted cells in tumors at which the MLPA assay was able to detect the presence of an NF1 deletion. This percentage was approximately 51%, similar to that of the qPCR assay.
To evaluate the ability of the qPCR assay to detect somatic deletions in dNFs and [SC.sup.NF1-/-] cells, we analyzed 16 DNA samples from dNFs and 5 [SC.sup.NF1-/-] samples bearing an NF1 somatic deletion. First, we used MMPAto calculate the percentage of DNA with 2 NF1 copies within tumor samples and cell cultures, as described elsewhere (28). The results with this technique also confirmed the presence of deletions in the NF1 region in these samples (28) (see Table 3 in the online Data Supplement).
[FIGURE 4 OMITTED]
The qPCR assay detected the presence of deletions in the NF1 region in all 16 dNF and [SC.sup.NF1-/-] samples tested that contained <56% of the NF1-nondeleted component (Fig. 5; see Fig. 2 in the online Data Supplement). When all loci known to be deleted in this sample set were considered, the qPCR assay showed 90.5% diagnostic sensitivity and 98.9% diagnostic specificity (see Table 2 in the online Data Supplement). The technique was still able to detect a deleted locus in all 4 tumor samples with >56% of NF1-nondeleted DNA, but the diagnostic sensitivity and specificity values were lower (see Table 2 in the online Data Supplement). The mean RCN value within the deleted loci in [SCNF.sup.1-/-] samples was 0.62 (0.11); this result probably denotes the presence of cells with NF1-nondeleted DNA in the SC cultures.
All qPCR results for the detection of somatic NF1 deletions in dNFs were confirmed with the results obtained with the parallel MLPA analysis (see Fig. 2 in the online Data Supplement).
[FIGURE 5 OMITTED]
Different techniques, including FISH, MLPA, and array comparative genomic hybridization, are currently being used to assess the presence of microdeletions involving the NF1 locus. A qPCR approach has also been developed to validate deletions involving specific exons within the NF1 gene that have previously been detected with MLPA analysis (26). In addition, qPCR has been used to detect low percentages of somatic NF1 point mutations (38); however, until the present study qPCR analysis had not been used to detect and distinguish between different types of NF1 microdeletions or to check for somatic second-hit deletions. We have developed a probe-based qPCR assay that detects both constitutional and somatic NF1 deletions in samples from NF1 patients by interrogating the copy number of an approximately 2.8-Mb region that includes the NF1 gene.
Our assay fulfills all of the essential aspects of the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines (39) and most of the desirable information (see Table 4 in the online Data Supplement). Our adherence to these criteria strengthens the reliability of the developed qPCR assayand the results we have obtained. The assay requires small amounts of DNA: Each reaction is performed in just 8 [micro]L, and only 15 ng per interrogated locus is required. qPCR reactions are highly efficient (E > 1.88) and amplify at similar rates with a large LDR. We used 5 ng DNA per PCR reaction, although smaller DNA quantities may be analyzed. Given all of the interrogated loci and replicates, a total of 210 ng is required per sample.
This assay enabled the detection of the 14 constitutional microdeletions and the 16 somatic deletions in dNFs containing <56% of NF1-nondeleted cells. The diagnostic sensitivity and specificity of our assay were near or at 100% when all of the interrogated loci were considered. For somatic deletions, the diagnostic sensitivity was around 90%, because the qPCR assay detected 2 copies for a few loci known to have a deletion. The presence of a deletion event in a dNF sample was considered, however, because most of the deleted loci within the 2.8-Mb NF1 region in that sample were detected as single copies. In addition, to increase diagnostic specificity and minimize the false-positive rate, we applied a stringent CI (99%) to assess for the absence of a deletion.
qPCR is a highly analytically sensitive, specific, and precise technique that has several advantages over other methodologies. It is cheap and fast. Determining the copy number status of different loci in several samples can be performed in just 3 h, including plate preparation, PCR amplification, and data analysis. Detecting copy number changes in samples of DNA extracted with different methodologies and kits or from different tissues and tumors can be challenging for many techniques, such as MLPA. qPCR, however, is very robust with respect to the DNA quality, thereby permitting the screening and comparison of DNA samples from different sources.
The reliable design of the qPCR assay we have described, along with the specific locations of the 11 interrogated loci within the NF1 region, allow accurate detection of Type-1, Type-2, and atypical NF1 microdeletions and are suitable for detecting the rare Type-3 microdeletions. The assay could be expanded, if required, to incorporate more loci for assessment. This qPCR assay is also capable of detecting deletions in the context of mosaicism, a feature important for constitutional Type-2 microdeletions and for somatic deletions found in dNFs.
We believe this qPCR assay could be incorporated into a genetic-testing setting as a useful diagnostic tool, either as a first screening step or as a validation technique for NF1 microdeletions (approximately 5% of NF1 cases). In addition, the assay allows the identification of somatic deletions in neurofibromas and other NF1 traits that require the double inactivation of the NF1 gene, when the deletion is present in at least 44% of the tissue sample.
Received August 16, 2012; accepted January 18, 2013.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Spanish Health Research Foundation: Instituto de Salud Carlos III (grant nos. PI081871, PI11/01609, and ISCIIIRTICC RD06/0020/1051), Government of Catalonia (grant no. 2009SGR290), and Asociacion Espanola Contra el Cancer (AECC). Expert Testimony: None declared. Patents: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Acknowledgments: We thank Dr. S. Villatoro for technical advice, H. Evans for manuscript correction, Serra laboratory members, NF ICO-IMPPC group members, and the Asociackm Espanola de Afectados de Neurofibromatosis for their constant support of our research.
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 Nonstandard abbreviations: NF1, neurofibromatosis type 1; dNF, dermal neurofibroma; REP, low-copy repeat; MPNST, malignant peripheral nerve sheath tumor; SC, Schwann cell; FISH, fluorescence in situ hybridization; MLPA, multiplex ligation-dependent probe amplification; SNP, single-nucleotide polymorphism; qPCR, quantitative real-time PCR; UPL, Universal ProbeLibrary (Roche); LDR, linear dynamic range; LINE, long interspersed repeat element; L1PA, LINE in the L1 family; Cq, quantification cycle; RQ, relative quantity; NRQ, normalized relative quantity; NF, normalization factor; RCN, relative copy number; RF, rescaling factor; MMPA, microsatellite multiplex PCR analysis; MIQE, minimum information for publication of quantitative real-time PCR experiments.
 Human genes: NF1, neurofibromin; SUZ12, suppressor of zeste 12 homolog (Drosophila); SUZ12P1, suppressor of zeste 12 homolog pseudogene 1; ADARB1, adenosine deaminase, RNA-specific, B1; TBC1D29, TBC1 domain family, member 29; CRLF3, cytokine receptor-like factor 3; RNF135, ring finger protein 135; UTP6, UTP6, small subunit (SSU) processome component, homolog (yeast); RHOT1, ras homolog family member T1; SSH2, slingshot homolog 2 (Drosophila); PSMD11, proteasome (prosome, macropain) 26S subunit, non-ATPase, 11; MAP2K4, mitogen-activated protein kinase kinase 4; OMG, oligodendrocyte myelin glycoprotein; EVI2BB, ecotropic viral integration site 2B; EVI2A, ecotropic viral integration site 2A; EFCAAB5, EF-hand calcium binding domain 5; NSRP1, nuclear speckle splicing regulatory protein 1; SLC6A4, solute carrier family 6 (neurotransmitter transporter, serotonin), member 4; BLMH, bleomycin hydrolase; TMIGD1, transmembrane and immunoglobulin domain containing 1; CPD, carboxypeptidase D; GOSR1, golgi SNAP receptor complex member 1; LRRC37BP1, leucine rich repeat containing 37B pseudogene 1; ATAD5, ATPase family, AAA domain containing 5; TEFM, transcription elongation factor, mitochondrial; ADAP2, ArfGAP with dual PH domains 2; RABB11FIP4, RAB11 family interacting protein 4 (class II); COPRS, coordinator of PRMT5, differentiation stimulator (formerly C17orf79); LRRC37B, leucine rich repeat containing 37B; RHBDL3, rhomboid, veinlet-like 3 (Drosophila); C17orf75, chromosome 17 open reading frame 75; ZNF207, zinc finger protein 207.
Ernest Terribas,  Carles Garcia-Linares,  Conxi Lazaro,  and Eduard Serra *
 Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain;  Molecular Diagnostics Unit, Hereditary Cancer Program, Catalan Institute of Oncology (ICO-IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.
* Address correspondence to this author at: Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Program on Hereditary Cancer, Carretera de Can Ruti, Caml de les Escoles s/n, 08916 Badalona, Barcelona, Spain. Fax 93-465-1472; e-mail firstname.lastname@example.org.
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|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Terribas, Ernest; Garcia-Linares, Carles; Lazaro, Conxi; Serra, Eduard|
|Date:||Jun 1, 2013|
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