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Abstract 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.

Abstract 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.

Hydrazone compounds represent an important area of research that includes, among others, synthetic approaches and biological studies. A series of 17 hydrazones have been synthesized by mechanochemical means. The fragments chosen were phenolic and furanyl aldehydes coupled with 12 heterocyclic hydrazines or hydrazinamides. All compounds can be obtained quantitatively when operating on a planetary ball mill and a maximum reaction time of 180 min (6 cycles of 30 min each). Complete spectroscopic analyses of hydrazones revealed eight compounds ( 3 - 5 , 8 - 11 , 16 ) present in one geometric form, six compounds ( 1 , 2 , 13 - 15 ) present in two isomeric forms, and three compounds ( 6 , 7 , 12 ) where one rotation is restricted giving rise to two different forms. The single crystal X-ray structure of one of the hydrazones bearing the isoniazid fragment ( 8 ) indicates a crystal lattice consisting of two symmetry-independent molecules with different geometries. All compounds obtained were tested for anti-infectious and antibacterial activities. Four compounds ( 1 , 3 , 5 and 8 ) showed good activity against Mycobacterium tuberculosis , and one ( 7 ) was very potent against Staphylococcus aureus . Most interesting, this series of compounds displayed very promising antileishmanial activity. Among all, compound 9 exhibited an IC 50 value of 0.3 µM on the Leishmania donovani intramacrophage amastigote in vitro model and a good selectivity index, better than miltefosine, making it worth evaluating in vivo.

Photo-iniferter reversible addition-fragmentation chain transfer (PI-RAFT) polymerization of N-vinylformamide (NVF) is demonstrated by using purple light. PNVFs with predetermined molar masses and narrow molar mass distributions are obtained. High RAFT chain-end fidelity is confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS), and chain extension experiment. To demonstrate the potential of this approach, an original poly(N-vinylpyrrolidone)-b-poly(N-vinylformamide) (PVP-b-PNVF) diblock copolymer is synthesized and characterized by aqueous size-exclusion chromatography (SEC), asymmetric flow field-flow fractionation (A4F), and 1 H diffusion-ordered spectroscopy nuclear magnetic resonance ( 1 H DOSY NMR). Finally, selective hydrolysis of PNVF block to corresponding pH-responsive poly(N-vinylpyrrolidone)-b-poly(N-vinylformamide) (PVP-b-PVAm) is performed., (© 2022 The Authors. Macromolecular Rapid Communications published by Wiley-VCH GmbH.)

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes., Competing Interests: Declaration of interests D.S.-L. and the Institut Curie have received honoraria for her participation in education meetings organized by AstraZeneca or Tesaro. The remaining authors declare no conflict of interest., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Introduction: Apoptosis deregulation have been associated to tumorigenesis process and was highlighted as a prominent hallmark of cancer. Several mutations have been reported in several forms of Blood cancer. However, it has never been investigated in familial aggregations of hematological malignancies., Methods: In this study, we performed a mutational analysis by sequencing the entire coding regions in four key apoptotic genes FAS, FASLG, CASP8 and CASP10 in 92 independent families belonging to French and Tunisian populations and diagnosed with several forms of familial hematological malignancies., Results: We report 15 genetic variations among which 7 were previously reported in several form of cancers and have a potential effect on gene expression. Particularly, the CASP8 variants p.Asp302His and p.Lys337Lys were detected in 15% and 10% of our group of patients respectively and were previously reported in association to breast cancer and to breast cancer susceptibility., Discussion: In this study, we do not report the underlining deleterious mutations in familial hematological malignancies, but we describe some variants with potential risk of developing blood cancer. To gain further insights on the association between apoptosis pathway deregulation and familial hematological malignancies, more apoptotic genes should be investigated., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families., Methods: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing., Results: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1 , fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin., Conclusions: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

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., (© 2021 Wiley Periodicals LLC.)

BRCA2 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 BRCA2 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 BRCA2 exon 3 ( BRCA2 e3) as a model for calibrating variant-induced spliceogenicity and estimating thresholds for BRCA2 haploinsufficiency. In silico 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 BRCA2 transcripts. Of 100 BRCA2 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 BRCA2 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 BRCA2 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 BRCA2 exhibited either null or hypomorphic phenotypes. Overall, our findings are likely to have broader implications on the interpretation of BRCA2 variants affecting the splicing pattern of other essential exons. SIGNIFICANCE: These findings demonstrate that BRCA2 tumor suppressor function tolerates substantial reduction in full-length transcripts, helping to determine the pathogenicity of BRCA2 leaky splicing variants, some of which may not increase cancer risk., (©2020 American Association for Cancer Research.)

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 BRCA2 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 ( n = 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., (©2020 American Association for Cancer Research.)

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/).

Intensive use of chlorinated pesticides from the 1960s to the 1990s has resulted in a diffuse contamination of soils and surface waters in the banana-producing areas of the French West Indies. The purpose of this research was, for the first time, to examine the degradation of two of these persistent pollutants - chlordecone (CLD) and beta-hexachlorocyclohexane (β-HCH) in 1 mg L -1 synthetic aqueous solutions by means of photolysis, (photo-) Fenton oxidation and ozonation processes. Fenton oxidation is not efficient for CLD and yields less than 15% reduction of β-HCH concentration in 5 h. Conversely, both molecules can be quantitatively converted under UV-Vis irradiation reaching 100% of degradation in 5 h, while combination with hydrogen peroxide and ferrous iron does not show any significant improvement except in high wavelength range (>280 nm). Ozonation exhibits comparable but lower degradation rates than UV processes. Preliminary identification of degradation products indicated that hydrochlordecone was formed during photo-Fenton oxidation of CLD, while for β-HCH the major product peak exhibited C 3 H 3 Cl 2 as most abundant fragment.

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.

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., (Copyright © 2016 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

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.

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.

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.

Sequencing mitochondrial DNA hypervariable regions I and II (HVI and HVII) is useful in forensic missing person and unidentified remains cases. Improvements in ease and sensitivity of testing will yield results from more samples in a timely fashion. Routinely, amplification of HVI and HVII is followed by Sanger sequencing using the BigDye(®) Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) using 4 μL of ready reaction mix (RRM). Each sequencing reaction is then purified through column filtration before capillary electrophoresis. Using lower amounts of RRM (2 μL or 1 μL) and purification using BigDye(®) XTerminator(™) (Applied Biosystems) instead of columns showed no loss of sequence length and increased the quality and the sensitivity of testing, allowing HVI and HVII typing from mitochondrial genome equivalent to 125 fg of nuclear DNA, or 100 pg of HVI/HVII amplicons. Using this methodology, testing can be completed in 1 day, and the cost of testing is reduced., (© 2014 American Academy of Forensic Sciences.)

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.

Arabidopsis Fused kinase TWO-IN-ONE (TIO) controls phragmoplast expansion through its interaction with the Kinesin-12 subfamily proteins that anchor the plus ends of interdigitating microtubules in the phragmoplast midzone. Previous analyses of loss-of-function mutants and RNA interference lines revealed that TIO positively controls both somatic and gametophytic cell cytokinesis; however, knowledge of the full spectrum of TIO functions during plant development remains incomplete. To characterize TIO functions further, we expressed TIO and a range of TIO variants under control of the TIO promoter in wild-type Arabidopsis plants. We discovered that TIO-overexpressing transgenic lines produce enlarged pollen grains, arising from incomplete cytokinesis during male meiosis, and show sporophytic abnormalities indicative of polyploidy. These phenotypes arose independently in TIO variants in which either gametophytic function or the ability of TIO to interact with Kinesin-12 subfamily proteins was abolished. Interaction assays in yeast showed TIO to bind to the AtNACK2/TETRASPORE, and plants doubly homozygous for kinesin-12a and kinesin-12b knockout mutations to produce enlarged pollen grains. Our results show TIO to dominantly inhibit male meiotic cytokinesis in a dosage-dependent manner that may involve direct binding to a component of the canonical NACK-PQR cytokinesis signaling pathway.

Background: Vanin-1 is an epithelial pantetheinase, which regulates intestinal inflammation in mouse. We investigated whether human VNN1 levels could be associated to the susceptibility to inflammatory bowel diseases (IBD) and explored the participation of PPARg to these processes., Methods: We studied VNN1 expression in colon biopsies from IBD patients. We investigated polymorphisms in the regulatory regions of the VNN1 gene and examined their genetic association with the disease. Functional relevance of these single-nucleotide polymorphisms (SNPs) was assayed, and we tested PPARg in nuclear complexes associated with specific VNN1 polymorphic sequences. In mouse, we examined Vanin-1 expression in gut and feces during dextran sulfate sodium-induced colitis and assayed the effect of PPARg on Vanin-1 regulation., Results: VNN1 is expressed by enterocytes and is upregulated in IBD. Three SNPs are statistically associated to IBD. The regions containing these SNPs specifically bind nuclear complexes and are correlated with the VNN1 transcript abundance in colon in an allele-dependent manner. One rare SNP is associated to severe ulcerative colitis with strong VNN1 and dropped PPARg levels. PPARg is involved in nuclear complexes that bound to VNN1 regulatory sites. Similarly, Vanin-1 is tightly regulated in the mouse gut in normal and colitis conditions and PPARg regulates its expression., Conclusions: VNN1 is a marker for IBD. Polymorphic positions in the VNN1 locus are direct targets for nuclear factors that might regulate the level of VNN1 in colon, and this could be linked to IBD susceptibility. It is hoped that modulating locally VNN1 expression or activity can be exploited to develop future therapeutic strategies against IBD.

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., (Copyright © 2013 S. Karger AG, Basel.)

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., (© 2012 Wiley Periodicals, Inc.)

Coordination of a cyclometallated Pt(II) complex (1) to an amyloid-β peptide was probed by NMR and ESI-MS. Furthermore, EPR showed that binding of 1 to the Cu(II)-amyloid-β species resulted in a reshuffling of the Cu(II) coordination sphere, which was absent or lower for the sister non cyclometallated Pt(II) complexes.

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.

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.

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.

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.

Asymmetric cell division at pollen mitosis I (PMI) is required to specify the differential fate of the daughter vegetative and generative cells. Cytokinesis at PMI displays specialized features, and it has been suggested that there might be distinct molecular pathways underpinning different modes of cytokinesis in plants. Activation of the NACK-PQR MAP kinase signaling pathway, which is essential for somatic cell cytokinesis in tobacco, depends upon the NACK1 and NACK2 kinesin-related proteins. Their Arabidopsis orthologs, HINKEL (HIK) and TETRASPORE (TES), were reported to be essential for cytokinesis in somatic cells and in microsporocytes, respectively. More recently, HIK and TES were shown to have a functionally redundant role in female gametophytic cytokinesis. We report here that HIK and TES are co-expressed in microspores and developing pollen, and, through analysis of microspore and pollen development in double heterozygote mutants, the occurrence of cell plate expansion defects during cytokinesis at PMI. The data demonstrate a functionally redundant role for HIK and TES in cell plate expansion during male gametophytic cytokinesis, extending the concept that different modes of cytokinesis are executed by a common signaling pathway, but reinforcing the individuality of gametophytic cytokinesis in its requirement for either TES or HIK.

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.

Cervical cancer (CC) cells exhibit complex karyotypic alterations, which is consistent with deregulation of numerous critical genes in its formation and progression. To characterize this karyotypic complexity at the molecular level, we used cDNA array comparative genomic hybridization (aCGH) to analyze 29 CC cases and identified a number of over represented and deleted genes. The aCGH analysis revealed at least 17 recurrent amplicons and six common regions of deletions. These regions contain several known tumor-associated genes, such as those involved in transcription, apoptosis, cytoskeletal remodeling, ion-transport, drug metabolism, and immune response. Using the fluorescence in situ hybridization (FISH) approach we demonstrated the presence of high-level amplifications at the 8q24.3, 11q22.2, and 20q13 regions in CC cell lines. To identify amplification-associated genes that correspond to focal amplicons, we examined one or more genes in each of the 17 amplicons by Affymetrix U133A expression arrays and semiquantitative reverse-transcription PCR (RT-PCR) in 31 CC tumors. This analysis exhibited frequent and robust upregulated expression in CC relative to normal cervix for genes EPHB2 (1p36), CDCA8 (1p34.3), AIM2 (1q22-23), RFC4, MUC4, and HRASLS (3q27-29), SKP2 (5p12-13), CENTD3 (5q31.3), PTK2, RECQL4 (8q24), MMP1 and MMP13 (11q22.2), AKT1 (14q32.3), ABCC3 (17q21-22), SMARCA4 (19p13.3) LIG1 (19q13.3), UBE2C (20q13.1), SMC1L1 (Xp11), KIF4A (Xq12), TMSNB (Xq22), and CSAG2 (Xq28). Thus, the gene dosage and expression profiles generated here have enabled the identification of focal amplicons characteristic for the CC genome and facilitated the validation of relevant genes in these amplicons. These data, thus, form an important step toward the identification of biologically relevant genes in CC pathogenesis. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat., ((c) 2007 Wiley-Liss, Inc.)

Metastatic renal cell carcinoma (RCC) is refractory to therapy; however, 10%-20% of patients respond favorably with interferon-alpha (IFN-alpha) treatment. To understand the molecular basis of response to IFN-alpha therapy, we performed global gene expression analysis of sensitive and resistant RCC cell lines in the absence and in the presence of IFN-alpha, using high-density oligonucleotide arrays to detect differentially expressed genes. In the absence of IFN-alpha, no significant differences in gene expression were observed between six sensitive and six resistant cell lines. Gene expression analysis following a time course of IFN-alpha2b treatment in one sensitive (SK-RC-17) and one resistant (SK-RC-12) cell line revealed that 484 and 354 transcripts, respectively, were modulated. A considerable number of these transcripts were similarly modulated between the two cell types that included several known targets of IFN signaling associated with antiviral and immunomodulatory activity. A further analysis of gene expression pattern in response to IFN revealed that several transcripts associated with proapoptotic function were upregulated in the sensitive cells. In the resistant cells, transcripts associated with cell survival and proliferation were induced, and key apoptotic molecules were suppressed. This study suggests that the IFN-alpha response of individual RCC tumors is determined by the expression pattern of genes in the apoptosis vs. survival and proliferation pathways rather than by alterations in expression of one or more individual genes.

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.

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.

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.

We performed parallel array comparative genomic hybridization and array expression analysis of the 12p11-p12 amplicon in human testicular seminomas and an ovarian carcinoma cell line using an expressed sequence tags (ESTs) array spotted with 8254 ESTs. The data were normalized using a robust statistical modeling and the significance inferred from the local SD. We identified two ESTs within the chromosomal amplicon that were amplified and overexpressed in > or =75-100 percent of analyzed tumors with the 12p11-p12 amplicon. These sequences, belonging to coding regions of two novel genes designated here as GCT1 and GCT2, were broadly expressed in a panel of human tissues, including testis and ovary. GCT1 and GCT2 were overexpressed in 92 and 71 percent, respectively, of a panel of seminomas tested. Combined array comparative genomic hybridization and array expression analysis is a valid approach for gene discovery in large chromosomal amplicons.

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.

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.

Objective: Serum CH50 and C4 levels are usually normal or elevated in rheumatoid arthritis (RA) but are classically decreased in patients with serious extra-articular manifestations (SEAMs) of the disease. The objective of this study was to evaluate whether complement assays are useful in diagnosing or predicting SEAMs of RA., Methods: First, a cross-sectional study of 405 patients admitted for RA compared patients with and without hypocomplementemia. Then, a retrospective longitudinal design was used to investigate within-patient complement level variations overtime., Results: In the univariate analysis, patients with low CH50 and C4 levels were more likely to have vasculitis and/or cryoglobulinemia than those with normal CH50 and C4 levels, and nodules were more common in the patients with low than with normal C4 levels. In a multivariate model based on symptoms, low C4 was associated with vasculitis and pleurisy and low CH50 with vasculitis. However, these associations were too weak to make CH50 and C4 determination useful for detecting SEAMs, and the within-subject variations in patients with SEAMs limited the predictive value of these assays., Conclusion: Hypocomplementemia is of limited usefulness for detecting or predicting SEAMs.

In an attempt to further increase transgene expression levels in plants over and above the enhancement obtained with a 5' untranslated leader intron, three different maize introns were inserted at three different positions within the coding sequence of the luciferase reporter gene. Constructs were transformed into maize (Black Mexican Sweet) cells and protoplasts, and their activity determined. Although all introns tested were correctly spliced, only one of them in a particular position was able to enhance gene expression. Correct splicing sites were used for intron removal and the quantity of luciferase mRNA produced did not differ significantly. These data indicate that both the position and the sequence of an intron have marked effects on expression levels, suggesting that nuclear processing of the pre-mRNA determines final expression levels through the structure of the mRNP.

Mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) have been found to be a cause of Rett syndrome (RTT). In order to provide further insights into the distribution and the spectrum of mutations, we investigated, in addition to the whole coding sequence, a phylogenetically conserved sequence within the 3' untranslated region (3' UTR) of the MECP2 gene for 55 sporadic RTT, including 47 typical and 8 nonclassical cases. We have developed an approach based on conformation-sensitive gel electrophoresis, sequence analysis and, for the first time, Southern blot analysis. Mutation detection, including unreported gross DNA rearrangements, was achieved in 79% of classical RTT and 25% of nonclassical RTT patients. The high prevalence of recurrent mutations allows us to propose a molecular diagnosis strategy for RTT.

Acute promyelocytic leukaemia (APL) exhibits a characteristic t(15;17) translocation that fuses the promyelocytic leukaemia (PML) gene on 15q22 to the retinoic acid receptor alpha (RARA) gene on 17q12-q21.1. In a small subset of acute promyelocytic-like leukaemias (APL-L), RARA is fused to a different partner: the pro-myelocytic leukaemia zinc finger (PLZF) gene on 11q23, the nucleophosmin (NPM) gene on 5q35 or the nuclear mitotic apparatus (NuMA) gene on 11q13. We report on the molecular characterization of a RARA gene re-arrangement in a patient with APL-L and demonstrate that the signal transducer and activator of transcription STAT5b gene is fused with RARA. STAT5b belongs to the janus kinase (JAK)-STAT signalling pathway. Remarkably, the STAT5b component of the chimeric protein is delocalized from the cytoplasm to the nucleus, where it displays a microspeckled pattern. Therefore, unusual features of this APL-L might result from dysregulation of the JAK/STAT5 signal transducing pathways in the patient leukaemic cells. In this study, we identified STAT5b as a new gene fused to RARA in leukaemia; this is the first human tumour bearing a structurally abnormal STAT gene.

Insulin sensitivity of kininogen-deficient rats was compared with that of normal rats using euglycaemic hyperinsulinaemic glucose clamping. Anaesthetized animals were infused with 2-50 mU kg-1 min-1 of insulin and the glucose infusion rates needed to maintain euglycaemia were determined. Maximum glucose uptake, insulin sensitivity index and insulin clearance were reduced in kininogen-deficient rats. Captopril increased the amount of glucose needed to maintain euglycaemia during infusion of 2 and 10 mU kg-1 min-1 of insulin in normal rats, but had no effect in kininogen-deficient rats. Anaesthetized rats of both strains were given an intraperitoneal injection of glucose and the evolution of blood glucose was followed for 120 min. The peak increase was higher in kininogen-deficient rats. Similar larger increases in blood glucose were observed after glucose injection in normal rats previously treated with HOE 140, a bradykinin B2 receptor antagonist. After glucose injection, plasma insulin increased in both groups of rats but reached lower levels in kininogen-deficient animals. These results suggest that bradykinin is involved not only in the clearance of glucose and insulin by the tissues during insulin infusion but also that bradykinin can affect the release of insulin after a glucose load.