Publications by authors named "Violaine Bourdon"

26 Publications

  • Page 1 of 1

Bayesian predictive model to assess BRCA2 mutational status according to clinical history: Early onset, metastatic phenotype or family history of breast/ovary cancer.

Prostate 2021 Feb 18. Epub 2021 Feb 18.

GRC n°5 Predictive Onco-Urology, Tenon Hospital, AP-HP, Sorbonne University, Paris, France.

Background: Mutations of the BRCA2 gene are the most frequent alterations found in germline DNA from men with prostate cancer (PrCa), but clinical parameters that could better orientate for BRCA2 mutation screening need to be established.

Methods: Germline DNA from 325 PrCa patients (median age at diagnosis: 57 years old) was screened for BRCA2 mutation. The mutation frequency was compared between three subgroups: patients with an age at diagnosis at 55 years old and under (Group I); a personal or family history of breast, uterine or ovarian cancer (Group II); or a metastatic disease (Group III). Frequency of BRCA2 mutations was established for each combination of phenotypes, and compared between patients meeting or not the criteria for each subgroup using Fisher's exact test. Mutual information, direct effect, elasticity and contribution to the mutational status of each phenotype, taking into account overlap between subgroups, were also estimated using Bayesian algorithms.

Results: The proportion of BRCA2 mutation was 5.9% in Group I, 10.9% in Group II and 6.9% in Group III. The frequency of BRCA2 mutation was significantly higher among patients of Group II (p = .006), and reached 15.6% among patients of this group who presented a metastatic disease. Mutual information, direct effect, elasticity and contribution to the mutational status were the highest for phenotype II. Fifteen (71.4%) of the 21 BRCA2 mutation carriers had an aggressive form of the disease. Four (19%) of them died from PrCa after a median follow-up duration of 64.5 months.

Conclusions: Our results showed that a higher frequency of BRCA2 mutation carriers is observed, not only among PrCa patients with young onset or a metastatic disease, but also with a personal or a familial history of breast cancer.
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http://dx.doi.org/10.1002/pros.24109DOI Listing
February 2021

Calibration of Pathogenicity Due to Variant-Induced Leaky Splicing Defects by Using Exon 3 as a Model System.

Cancer Res 2020 09 8;80(17):3593-3605. Epub 2020 Jul 8.

Inserm U1245, UNIROUEN, Normandie University, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

is a clinically actionable gene implicated in breast and ovarian cancer predisposition that has become a high priority target for improving the classification of variants of unknown significance (VUS). Among all VUS, those causing partial/leaky splicing defects are the most challenging to classify because the minimal level of full-length (FL) transcripts required for normal function remains to be established. Here, we explored exon 3 (e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for haploinsufficiency. predictions, minigene splicing assays, patients' RNA analyses, a mouse embryonic stem cell (mESC) complementation assay and retrieval of patient-related information were combined to determine the minimal requirement of FL transcripts. Of 100 e3 variants tested in the minigene assay, 64 were found to be spliceogenic, causing mild to severe RNA defects. Splicing defects were also confirmed in patients' RNA when available. Analysis of a neutral leaky variant (c.231T>G) showed that a reduction of approximately 60% of FL transcripts from a mutant allele does not cause any increase in cancer risk. Moreover, data obtained from mESCs suggest that variants causing a decline in FL with approximately 30% of wild-type are not pathogenic, given that mESCs are fully viable and resistant to DNA-damaging agents in those conditions. In contrast, mESCs producing lower relative amounts of FL exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of leaky splicing variants, some of which may not increase cancer risk.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484206PMC
September 2020

Skipping Nonsense to Maintain Function: The Paradigm of Exon 12.

Cancer Res 2020 04 11;80(7):1374-1386. Epub 2020 Feb 11.

Normandie Univ, UNIROUEN, Inserm U1245, Normandy Centre for Genomic and Personalized Medicine, Rouen, France.

Germline nonsense and canonical splice site variants identified in disease-causing genes are generally considered as loss-of-function (LoF) alleles and classified as pathogenic. However, a fraction of such variants could maintain function through their impact on RNA splicing. To test this hypothesis, we used the alternatively spliced exon 12 (E12) as a model system because its in-frame skipping leads to a potentially functional protein. All E12 variants corresponding to putative LoF variants or predicted to alter splicing ( = 40) were selected from human variation databases and characterized for their impact on splicing in minigene assays and, when available, in patient lymphoblastoid cell lines. Moreover, a selection of variants was analyzed in a mouse embryonic stem cell-based functional assay. Using these complementary approaches, we demonstrate that a subset of variants, including nonsense variants, induced in-frame E12 skipping through the modification of splice sites or regulatory elements and, consequently, led to an internally deleted but partially functional protein. These data provide evidence, for the first time in a cancer-predisposition gene, that certain presumed null variants can retain function due to their impact on splicing. Further studies are required to estimate cancer risk associated with these hypomorphic variants. More generally, our findings highlight the need to exercise caution in the interpretation of putative LoF variants susceptible to induce in-frame splicing modifications. SIGNIFICANCE: This study presents evidence that certain presumed loss-of-function variants in a cancer predisposition gene can retain function due to their direct impact on RNA splicing.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2491DOI Listing
April 2020

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort.

Nucleic Acids Res 2018 09;46(15):7913-7923

Inserm U830, Institut Curie Centre de Recherches, 75005 Paris, France.

Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5' and 3' consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).
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http://dx.doi.org/10.1093/nar/gky372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125621PMC
September 2018

GATA2 gene analysis in several forms of hematological malignancies including familial aggregations.

Ann Hematol 2017 Oct 27;96(10):1635-1639. Epub 2017 Jul 27.

UR "Biologie moléculaire des leucémies et lymphomes", Laboratoire de Biochimie, Faculté de Médecine de Sousse, Université de Sousse, Avenue Mohamed Karoui, 4000, Sousse, Tunisia.

The genetic predisposition to familial hematological malignancies has been previously reported highlighting inherited gene mutations. Several genes have been reported but genetic basis remains not well defined. In this study, we extended our investigation to a potential candidate GATA2 gene which was analyzed by direct sequencing in 119 cases including familial aggregations with a variety of hematological malignancies and sporadic acute leukemia belonging to Tunisian and French populations. We reported a deleterious p.Arg396Gln GATA2 mutation in one patient diagnosed with both sporadic acute myeloid leukemia (AML) and breast cancer. We also reported several GATA2 variations in familial cases. The absence of deleterious mutations in this large cohort of familial aggregations of hematological malignancies may strengthen the hypothesis that GATA2 mutations are an important predisposing factor, although as a secondary genetic event, required for the development of overt malignant disease.
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http://dx.doi.org/10.1007/s00277-017-3076-9DOI Listing
October 2017

ARLTS1, potential candidate gene in familial aggregation of hematological malignancies.

Bull Cancer 2017 Feb 17;104(2):123-127. Epub 2016 Nov 17.

Université de Sousse, faculté de médecine de Sousse, laboratoire de Biochimie, UR « biologie moléculaire des leucémies et lymphomes », avenue Mohamed Karoui, 4000 Sousse, Tunisia.

Introduction: Genetic predisposition to familial hematological malignancies was previously described through several epidemiological analyses, but the genetic basis remains unclear. The tumor-suppressor ARLTS1 gene was previously described in sporadic hematological malignancies and familial cancer context.

Methods: In this study, we sequence the ARLTS1 gene in 100 patients belonging to 88 independent Tunisian and French families.

Results: After gene sequencing, we report 8 genetic variations, most of which were previously reported in several cancer forms. The most common variants were W149X and C148R and were previously associated to B-cell chronic lymphocytic leukemia and to high-risk of familial breast cancer.

Conclusions: These results emphasize the fact that ARLTS1 gene mutations can be considered as a potential predisposing factor in familial hematological malignancies and other several cancer forms.
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http://dx.doi.org/10.1016/j.bulcan.2016.10.016DOI Listing
February 2017

Familial hematological malignancies: new IDH2 mutation.

Ann Hematol 2016 Dec 3;95(12):1943-1947. Epub 2016 Sep 3.

UR "Biologie moléculaire des leucémies et lymphomes," Laboratoire de Biochimie, Faculté de Médecine de Sousse, Université de Sousse - Tunisie, Avenue Mohamed Karoui, 4002, Sousse, Tunisia.

Isocitrate dehydrogenase IDH 1 and IDH 2 mutations were reported in several cancer forms, especially in hematological malignancies, but were never been investigated in familial aggregation. The aim of this study is to determine whether germline isocitrate dehydrogenase genes mutations are involved.We targeted IDH1 and IDH2 genes in 104 familial cases belonging to Tunisian and French populations, including several forms of hematological malignancies and cosegregated solid tumors.We report one IDH1 variant: c.315 G>T, p.Gly105Gly in 15 % of cases, which was assigned to the worst outcome in several studies. Three IDH2 variants were found, among them, one intronic substitution c.543+45 G>A (rs142033117) and two new variants not previously described: c.389 A>T, p.Lys130Met and c.414 T>C, p.Thr138Thr. The p.Lys130Met was found in one case diagnosed with Waldenstrom's disease with familial history of cancer. The enrolled in silico analysis, the functional study, and the absence of this variant in control population strengthen the hypothesis of its deleterious effect.From an extended number of candidate genes analyzed in familial hematological malignancies, IDH2 might be considerably involved since we reported a potential damaging effect.
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http://dx.doi.org/10.1007/s00277-016-2813-9DOI Listing
December 2016

Mutational analysis of TP53 gene in Tunisian familial hematological malignancies and sporadic acute leukemia cases.

Fam Cancer 2017 01;16(1):153-157

RU "Molecular Biology of Leukemias and Lymphomas", Laboratory of Biochemistry, Faculty of Medicine of Sousse, University of Sousse, Avenue Mohamed Karoui, 4000, Sousse, Tunisia.

Mutations are responsible for familial cancer syndromes which account for approximately 5-10 % of all types of cancers. Familial cancers are often caused by genetic alterations occurring either in tumor suppressor or genomic stability genes such as TP53. In this study, we have analyzed the TP53 gene by direct sequencing approach, in a panel of 18 Tunisian familial hematological malignancies cases including several forms of leukemia, lymphoma and myeloid syndrome and 22 cases of sporadic acute leukemia. In one familial case diagnosed with acute lymphoblastic leukemia, we reported an intronic substitution 559+1 G>A which may disrupt the splice site and impact the normal protein function. Most of the deleterious mutations (Arg158His; Pro282Trp; Thr312Ser) as classified by IARC data base, were commonly reported in ALL cases studied here. The cosegregation of the two variants rs1042522 and rs1642785 was observed in most patients which may be in favor of the presence of linkage disequilibrium. The most defined TP53 mutations found here were identified in acute lymphoblastic leukemia context whereas only 3 % of mutations have been in previous studies. The cosegregation of the two recurrent variant rs1042522 and rs1642785 should be further confirmed.
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http://dx.doi.org/10.1007/s10689-016-9931-3DOI Listing
January 2017

Mutational analysis of JAK2, CBL, RUNX1, and NPM1 genes in familial aggregation of hematological malignancies.

Ann Hematol 2016 Jun 23;95(7):1043-50. Epub 2016 Apr 23.

UR "Biologie moléculaire des leucémies et lymphomes", Laboratoire de Biochimie, Faculté de Médecine de Sousse, Université de Sousse, Avenue Mohamed Karoui, 4000, Sousse, Tunisia.

Familial aggregation of hematological malignancies has been reported highlighting inherited genetic predisposition. In this study, we targeted four candidate genes: JAK2 and RUNX1 genes assuring a prominent function in hematological process and CBL and NPM1 as proto-oncogenes. Their disruption was described in several sporadic hematological malignancies. The aim of this study is to determine whether JAK2, CBL, RUNX1, and NPM1 germline genes mutations are involved in familial hematological malignancies. Using direct sequencing, we analyzed JAK2 (exons 12 and 14); CBL (exons 7, 8 and 9); NPM1 (exon 12) and the entire RUNX1 in 88 independent families belonging to Tunisian and French populations. Twenty-one sporadic acute leukemias were included in this study. We reported a heterozygous intronic c.1641 + 6 T > C JAK2 variant (rs182123615) found in two independent familial cases diagnosed with gastric lymphoma and Hodgkin lymphoma. The in silico analysis suggested a potential impact on splicing, but the functional splicing minigene reporter assay on rs182123615 variant showed no aberrant transcripts. In one sporadic acute myeloblastic leukemia, we reported an insertion 846 in. TGTT in exon 12 of NPM1 gene that may impact the normal reading frame. The rs182123615 JAK2 variant was described in several contexts including myeloproliferative neoplasms and congenital erythrocytosis and was supposed to be pathogenic. Through this current study, we established the assessment of pathogenicity of rs182123615 and we classified it rather as rare polymorphism.
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http://dx.doi.org/10.1007/s00277-016-2678-yDOI Listing
June 2016

About sequence quality: impact on clinical applications.

Genet Test Mol Biomarkers 2014 May 12;18(5):299-305. Epub 2014 Mar 12.

1 Laboratoire d'oncogénétique moléculaire, Institut Paoli-Calmettes , Marseille, France .

Sanger DNA sequencing is a robust and flexible technology and should have a crucial role in clinical practice for a long time. Nevertheless, in routine application of DNA sequencing, we are regularly confronted with sequence data quality problems. Surprisingly, we found that the definition of sequence quality is fuzzy and too empirical for many clinical applications. There are few studies or guidelines that directly address quality issues for Sanger sequencing in clinical situations. In addition, these use several combined parameters to ensure the sequence quality; this is too complicated to apply for daily use. Our heuristic analysis of nearly 46,000 sequence traces demonstrated that a combination of three basic parameters (average quality value, average sequence intensity, and electropherogram profile) was necessary and sufficient to determine accurately the quality of any sequence, even when deletions, insertions, and/or repeat sequence regions were present in a sequence trace. Therefore, we propose a simple and practical method with a diagram and decision making table for sequence quality determination in clinical sequencing.
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http://dx.doi.org/10.1089/gtmb.2013.0435DOI Listing
May 2014

Prediction of BRCA1 germ-line mutation status in patients with breast cancer using histoprognosis grade, MS110, Lys27H3, vimentin, and KI67.

Pathobiology 2013 23;80(5):219-27. Epub 2013 Apr 23.

Department of Cancer Genetics/CIC-P Inserm 9502, Paoli Calmettes Institute, University of Aix-Marseille II, Marseille, France.

Family structure, lack of reliable information, cost, and delay are usual concerns when deciding to perform BRCA analyses. Testing breast cancer tissues with four antibodies (MS110, lys27H3, vimentin, and KI67) in addition to grade evaluation enabled us to rapidly select patients for genetic testing identification. We constituted an initial breast cancer tissue microarray, considered as a learning set, comprising 27 BRCA1 and 81 sporadic tumors. A second independent validation set of 28 BRCA1 tumors was matched to 28 sporadic tumors using the same original conditions. We investigated morphological parameters and 21 markers by immunohistochemistry. A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility. In the initial set, univariate analyses identified 11 markers significantly associated with BRCA1 status. Then, the best multivariate model comprised only grade 3, MS110, Lys27H3, vimentin, and KI67. When applied to the validation set, BRCA1 tumors were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles. This study offers a new rapid and cost-effective method for the prescreening of patients at high risk of being BRCA1 mutation carriers, to guide genetic testing, and finally to provide appropriate preventive measures, advice, and treatments including targeted therapy to patients and their families.
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http://dx.doi.org/10.1159/000339432DOI Listing
January 2014

Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

Hum Mutat 2012 Aug 11;33(8):1228-38. Epub 2012 May 11.

Service de Génétique, Institut Curie et Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.
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http://dx.doi.org/10.1002/humu.22101DOI Listing
August 2012

Molecular study of the perforin gene in familial hematological malignancies.

Hered Cancer Clin Pract 2011 Sep 21;9(1). Epub 2011 Sep 21.

Département d'Oncologie Génétique, de Prévention et Dépistage, Institut Paoli-Calmettes, 232 Boulevard Sainte Marguerite, Marseille, 13009, France.

Perforin gene (PRF1) mutations have been identified in some patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH) and in patients with lymphoma. The aim of the present study was to determine whether patients with a familial aggregation of hematological malignancies harbor germline perforin gene mutations. For this purpose, 81 unrelated families from Tunisia and France with aggregated hematological malignancies were investigated. The variants detected in the PRF1 coding region amounted to 3.7% (3/81). Two of the three variants identified were previously described: the p.Ala91Val pathogenic mutation and the p.Asn252Ser polymorphism. A new p.Ala 211Val missense substitution was identified in two related Tunisian patients. In order to assess the pathogenicity of this new variation, bioinformatic tools were used to predict its effects on the perforin protein structure and at the mRNA level. The segregation of the mutant allele was studied in the family of interest and a control population was screened. The fact that this variant was not found to occur in 200 control chromosomes suggests that it may be pathogenic. However, overexpression of mutated PRF1 in rat basophilic leukemia cells did not affect the lytic function of perforin differently from the wild type protein.
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http://dx.doi.org/10.1186/1897-4287-9-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197553PMC
September 2011

SMARCB1/INI1 germline mutations contribute to 10% of sporadic schwannomatosis.

BMC Neurol 2011 Jan 24;11. Epub 2011 Jan 24.

Laboratory of Molecular Oncogenetics, Institut Paoli-Calmettes, Marseille, France.

Background: Schwannomatosis is a disease characterized by multiple non-vestibular schwannomas. Although biallelic NF2 mutations are found in schwannomas, no germ line event is detected in schwannomatosis patients. In contrast, germline mutations of the SMARCB1 (INI1) tumor suppressor gene were described in familial and sporadic schwannomatosis patients.

Methods: To delineate the SMARCB1 gene contribution, the nine coding exons were sequenced in a series of 56 patients affected with a variable number of non-vestibular schwannomas.

Results: Nine variants scattered along the sequence of SMARCB1 were identified. Five of them were classified as deleterious. All five patients carrying a SMARCB1 mutation had more multiple schwannomas, corresponding to 10.2% of patients with schwannomatosis. They were also diagnosed before 35 years of age.

Conclusions: These results suggest that patients with schwannomas have a significant probability of carrying a SMARCB1 mutation. Combined with data available from other studies, they confirm the clinical indications for genetic screening of the SMARCB1 gene.
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http://dx.doi.org/10.1186/1471-2377-11-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037869PMC
January 2011

Germline APC mutation spectrum derived from 863 genomic variations identified through a 15-year medical genetics service to French patients with FAP.

J Med Genet 2010 Oct 3;47(10):721-2. Epub 2010 Aug 3.

Centre de Recherche en Cancérologie de Marseille INSERM UMR891, Marseille, France.

Heterozygous APC germline alteration is responsible for familial adenomatous polyposis, a colon cancer predisposition with almost complete penetrance. Point mutations generally lead to truncated proteins or no protein at all. They mainly involve exon 3 to codon 1700 (exon 15). The work presented here delineates precisely the APC mutation spectrum from 15 years of systematic molecular screening which identified 863 independent alterations in the French population.
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http://dx.doi.org/10.1136/jmg.2010.078964DOI Listing
October 2010

High frequency of RUNX1 biallelic alteration in acute myeloid leukemia secondary to familial platelet disorder.

Blood 2009 May 8;113(22):5583-7. Epub 2009 Apr 8.

Department of Hematology, Biology and Pathology Center, Centre Hospitalier Régional Universitaire (CHRU) of Lille, Lille, France.

Familial platelet disorder (FPD), a rare autosomal dominant disorder characterized by quantitative and qualitative platelet abnormalities, is considered as a model of genetic predisposition to acute myeloid leukemia (AML). So far, monoallelic RUNX1 germline mutations have been found in 19 of 20 families with reported FPD, and the analysis of blast cells from only 5 patients at acute leukemia (AL) stage has shown no additional RUNX1 abnormality. Here, we performed RUNX1 analysis at constitutional and somatic levels in 8 persons with FPD who developed AL from 4 independent families. In addition to the germline RUNX1 mutation, we identified a second RUNX1 alteration in 6 AML cases (acquired point mutations in 4 cases and duplication of the altered RUNX1 allele associated with acquired trisomy 21 in 2 other cases). Although haploinsufficiency of RUNX1 causes FPD, our findings suggest that a second genetic event involving RUNX1 is often associated with progression to AML.
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http://dx.doi.org/10.1182/blood-2008-07-168260DOI Listing
May 2009

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.

Hum Mutat 2009 Jun;30(6):867-75

Centre René Huguenin, Laboratoire d'Oncogénétique, Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U735, Saint-Cloud, France.

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.
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http://dx.doi.org/10.1002/humu.20947DOI Listing
June 2009

Age-Dependent Cancer Risk Is Not Different in between MSH2 and MLH1 Mutation Carriers.

J Cancer Epidemiol 2009 8;2009:791754. Epub 2009 Mar 8.

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 891, Centre de Recherches en Cancérologie de Marseille, 13009 Marseille, France.

Lynch syndrome is mostly characterized by early-onset colorectal and endometrial adenocarcinomas. Over 90% of the causal mutations occur in two mismatch repair genes, MSH2 and MLH1. The aim of this study was to evaluate the age-dependent cancer risk in MSH2 or MLH1 mutation carriers from data of DNA diagnostic laboratories. To avoid overestimation, evaluation was based on the age-dependent proportion of mutation carriers in asymptomatic first-degree relatives of identified mutation carriers. Data from 859 such eligible relatives were collected from 8 centers; 387 were found to have inherited the mutation from their relatives. Age-dependent risks were calculated either using a nonparametric approach for four discrete age groups or assuming a modified Weibull distribution for the dependence of risk on age. Cancer risk was estimated starting at 28 (25-32 0.68 confidence interval) and to reach near 0.70 at 70 years. The risks were very similar for MSH2 and MLH1 mutation carriers. Although not statistically significant, the risk in males appeared to precede that for females by ten years. This difference needs to be investigated on a larger dataset. If confirmed, this would indicate that the onset of the colonoscopic surveillance may be different in male and female mutation carriers.
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http://dx.doi.org/10.1155/2009/791754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2859022PMC
July 2011

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers.

Am J Hum Genet 2008 Apr 20;82(4):937-48. Epub 2008 Mar 20.

Cancer Research UK, Genetic Epidemiology Unit, Department of Public Health and Primary Care, University of Cambridge, UK.

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.
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http://dx.doi.org/10.1016/j.ajhg.2008.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2427217PMC
April 2008

Significant contribution of germline BRCA2 rearrangements in male breast cancer families.

Cancer Res 2004 Nov;64(22):8143-7

Institut National de la Santé et de la Recherche Médicale (INSERM) U614, Faculty of Medicine, IFRMP, University of Rouen, Rouen, France.

Although screening for large deletions or duplications of the BRCA1 gene is becoming a routine component of the molecular diagnosis of familial breast cancer, little is known about the occurrence of such rearrangements in the BRCA2 gene. Because of the high frequency of BRCA2 mutations in breast cancer families with at least one case of male breast cancer, we selected a cohort of 39 such families, tested negative for mutations in the coding regions of BRCA1 and BRCA2, and developed an assay for BRCA2 rearrangements, based on quantitative multiplex PCR of short fluorescent fragments (QMPSF). We found three rearrangements: (1) a deletion of exons 12 and 13; (2) a duplication of exons 1 and 2; and (3) a complete deletion of BRCA2. We determined the boundaries of the deletion of exons 12 and 13, showing that it resulted from an unequal recombination between Alu sequences. We mapped the complete BRCA2 deletion, which extends over at least 298 kb and showed that it does not affect APRIN/AS3, previously characterized as a tumor suppressor gene, but it comprises several loci corresponding to proven or putative transcripts of unknown functional significance. These data suggest that screening for BRCA2 rearrangements should be done, especially in male breast cancer families tested negative for BRCA1 and BRCA2 mutations.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-2467DOI Listing
November 2004

Chromosome imbalances in oligodendroglial tumors detected by comparative genomic hybridization.

Ann Genet 2004 Apr-Jun;47(2):105-11

Laboratoire de Génétique-EA3441, CHU Nancy-Brabois, avenue du Morvan, 54111 Vandoeuvre les Nancy, France.

Seven well-differentiated oligodendrogliomas, 16 anaplastic oligodendrogliomas and two cases of oligoastrocytomas were investigated by comparative genomic hybridization (CGH) on frozen tissue samples. The most frequent losses found involved 1p and 19q in 32% of cases. Loss of 9p was observed during malignant progression in 25% of anaplastic oligodendrogliomas. In two anaplastic oligodendrogliomas gain of 1q was found. The frequent losses of chromosome 16 and 22 have not been reported previously. These results underscore that CGH is a powerful tool for the classification of gliomas complementing the traditional histopathological approach.
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http://dx.doi.org/10.1016/j.anngen.2003.10.002DOI Listing
October 2005

MECP2 mutations or polymorphisms in mentally retarded boys: diagnostic implications.

Mol Diagn 2003 ;7(1):3-7

Laboratory of Medical Genetics, EA 3441, CHU-Brabois, Vandoeuvre-Lès Nancy, France.

Background: Among the well characterized X-linked conditions causing mental retardation, mutations in the methyl-CpG-binding protein 2 gene (MECP2) in Xq28 have been found in up to 85% of patients with Rett syndrome, a neurologic disorder which, in addition to other symptoms, severely affects higher cognitive functions in females. Mutations in the MECP2 gene are involved in a broad spectrum of phenotypes from classical Rett syndrome to mild intellectual difficulties in females and neonatal encephalopathy in males. Recently, mutations in the MECP2 gene were reported in males with non-specific mental retardation suggesting that defects in MECP2 could be responsible for up to 2% of X-linked mental retardation.

Methods: We screened by denaturing high-pressure liquid chromatography the entire coding region and flanking intronic sequences of the MECP2 gene in a cohort of 354 mentally retarded males found negative for an expansion across the FRAXA CGG repeat and in a family in which a boy and his sister were mentally retarded.

Results: We identified mainly silent polymorphisms within the MECP2 gene, together with four sequence alterations of unknown significance, i.e. three missense mutations (T197M, T228S, and P376S) and one substitution at position -19 in intron 3 (378-19delT). Further familial investigations allowed us to ruled out a pathogenic effect for the intronic variant, the T228S and the P376S missense mutations.

Conclusions: These results confirm that MECP2 mutations in males are far more rare than initially thought and call for a careful evaluation of the pathogenicity of the MECP2 missense mutations identified in mentally retarded males before genetic counseling is proposed to the relatives.
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http://dx.doi.org/10.1007/BF03260014DOI Listing
September 2005

Spectrum of MECP2 mutations in Rett syndrome.

Genet Test 2002 ;6(1):1-6

INSERM U129-ICGM, Faculté de Médecine Cochin, 75014 Paris, France.

Mutations in the MECP2 (Methyl-CpG-binding protein) gene recently have been reported to cause Rett syndrome (RTT), an X-linked progressive encephalopathy. We have collected the results of MECP2 analysis conducted in four laboratories in France. A total of 301 RTT alleles have been analyzed, demonstrating a total of 69 different mutations so far observed and accounting for 64% of MECP2 genes in RTT patients living in France. R168X (11.5%) is the most common of MECP2 mutations, followed by R255X (10.9%), R270X (10.5%), T158M (7.8%), and R306C (6.8%). Only 10 mutations had a relative frequency > 2%. A total of 59 mutations were found in a small number of RTT alleles (from 1 to 2). These data demonstrate the high allelic heterogeneity of RTT in France and provide information relevant to the development of strategies for molecular diagnosis and genetic counseling in RTT families.
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http://dx.doi.org/10.1089/109065702760093843DOI Listing
December 2002

T-cell immune constitution after peripheral blood mononuclear cell transplantation in complete DiGeorge syndrome.

Br J Haematol 2002 Jun;117(4):899-906

Unité de Thérapie cellulaire et Tissus, CHU de Nancy, UMR CNRS 7563, Allée du Morvan, 54511 Vandoeuvre-lès-Nancy, France.

Complete DiGeorge syndrome (cDGS) is a congenital disorder characterized by typical facies, thymic aplasia, susceptibility to infections, hypoparathyroidism and conotruncal cardiac defect. Fetal thymus or post-natal thymus tissue transplantations and human leucocyte antigen (HLA)-genoidentical bone marrow transplantations were followed in a few cases by immune reconstitution. More recently, a peripheral blood mononuclear cell transplantation (PBMCT) was performed with an HLA-genoidentical donor and followed by a partial T-cell engraftment and immune reconstitution. We report a boy with cDGS, without cardiac defect, who suffered recurrent severe infections. At the age of 4 years, he underwent PBMCT from his HLA-genoidentical sister. He received no conditioning regimen, but graft-versus-host disease (GVHD) prophylaxis was with oral cyclosporin A and mycophenolate mofetil. Toxicity was mild, with grade I acute GVHD. The patient is currently 2.5 years post-PBMCT with excellent clinical performances. Mixed chimaerism can only be observed on the T-cell population (50% donor T cells). T-lymphocyte count fluctuated (CD3 more than 400 x 10(6)/l at d 84 and CD4 more than 200 x 10(6)/l at d 46). Exclusive memory phenotype T cells and absence of new thymic emigrants suggest expansion of infused T cells. T-cell mitogen and tetanus antigen responses normalized a few months after transplantation. After immunizations, specific antibodies were produced. PBMCT from an HLA identical sibling could be an efficient treatment of immune deficiency in cDGS.
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http://dx.doi.org/10.1046/j.1365-2141.2002.03496.xDOI Listing
June 2002