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1.
Clin Chem ; 65(9): 1153-1160, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31292136

RESUMO

BACKGROUND: Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS: We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS: UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS: The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

2.
J Med Genet ; 56(8): 526-535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30923172

RESUMO

BACKGROUND: Balanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing to characterise breakpoints of balanced chromosomal rearrangements at the molecular level in patients with intellectual disability and/or congenital anomalies. METHODS: Breakpoints were characterised by a paired-end low depth whole genome sequencing (WGS) strategy and validated by Sanger sequencing. Expression study of disrupted and neighbouring genes was performed by RT-qPCR from blood or lymphoblastoid cell line RNA. RESULTS: Among the 55 patients included (41 reciprocal translocations, 4 inversions, 2 insertions and 8 complex chromosomal rearrangements), we were able to detect 89% of chromosomal rearrangements (49/55). Molecular signatures at the breakpoints suggested that DNA breaks arose randomly and that there was no major influence of repeated elements. Non-homologous end-joining appeared as the main mechanism of repair (55% of rearrangements). A diagnosis could be established in 22/49 patients (44.8%), 15 by gene disruption (KANSL1, FOXP1, SPRED1, TLK2, MBD5, DMD, AUTS2, MEIS2, MEF2C, NRXN1, NFIX, SYNGAP1, GHR, ZMIZ1) and 7 by position effect (DLX5, MEF2C, BCL11B, SATB2, ZMIZ1). In addition, 16 new candidate genes were identified. Systematic gene expression studies further supported these results. We also showed the contribution of topologically associated domain maps to WGS data interpretation. CONCLUSION: Paired-end WGS is a valid strategy and may be used for structural variation characterisation in a clinical setting.

3.
Am J Hum Genet ; 104(2): 319-330, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30639322

RESUMO

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.


Assuntos
Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Mutação Puntual , Fatores de Transcrição/genética , Alelos , Animais , Criança , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Camundongos , Síndrome , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
4.
Prenat Diagn ; 36(6): 523-9, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27018091

RESUMO

OBJECTIVE: Sex chromosome aneuploidies are frequently detected fortuitously in a prenatal diagnosis. Most cases of 47, XXX and 47, XYY syndromes are diagnosed in this context, and parents are thus faced with an unexpected situation. The objective of the present study was to characterize a French cohort of prenatally diagnosed cases of 47, XXX and 47, XYY and to evaluate the termination of pregnancy (TOP) rate before and after France's implementation of multidisciplinary centres for prenatal diagnosis in 1997. METHODS: This retrospective study identified respectively 291 and 175 cases of prenatally diagnosed 47, XXX and 47, XYY between 1976 and 2012. For each case, the indication, maternal age, karyotype and outcome were recorded. RESULTS: Most diagnoses of the two conditions were fortuitous. The occurrence of 47, XXX was associated with advanced maternal age. The overall TOP rate was higher for 47, XXX (22.9%) than for 47, XYY (14.6%), although this difference was not statistically significant. However, the TOP rates fell significantly after 1997 (from 41.1% to 11.8% for 47, XXX and from 25.8% to 6.7% for 47, XYY). CONCLUSION: The TOP rates after prenatal diagnoses of 47, XXX and 47, XYY fell significantly after 1997, following France's implementation of multidisciplinary centres for prenatal diagnosis. © 2016 John Wiley & Sons, Ltd.


Assuntos
Aborto Induzido/estatística & dados numéricos , Aborto Espontâneo/epidemiologia , Resultado da Gravidez/epidemiologia , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/epidemiologia , Transtornos dos Cromossomos Sexuais/epidemiologia , Cariótipo XYY/epidemiologia , Aborto Induzido/tendências , Adulto , Amniocentese , Amostra da Vilosidade Coriônica , Cromossomos Humanos X , Estudos de Coortes , Feminino , Morte Fetal , França/epidemiologia , Humanos , Idade Materna , Gravidez , Diagnóstico Pré-Natal , Estudos Retrospectivos , Aberrações dos Cromossomos Sexuais , Transtornos dos Cromossomos Sexuais/diagnóstico , Transtornos dos Cromossomos Sexuais/diagnóstico por imagem , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/diagnóstico , Transtornos do Cromossomo Sexual no Desenvolvimento Sexual/diagnóstico por imagem , Trissomia/diagnóstico , Cariótipo XYY/diagnóstico , Cariótipo XYY/diagnóstico por imagem
5.
Eur J Hum Genet ; 24(6): 830-7, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26395556

RESUMO

The increasing use of array-CGH in malformation syndromes with intellectual disability could lead to the description of new contiguous gene syndrome by the analysis of the gene content of the microdeletion and reverse phenotyping. Thanks to a national and international call for collaboration by Achropuce and Decipher, we recruited four patients carrying de novo overlapping deletions of chromosome 9q33.3q34.11, including the STXBP1, the LMX1B and the ENG genes. We restrained the selection to these three genes because the effects of their haploinsufficency are well described in the literature and easily recognizable clinically. All deletions were detected by array-CGH and confirmed by FISH. The patients display common clinical features, including intellectual disability with epilepsy, owing to the presence of STXBP1 within the deletion, nail dysplasia and bone malformations, in particular patellar abnormalities attributed to LMX1B deletion, epistaxis and cutaneous-mucous telangiectasias explained by ENG haploinsufficiency and common facial dysmorphism. This systematic analysis of the genes comprised in the deletion allowed us to identify genes whose haploinsufficiency is expected to lead to disease manifestations and complications that require personalized follow-up, in particular for renal, eye, ear, vascular and neurological manifestations.


Assuntos
Deleção Cromossômica , Anormalidades Craniofaciais/genética , Endoglina/genética , Epilepsia/genética , Deficiência Intelectual/genética , Proteínas com Homeodomínio LIM/genética , Proteínas Munc18/genética , Fatores de Transcrição/genética , Adolescente , Criança , Cromossomos Humanos Par 9/genética , Anormalidades Craniofaciais/diagnóstico , Epilepsia/diagnóstico , Feminino , Haploinsuficiência , Humanos , Deficiência Intelectual/diagnóstico , Masculino , Fenótipo , Síndrome
6.
Ann Pathol ; 34(3): 223-7, 2014 Jun.
Artigo em Francês | MEDLINE | ID: mdl-24950872

RESUMO

We report on a 51-year-old woman who presented with a cervical spinal cord tumor clinically suspected to be a metastasis. Histological examination revealed an anaplastic meningioma containing epithelial nests arranged in a gland-like pattern suggestive of adenocarcinoma. This component strongly expressed cytokeratins whereas the meningothelial component was vimentin--epithelial membrane antigen--and progesterone receptor-immunoreactive, suggesting either anaplastic meningioma with adenocarcinoma-like metaplasia, or adenocarcinoma metastasis in a meningioma, but the search for a primitive neoplasia including thoracic-abdominal-pelvic computed tomography and mammography was negative. Anaplastic meningiomas with adenocarcinoma-like metaplasia are uncommon lesions, 4 cases having been reported in the literature so far. Their immunohistochemical and chromosomal characteristics are similar to those observed in secretory meningiomas. When available, fluorescence in situ hybridization detects the same chromosomal alterations in the two components, confirming a common clonal origin. This observation demonstrates the necessity to perform the correct diagnosis of malignant meningioma with adenocarcinomatous metaplasia, whose prognosis and treatment radically differ from those of metastatic adenocarcinoma located in a meningioma.


Assuntos
Neoplasias Meníngeas/patologia , Meningioma/patologia , Adenocarcinoma/diagnóstico , Antiporters/análise , Biomarcadores Tumorais/análise , Neoplasias da Mama , Carcinoma/diagnóstico , Carcinoma/secundário , Células Clonais/patologia , Diagnóstico Diferencial , Feminino , Humanos , Hibridização in Situ Fluorescente , Queratina-7/análise , Imagem por Ressonância Magnética , Neoplasias Meníngeas/química , Neoplasias Meníngeas/complicações , Neoplasias Meníngeas/diagnóstico , Meningioma/química , Meningioma/complicações , Meningioma/diagnóstico , Metaplasia , Pessoa de Meia-Idade , Mucina-1/análise , Receptores de Progesterona/análise , Compressão da Medula Espinal/etiologia , Vimentina/análise
7.
Cell Transplant ; 23(12): 1475-87, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25565635

RESUMO

Genetic alterations have recently been described as emerging during the culture of embryonic stem cells or induced pluripotent stem cells, raising concerns about their safety in future clinical use. Myoblasts are adult stem cells with important therapeutic potential that have been used in clinical trials for almost 20 years, but their genome integrity has not yet been established. Here we produced 10 human myoblast preparations and investigated their genomic stability. At the third passage, half of the preparations had a normal karyotype and half showed one to four alterations/30 metaphases. Chromosome 2 trisomy was found in 1-2/30 metaphases and/or 2/100 nuclei by FISH in 3/10 samples, and there was no other recurrent anomaly. When prolonging cultures, these erratic abnormalities were never associated with a growth advantage. Cellular senescence was manifested in all samples by growth arrest before passage 15. Expression of TERT was always negative. Molecular analysis of individual p53 transcripts did not reveal tumorigenic mutations. CGH array (10 samples) and exome sequencing (one sample) failed to detect copy number variations or accumulation of mutations, respectively. Myoblasts did not grow either in soft agar or in vivo after injection in immunodeficient mice. Hence, occasional genomic abnormalities may occur during myoblast culture but are not associated with risk of transformation.


Assuntos
Transformação Celular Neoplásica , Instabilidade Cromossômica , Mioblastos/metabolismo , Mioblastos/patologia , Animais , Carcinogênese , Proliferação de Células , Células Cultivadas , Senescência Celular , Hibridização Genômica Comparativa , Feminino , Dosagem de Genes , Humanos , Imunofenotipagem , Cariotipagem , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Fenótipo
8.
Arch Gen Psychiatry ; 66(9): 947-56, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19736351

RESUMO

CONTEXT: Results of comparative genomic hybridization studies have suggested that rare copy number variations (CNVs) at numerous loci are involved in the cause of mental retardation, autism spectrum disorders, and schizophrenia. OBJECTIVES: To provide an estimate of the collective frequency of a set of recurrent or overlapping CNVs in 3 different groups of cases compared with healthy control subjects and to assess whether each CNV is present in more than 1 clinical category. DESIGN: Case-control study. SETTING: Academic research. PARTICIPANTS: We investigated 28 candidate loci previously identified by comparative genomic hybridization studies for gene dosage alteration in 247 cases with mental retardation, in 260 cases with autism spectrum disorders, in 236 cases with schizophrenia or schizoaffective disorder, and in 236 controls. MAIN OUTCOME MEASURES: Collective and individual frequencies of the analyzed CNVs in cases compared with controls. RESULTS: Recurrent or overlapping CNVs were found in cases at 39.3% of the selected loci. The collective frequency of CNVs at these loci is significantly increased in cases with autism, in cases with schizophrenia, and in cases with mental retardation compared with controls (P < .001, P = .01, and P = .001, respectively, Fisher exact test). Individual significance (P = .02 without correction for multiple testing) was reached for the association between autism and a 350-kilobase deletion located at 22q11 and spanning the PRODH and DGCR6 genes. CONCLUSIONS: Weakly to moderately recurrent CNVs (transmitted or occurring de novo) seem to be causative or contributory factors for these diseases. Most of these CNVs (which contain genes involved in neurotransmission or in synapse formation and maintenance) are present in the 3 pathologic conditions (schizophrenia, autism, and mental retardation), supporting the existence of shared biologic pathways in these neurodevelopmental disorders.


Assuntos
Transtorno Autístico/genética , Deficiência Intelectual/genética , Esquizofrenia/genética , Adolescente , Adulto , Transtorno Autístico/diagnóstico , Estudos de Casos e Controles , Mapeamento Cromossômico/estatística & dados numéricos , Hibridização Genômica Comparativa/estatística & dados numéricos , Feminino , Dosagem de Genes/genética , Frequência do Gene , Genótipo , Humanos , Hibridização in Situ Fluorescente/estatística & dados numéricos , Deficiência Intelectual/diagnóstico , Masculino , Neurogênese , Análise de Sequência com Séries de Oligonucleotídeos , Prolina/sangue , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/genética , Esquizofrenia/diagnóstico
10.
Eur J Hum Genet ; 14(9): 1009-17, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16773131

RESUMO

In contrast to the numerous well-known microdeletion syndromes, only a few microduplications have been described, and this discrepancy may be due in part to methodological bias. In order to facilitate the detection of genomic microdeletions and microduplications, we developed a new assay based on QMPSF (Quantitative Multiplex PCR of Short fluorescent Fragments) able to explore simultaneously 12 candidate loci involved in mental retardation (MR) and known to be the target of genomic rearrangements. We first screened 153 patients with MR and facial dysmorphism associated with malformations, or growth anomalies, or familial history, with cytogenetically normal chromosomes, and the absence of FRAXA mutation and subtelomeric rearrangements. In this series, we found a 5q35 deletion removing the NSD1 gene in a patient with severe epilepsy, profound MR and, retrospectively, craniofacial features of Sotos syndrome. In a second series, we screened 140 patients with MR and behaviour disturbance who did not fulfil the de Vries criteria for subtelomeric rearrangements and who had a normal karyotype and no detectable FRAXA mutation. We detected a 22q11 deletion in a patient with moderate MR, obesity, and facial dysmorphism and a 4 Mb 17p11 duplication in a patient with moderate MR, behaviour disturbance, strabismus, and aspecific facial features. This new QMPSF assay can be gradually upgraded to include additional loci involved in newly recognised microduplication/microdeletion syndromes, and should facilitate wide screenings of patients with idiopathic MR and provide better estimates of the microduplication frequency in the MR population.


Assuntos
Deleção Cromossômica , Duplicação Gênica , Deficiência Intelectual/genética , Reação em Cadeia da Polimerase/métodos , Pré-Escolar , Aberrações Cromossômicas , Cromossomos Humanos Par 17 , Feminino , Síndrome do Cromossomo X Frágil/genética , Rearranjo Gênico , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas Nucleares/genética , Reprodutibilidade dos Testes , Telômero/genética
11.
Am J Med Genet A ; 134(4): 439-42, 2005 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-15810003

RESUMO

Holt-Oram syndrome, the major "heart-hand" syndrome is defined by the association of radial defects or triphalangeal thumbs and septal heart defects. The transmission is autosomal dominant and the causative gene has been shown to be TBX5, located on 12q24.1, which encodes a transcription factor. Genetic heterogeneity has been suggested by several reports. We identified a 14(q23.3 approximately 24.2q31.1) deletion in a boy presenting severe bilateral asymmetrical radial aplasia, congenital heart defects, and developmental delay. This deletion, whose size could be estimated to be 9.6-13.7 Mb, was shown to be inherited via his mother's interchromosomal insertion. This is the second report of a chromosome 14 interstitial deletion associated with clinical features of Holt-Oram syndrome. These observations suggest the existence of a new "heart-hand" locus on chromosome 14q.


Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Cromossomos Humanos Par 14/genética , Deformidades Congênitas da Mão/patologia , Cardiopatias Congênitas/patologia , Anormalidades Múltiplas/patologia , Bandeamento Cromossômico , Evolução Fatal , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Cariotipagem , Masculino , Síndrome
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