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1.
Int J Mol Sci ; 22(19)2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34638739

RESUMO

Numerical chromosomal aberrations in sperm are considered to be a major factor in infertility, early pregnancy loss and syndromes with developmental and cognitive disabilities in mammals, including primates. Despite numerous studies in human and farm animals, the incidence and importance of sperm aneuploidies in non-human primate remains mostly undetermined. Here we investigated the incidence and distribution of sperm aneuploidy in chimpanzees (Pan troglodytes), the species closest to human. We identify evolutionary conserved DNA sequences in human and chimpanzee and selected homologous sub-telomeric regions for all chromosomes to build custom probes and perform sperm-FISH analysis on more than 10,000 sperm nuclei per chromosome. Chimpanzee mean autosomal disomy rate was 0.057 ± 0.02%, gonosomes disomy rate was 0.198% and the total disomy rate was 1.497%. The proportion of X or Y gametes was respectively 49.94% and 50.06% for a ratio of 1.002 and diploidy rate was 0.053%. Our data provide for the first time an overview of aneuploidy in non-human primate sperm and shed new insights into the issues of aneuploidy origins and mechanisms.


Assuntos
Aneuploidia , Cromossomos de Mamíferos/genética , Hibridização in Situ Fluorescente , Espermatozoides , Animais , Humanos , Masculino , Pan troglodytes
2.
Am J Hum Genet ; 108(10): 1907-1923, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34597585

RESUMO

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.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Predisposição Genética para Doença , Variação Genética , Neoplasias Ovarianas/patologia , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Feminino , Testes Genéticos , Genótipo , Humanos , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/genética
3.
Cancers (Basel) ; 13(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34359559

RESUMO

Assessment of age-dependent cancer risk for carriers of a predicted pathogenic variant (PPV) is often hampered by biases in data collection, with a frequent under-representation of cancer-free PPV carriers. TUMOSPEC was designed to estimate the cumulative risk of cancer for carriers of a PPV in a gene that is usually tested in a hereditary breast and ovarian cancer context. Index cases are enrolled consecutively among patients who undergo genetic testing as part of their care plan in France. First- and second-degree relatives and cousins of PPV carriers are invited to participate whether they are affected by cancer or not, and genotyped for the familial PPV. Clinical, family and epidemiological data are collected, and all data including sequencing data are centralized at the coordinating centre. The three-year feasibility study included 4431 prospective index cases, with 19.1% of them carrying a PPV. When invited by the coordinating centre, 65.3% of the relatives of index cases (5.7 relatives per family, on average) accepted the invitation to participate. The study logistics were well adapted to clinical and laboratory constraints, and collaboration between partners (clinicians, biologists, coordinating centre and participants) was smooth. Hence, TUMOSPEC is being pursued, with the aim of optimizing clinical management guidelines specific to each gene.

4.
Hum Genet ; 140(9): 1367-1377, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34255152

RESUMO

Spermatozoa are polarized cells with a head and a flagellum joined together by the connecting piece. Flagellum integrity is critical for normal sperm function, and flagellum defects consistently lead to male infertility. Multiple morphological abnormalities of the flagella (MMAF) is a distinct sperm phenotype consistently leading to male infertility due to a reduced or absent sperm motility associated with severe morphological and ultrastructural flagellum defects. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analyzed remain unresolved, suggesting that many yet uncharacterized gene defects account for this phenotype. By performing a retrospective exome analysis of the unsolved cases from our initial cohort of 167 infertile men with a MMAF phenotype, we identified one individual carrying a homozygous frameshift variant in CFAP206, a gene encoding a microtubule-docking adapter for radial spoke and inner dynein arm. Immunostaining experiments in the patient's sperm cells demonstrated the absence of WDR66 and RSPH1 proteins suggesting severe radial spokes and calmodulin and spoke-associated complex defects. Using the CRISPR-Cas9 technique, we generated homozygous Cfap206 knockout (KO) mice which presented with male infertility due to functional, structural and ultrastructural sperm flagellum defects associated with a very low rate of embryo development using ICSI. Overall, we showed that CFAP206 is essential for normal sperm flagellum structure and function in human and mouse and that bi-allelic mutations in CFAP206 cause male infertility in man and mouse by inducing morphological and functional defects of the sperm flagellum that may also cause ICSI failures.


Assuntos
Proteínas do Citoesqueleto , Mutação da Fase de Leitura , Homozigoto , Infertilidade Masculina , Cauda do Espermatozoide/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Masculino , Camundongos
5.
Microorganisms ; 9(6)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204665

RESUMO

The implementation of MALDI-TOF MS in medical microbiology laboratories has revolutionized practices and significantly reduced turnaround times of identification processes. However, although bacteriology quickly benefited from the contributions of this technique, adjustments were necessary to accommodate the specific characteristics of fungi. MALDI-TOF MS is now an indispensable tool in clinical mycology laboratories, both for the identification of yeasts and filamentous fungi, and other innovative uses are gradually emerging. Based on the practical experience of our medical mycology laboratory, this review will present the current uses of MALDI-TOF MS and the adaptations we implemented, to allow their practical execution in a daily routine. We will also introduce some less mainstream applications, like those for fungemia, or even still under development, as is the case for the determination of sensitivity to antifungal agents or typing methods.

6.
J Med Genet ; 57(10): 708-716, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32161152

RESUMO

BACKGROUND: Multiple morphological abnormalities of the flagella (MMAF) consistently lead to male infertility due to a reduced or absent sperm motility defined as asthenozoospermia. Despite numerous genes recently described to be recurrently associated with MMAF, more than half of the cases analysed remain unresolved, suggesting that many yet uncharacterised gene defects account for this phenotype METHODS: Exome sequencing was performed on 167 infertile men with an MMAF phenotype. Immunostaining and transmission electron microscopy (TEM) in sperm cells from affected individuals were performed to characterise the ultrastructural sperm defects. Gene inactivation using RNA interference (RNAi) was subsequently performed in Trypanosoma. RESULTS: We identified six unrelated affected patients carrying a homozygous deleterious variants in MAATS1, a gene encoding CFAP91, a calmodulin-associated and spoke-associated complex (CSC) protein. TEM and immunostaining experiments in sperm cells showed severe central pair complex (CPC) and radial spokes defects. Moreover, we confirmed that the WDR66 protein is a physical and functional partner of CFAP91 into the CSC. Study of Trypanosoma MAATS1's orthologue (TbCFAP91) highlighted high sequence and structural analogies with the human protein and confirmed the axonemal localisation of the protein. Knockdown of TbCFAP91 using RNAi impaired flagellar movement led to CPC defects in Trypanosoma as observed in humans. CONCLUSIONS: We showed that CFAP91 is essential for normal sperm flagellum structure and function in human and Trypanosoma and that biallelic variants in this gene lead to severe flagellum malformations resulting in astheno-teratozoospermia and primary male infertility.


Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/genética , Infertilidade Masculina/genética , Anormalidades Múltiplas/patologia , Animais , Astenozoospermia/patologia , Axonema/genética , Axonema/ultraestrutura , Homozigoto , Humanos , Infertilidade Masculina/patologia , Masculino , Mutação/genética , Motilidade Espermática/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Cauda do Espermatozoide/ultraestrutura , Espermatozoides/patologia , Espermatozoides/ultraestrutura , Trypanosoma/genética , Sequenciamento Completo do Exoma
7.
Proteomics Clin Appl ; 14(5): e1900116, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32198817

RESUMO

PURPOSE: Glioblastoma is one of the most aggressive primary brain cancers. The precise grading of tumors is important to adopt the best follow-up treatment but complementary methods to histopathological diagnosis still lack in achieving an unbiased and reliable classification. EXPERIMENTAL DESIGN: To progress in the field, a rapid Matrix Assisted Laser Desorption Ionization - Time of Flight Mass spectrometry (MALDI-TOF MS) protocole, devised for the identification and taxonomic classification of microorganisms and based on the analysis of whole cell extracts, was applied to glioma cell lines. RESULTS: The analysis of different human glioblastoma cell lines permitted to identify distinct proteomic profiles thus demonstrating the ability of MALDI-TOF to distinguish different malignant cell types. CONCLUSIONS AND CLINICAL RELEVANCE: In the study, the authors showed the ability of MALDI-TOF profiling to discriminate glioblastoma cell lines, demonstrating that this technique could be used in complement to histological tumor classification. The proposed procedure is rapid and inexpensive and could be used to improve brain tumors classification and help propose a personalized and more efficient treatment.


Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/metabolismo , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias Encefálicas/classificação , Linhagem Celular Tumoral , Diagnóstico Diferencial , Humanos , Medicina de Precisão , Fatores de Tempo
8.
Hum Reprod ; 34(10): 2071-2079, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31621862

RESUMO

The use of high-throughput sequencing techniques has allowed the identification of numerous mutations in genes responsible for severe astheno-teratozoospermia due to multiple morphological abnormalities of the sperm flagella (MMAF). However, more than half of the analysed cases remain unresolved suggesting that many yet uncharacterised gene defects account for this phenotype. Based on whole-exome sequencing data from a large cohort of 167 MMAF-affected subjects, we identified two unrelated affected individuals carrying a homozygous deleterious mutation in CFAP70, a gene not previously linked to the MMAF phenotype. One patient had a homozygous splice variant c.1723-1G>T, altering a consensus splice acceptor site of CFAP70 exon 16, and one had a likely deleterious missense variant in exon 3 (p.Phe60Ile). The CFAP70 gene encodes a regulator protein of the outer dynein arms (ODA) strongly expressed in the human testis. In the sperm cells from the patient carrying the splice variant, immunofluorescence (IF) experiments confirmed the absence of the protein in the sperm flagellum. Moreover, IF analysis showed the absence of markers for the ODAs and the central pair complex of the axoneme. Interestingly, whereas CFAP70 staining was present in sperm cells from patients with mutations in the three other MMAF-related genes ARMC2, FSIP2 and CFAP43, we observed an absence of staining in sperm cells from patients mutated in the WDR66 gene, suggesting a possible interaction between two different axonemal components. In conclusion, this work provides the first evidence that loss of CFAP70 function causes MMAF and that ODA-related proteins may be crucial for the assembly and/or stability of the flagellum axoneme in addition to its motility.


Assuntos
Astenozoospermia/genética , Proteínas Associadas aos Microtúbulos/genética , Cauda do Espermatozoide/patologia , Astenozoospermia/diagnóstico , Astenozoospermia/patologia , Axonema/patologia , Análise Mutacional de DNA , Éxons/genética , Homozigoto , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Mutação de Sentido Incorreto , Sítios de Splice de RNA/genética , Índice de Gravidade de Doença , Sequenciamento Completo do Exoma
9.
Am J Hum Genet ; 104(2): 331-340, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30686508

RESUMO

Male infertility is a major health concern. Among its different causes, multiple morphological abnormalities of the flagella (MMAF) induces asthenozoospermia and is one of the most severe forms of qualitative sperm defects. Sperm of affected men display short, coiled, absent, and/or irregular flagella. To date, six genes (DNAH1, CFAP43, CFAP44, CFAP69, FSIP2, and WDR66) have been found to be recurrently associated with MMAF, but more than half of the cases analyzed remain unresolved, suggesting that many yet-uncharacterized gene defects account for this phenotype. Here, whole-exome sequencing (WES) was performed on 168 infertile men who had a typical MMAF phenotype. Five unrelated affected individuals carried a homozygous deleterious mutation in ARMC2, a gene not previously linked to the MMAF phenotype. Using the CRISPR-Cas9 technique, we generated homozygous Armc2 mutant mice, which also presented an MMAF phenotype, thus confirming the involvement of ARMC2 in human MMAF. Immunostaining experiments in AMRC2-mutated individuals and mutant mice evidenced the absence of the axonemal central pair complex (CPC) proteins SPAG6 and SPEF2, whereas the other tested axonemal and peri-axonemal components were present, suggesting that ARMC2 is involved in CPC assembly and/or stability. Overall, we showed that bi-allelic mutations in ARMC2 cause male infertility in humans and mice by inducing a typical MMAF phenotype, indicating that this gene is necessary for sperm flagellum structure and assembly.


Assuntos
Alelos , Astenozoospermia/genética , Astenozoospermia/patologia , Proteínas do Citoesqueleto/genética , Flagelos/genética , Mutação , Espermatozoides/anormalidades , Espermatozoides/patologia , Animais , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/deficiência , Humanos , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Camundongos , Proteínas dos Microtúbulos/deficiência , Proteínas
10.
Clin Genet ; 94(6): 575-580, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30221343

RESUMO

We report findings from a male fetus of 26 weeks' gestational age with severe isolated intrauterine growth restriction (IUGR). Chromosomal microarray analysis (CMA) on amniotic fluid cells revealed a 1.06-Mb duplication in 19q13.42 inherited from the healthy father. This duplication contains 34 genes including ZNF331, a gene encoding a zinc-finger protein specifically imprinted (paternally expressed) in the placenta. Study of the ZNF331 promoter by methylation-specific-multiplex ligation-dependent probe amplification showed that the duplicated allele was not methylated in the fetus unlike in the father's genome, suggesting both copies of the ZNF331 gene are expressed in the fetus. The anti-ZNF331 immunohistochemical analysis confirmed that ZNF331 was expressed at higher levels in renal and placental tissues from this fetus compared to controls. Interestingly, ZNF331 expression levels in the placenta have previously been reported to inversely correlate with fetal growth parameters. The original observation presented in this report showed that duplication of ZNF331 could be a novel genetic cause of isolated IUGR and underlines the usefulness of CMA to investigate the genetic causes of isolated severe IUGR.


Assuntos
Cromossomos Humanos Par 19 , Retardo do Crescimento Fetal/diagnóstico , Retardo do Crescimento Fetal/genética , Duplicação Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Impressão Genômica , Adulto , Biópsia , Proteínas de Ligação a DNA/genética , Epigênese Genética , Feminino , Estudos de Associação Genética/métodos , Testes Genéticos , Humanos , Imuno-Histoquímica , Proteínas de Neoplasias/genética , Gravidez , Ultrassonografia Pré-Natal
11.
Hum Reprod ; 33(10): 1973-1984, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137358

RESUMO

STUDY QUESTION: Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? SUMMARY ANSWER: WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. WHAT IS KNOWN ALREADY: The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. MAIN RESULTS AND THE ROLE OF CHANCE: We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). LIMITATIONS, REASONS FOR CAUTION: The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.


Assuntos
Cauda do Espermatozoide/patologia , Teratozoospermia/genética , Adulto , Estudos de Casos e Controles , Humanos , Infertilidade Masculina/genética , Masculino , Pessoa de Meia-Idade , Mutação , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cauda do Espermatozoide/ultraestrutura , Teratozoospermia/diagnóstico , Sequenciamento Completo do Exoma/métodos
12.
Artigo em Inglês | MEDLINE | ID: mdl-28674045

RESUMO

The emergence of fluoroquinolone (FQ)-resistant mutants of Legionella pneumophila in infected humans was previously reported using a next-generation DNA sequencing (NGS) approach. This finding could explain part of the therapeutic failures observed in legionellosis patients treated with these antibiotics. The aim of this study was to develop digital PCR (dPCR) assays allowing rapid and accurate detection and quantification of these resistant mutants in respiratory samples, especially when the proportion of mutants in a wild-type background is low. We designed three dPCRgyrA assays to detect and differentiate the wild-type and one of the three gyrA mutations previously described as associated with FQ resistance in L. pneumophila: at positions 248C→T (T83I), 259G→A (D87N), and 259G→C (D87H). To assess the performance of these assays, mixtures of FQ-resistant and -susceptible strains of L. pneumophila were analyzed, and the results were compared with those obtained with Sanger DNA sequencing and real-time quantitative PCR (qPCR) technologies. The dPCRgyrA assays were able to detect mutated gyrA sequences in the presence of wild-type sequences at up to 1:1,000 resistant/susceptible allele ratios. By comparison, Sanger DNA sequencing and qPCR were less sensitive, allowing the detection of gyrA mutants at up to 1:1 and 1:10 ratios, respectively. When testing 38 respiratory samples from 23 legionellosis patients (69.6% treated with an FQ), dPCRgyrA detected small amounts of gyrA mutants in four (10.5%) samples from three (13.0%) patients. These results demonstrate that dPCR is a highly sensitive alternative to quantify FQ resistance in L. pneumophila, and it could be used in clinical practice to detect patients that could be at higher risk of therapeutic failure.


Assuntos
Antibacterianos/farmacologia , DNA Girase/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/farmacologia , Legionella pneumophila/efeitos dos fármacos , Legionella pneumophila/genética , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , DNA Bacteriano/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Legionelose/microbiologia , Masculino , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , Análise de Sequência de DNA/métodos , Adulto Jovem
13.
J Med Genet ; 54(7): 502-510, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28270404

RESUMO

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) represent a significant healthcare burden since it is the primary cause of chronic kidney in children. CNVs represent a recurrent molecular cause of CAKUT but the culprit gene remains often elusive. Our study aimed to define the gene responsible for CAKUT in patients with an 1q23.3q24.1 microdeletion. METHODS: We describe eight patients presenting with CAKUT carrying an 1q23.3q24.1 microdeletion as identified by chromosomal microarray analysis (CMA). Clinical features were collected, especially the renal and urinary tract phenotype, and extrarenal features. We characterised PBX1 expression and localisation in fetal and adult kidneys using quantitative RT-PCR and immunohistochemistry. RESULTS: We defined a 276-kb minimal common region (MCR) that only overlaps with the PBX1 gene. All eight patients presented with syndromic CAKUT. CAKUT were mostly bilateral renal hypoplasia (75%). The most frequent extrarenal symptoms were developmental delay and ear malformations. We demonstrate that PBX1 is strongly expressed in fetal kidneys and brain and expression levels decreased in adult samples. In control fetal kidneys, PBX1 was localised in nuclei of medullary, interstitial and mesenchymal cells, whereas it was present in endothelial cells in adult kidneys. CONCLUSIONS: Our results indicate that PBX1 haploinsufficiency leads to syndromic CAKUT as supported by the Pbx1-null mice model. Correct PBX1 dosage appears to be critical for normal nephrogenesis and seems important for brain development in humans. CMA should be recommended in cases of fetal renal anomalies to improve genetic counselling and pregnancy management.


Assuntos
Haploinsuficiência/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Criança , Pré-Escolar , Feminino , Feto/metabolismo , Genoma Humano , Humanos , Lactente , Rim/anormalidades , Rim/embriologia , Rim/metabolismo , Rim/patologia , Masculino , Síndrome
14.
J Proteomics ; 152: 150-152, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-27989942

RESUMO

This study describes an innovative strategy for rapid detection and identification of bacteria causing endophthalmitis, combining the use of an automated blood culture system with MALDI-TOF mass spectrometry methodology. Using this protocol, we could identify 96% of 45 bacterial strains isolated from vitreous samples collected in acute post-operative endophthalmitis patients.


Assuntos
Bactérias/isolamento & purificação , Endoftalmite/diagnóstico , Endoftalmite/microbiologia , Complicações Pós-Operatórias/microbiologia , Técnicas Bacteriológicas/métodos , Ensaios de Triagem em Larga Escala , Humanos , Complicações Pós-Operatórias/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Corpo Vítreo/microbiologia
15.
Proteomics Clin Appl ; 11(5-6)2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27921389

RESUMO

PURPOSE: Fast species diagnosis has an important health care impact, as rapid and specific antibacterial therapy is of clear benefit for patient's outcome. Here, a new protocol for species identification directly from positive blood cultures is proposed. EXPERIMENTAL DESIGN: Four in-house protocols for bacterial identification by MS directly from clinical positive blood cultures evaluating two lytic agents, SDS and saponin, and two protein extraction schemes, fast (FP) and long (LP) are compared. One hundred and sixty-eight identification tests are carried out on 42 strains. RESULTS: Overall, there are correct identifications to the species level in 90% samples for the SDS-LP, 60% for the SDS-FP, 48% for the saponin LP, and 43% for the saponin FP. Adapted scores allowed 92, 86, 72, and 53% identification for SDS-LP, SDS-FP, saponin LP, and saponin FP, respectively. Saponin lysis is associated with a significantly lower score compared to SDS (0.87 [0.83-0.92], p-value < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: This study supports the use of SDS lysis instead of saponin lysis and the application of this rapid and cost-effective protocol in daily routine for microbiological agents implicated in septicemia.


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Hemocultura , Saponinas/farmacologia , Dodecilsulfato de Sódio/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Bactérias/citologia
16.
Genet Med ; 19(6): 701-710, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27906199

RESUMO

PURPOSE: To determine whether duplication of the ARID1A gene is responsible for a new recognizable syndrome. METHODS: We describe four patients with a 1p36.11 microduplication involving ARID1A as identified by array-comparative genomic hybridization . We performed comparative transcriptomic analysis of patient-derived fibroblasts using RNA sequencing and evaluated the impact of ARID1A duplication on the cell cycle using fluorescence-activated cell sorting. Functional relationships between differentially expressed genes were investigated with ingenuity pathway analysis (IPA). RESULTS: Combining the genomic data, we defined a small (122 kb), minimally critical region that overlaps the full ARID1A gene. The four patients shared a strikingly similar phenotype that included intellectual disability and microcephaly. Transcriptomic analysis revealed the deregulated expression of several genes previously linked to microcephaly and developmental disorders as well as the involvement of signaling pathways relevant to microcephaly, among which the polo-like kinase (PLK) pathway was especially notable. Cell-cycle analysis of patient-derived fibroblasts showed a significant increase in the proportion of cells in G1 phase at the expense of G2-M cells. CONCLUSION: Our study reports a new microduplication syndrome involving the ARID1A gene. This work is the first step in clarifying the pathophysiological mechanism that links changes in the gene dosage of ARID1A with intellectual disability and microcephaly.Genet Med advance online publication 01 December 2016.


Assuntos
Cromossomos Humanos Par 1 , Duplicação Gênica , Deficiência Intelectual/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Proteínas de Ligação a DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Síndrome
17.
Contrast Media Mol Imaging ; 11(6): 535-543, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27766757

RESUMO

Cellular MRI, which visualizes magnetically labelled cells (cells*), is an active research field for in vivo cell therapy and tracking. The simultaneous relaxation rate measurements (R2 *, R2 , R1 ) are the basis of a quantitative cellular MRI method proposed here. U937 cells were labelled with Molday ION Rhodamine B, a bi-functional superparamagnetic and fluorescent nanoparticle (U937*). U937* viability and proliferation were not affected in vitro. In vitro relaxometry was performed in a cell concentration range of [2.5 × 104 -108 ] cells/mL. These measurements show the existence of complementary cell concentration intervals where these rates vary linearly. The juxtaposition of these intervals delineates a wide cell concentration range over which one of the relaxation rates in a voxel of an in vivo image can be converted into an absolute cell concentration. The linear regime was found at high concentrations for R1 in the range of [106 - 2 × 108 ] cells/mL, at intermediate concentrations for R2 in [2.5 × 105 - 5 × 107 ] cells/mL and at low concentrations for R2 * in [8 × 104 - 5 × 106 ] cells/mL. In vivo relaxometry was performed in a longitudinal study, with labelled U937 cells injected into a U87 glioma mouse model. Using in vitro data, maps of in vivo U937* concentrations were obtained by converting one of the in vivo relaxation rates to cell concentration maps. MRI results were compared with the corresponding optical images of the same brains, showing the usefulness of our method to accurately follow therapeutic cell biodistribution in a longitudinal study. Results also demonstrate that the method quantifies a large range of magnetically labelled cells*. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Transplante de Células , Imageamento por Ressonância Magnética/métodos , Animais , Encéfalo/patologia , Contagem de Células , Movimento Celular , Fluorescência , Glioma/patologia , Xenoenxertos , Humanos , Magnetismo , Camundongos , Células U937/transplante
18.
Proc Natl Acad Sci U S A ; 112(25): E3207-15, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26056270

RESUMO

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFß family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Canal Arterial/fisiologia , Fator 2 de Diferenciação de Crescimento/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Canal Arterial/patologia , Fator 2 de Diferenciação de Crescimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
19.
J Clin Microbiol ; 53(5): 1761-4, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25762771

RESUMO

We developed an in-house assay for the direct identification, by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, of yeasts in blood culture. Sixty-one representative strains from 12 species were analyzed in spiked blood cultures. Our assay accurately identified 95 of 107 (88.8%) positive blood cultures and outperformed the commercial Sepsityper kit (81.7% identification).


Assuntos
Sangue/microbiologia , Fungemia/diagnóstico , Fungemia/microbiologia , Técnicas Microbiológicas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Leveduras/classificação , Leveduras/isolamento & purificação , Humanos , Sensibilidade e Especificidade , Leveduras/química
20.
Tumour Biol ; 35(7): 6221-33, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24633919

RESUMO

Among rodent models for brain tumors, the 9L gliosarcoma is one of the most widely used. Our 9L-European Synchrotron Radiation Facility (ESRF) model was developed from cells acquired at the Brookhaven National Laboratory (NY, USA) in 1997 and implanted in the right caudate nucleus of syngeneic Fisher rats. It has been largely used by the user community of the ESRF during the last decade, for imaging, radiotherapy, and chemotherapy, including innovative treatments based on particular irradiation techniques and/or use of new drugs. This work presents a detailed study of its characteristics, assessed by magnetic resonance imaging (MRI), histology, immunohistochemistry, and cytogenetic analysis. The data used for this work were from rats sampled in six experiments carried out over a 3-year period in our lab (total number of rats = 142). The 9L-ESRF tumors were induced by a stereotactic inoculation of 10(4) 9L cells in the right caudate nucleus of the brain. The assessment of vascular parameters was performed by MRI (blood volume fraction and vascular size index) and by immunostaining of vessels (rat endothelial cell antigen-1 and type IV collagen). Immunohistochemistry and regular histology were used to describe features such as tumor cell infiltration, necrosis area, nuclear pleomorphism, cellularity, mitotic characteristics, leukocytic infiltration, proliferation, and inflammation. Moreover, for each of the six experiments, the survival of the animals was assessed and related to the tumor growth observed by MRI or histology. Additionally, the cytogenetic status of the 9L cells used at ESRF lab was investigated by comparative genomics hybridization analysis. Finally, the response of the 9L-ESRF tumor to radiotherapy was estimated by plotting the survival curves after irradiation. The median survival time of 9L-ESRF tumor-bearing rats was highly reproducible (19-20 days). The 9L-ESRF tumors presented a quasi-exponential growth, were highly vascularized with a high cellular density and a high proliferative index, accompanied by signs of inflammatory responses. We also report an infiltrative pattern which is poorly observed on conventional 9 L tumor. The 9L-ESRF cells presented some cytogenetic specificities such as altered regions including CDK4, CDKN2A, CDKN2B, and MDM2 genes. Finally, the lifespan of 9L-ESRF tumor-bearing rats was enhanced up to 28, 35, and 45 days for single doses of 10, 20, and 2 × 20 Gy, respectively. First, this report describes an animal model that is used worldwide. Second, we describe few features typical of our model if compared to other 9L models worldwide. Altogether, the 9L-ESRF tumor model presents characteristics close to the human high-grade gliomas such as high proliferative capability, high vascularization and a high infiltrative pattern. Its response to radiotherapy demonstrates its potential as a tool for innovative radiotherapy protocols.


Assuntos
Neoplasias Encefálicas/genética , Gliossarcoma/genética , Neoplasias Experimentais/genética , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Proliferação de Células , Modelos Animais de Doenças , Gliossarcoma/patologia , Gliossarcoma/terapia , Humanos , Gradação de Tumores , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica , Ratos , Ratos Endogâmicos F344
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