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1.
Prenat Diagn ; 39(11): 986-992, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31273809

RESUMO

OBJECTIVE: Uniparental disomy (UPD) testing is currently recommended during pregnancy in fetuses carrying a balanced Robertsonian translocation (ROB) involving chromosome 14 or 15, both chromosomes containing imprinted genes. The overall risk that such a fetus presents a UPD has been previously estimated to be around ~0.6-0.8%. However, because UPD are rare events and this estimate has been calculated from a number of studies of limited size, we have reevaluated the risk of UPD in fetuses for whom one of the parents was known to carry a nonhomologous ROB (NHROB). METHOD: We focused our multicentric study on NHROB involving chromosome 14 and/or 15. A total of 1747 UPD testing were performed in fetuses during pregnancy for the presence of UPD(14) and/or UPD(15). RESULT: All fetuses were negative except one with a UPD(14) associated with a maternally inherited rob(13;14). CONCLUSION: Considering these data, the risk of UPD following prenatal diagnosis of an inherited ROB involving chromosome 14 and/or 15 could be estimated to be around 0.06%, far less than the previous estimation. Importantly, the risk of miscarriage following an invasive prenatal sampling is higher than the risk of UPD. Therefore, we do not recommend prenatal testing for UPD for these pregnancies and parents should be reassured.

2.
J Assist Reprod Genet ; 36(5): 973-978, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30850901

RESUMO

BACKGROUND: The prevalence of chromosomal translocations is 1/500 in the general population. While in the vast majority of cases, carriers have a normal phenotype; they can present with difficulty conceiving due to the presence of a proportion of unbalanced gametes as a consequence of abnormal chromosomal segregation during meiosis. Since complex translocations involve three or more chromosomes, meiotic segregation leads to a greater number of possible combinations which effectively complicate both their study and therapeutic care. CASE PRESENTATION: We report on the case of a male carrier of a complex homogeneous double Robertsonian translocation: 44, XY, der(13;14)(q10;q10),der(21;22)(q10;q10). We studied his meiotic segregation by FISH on spermatozoa from the initial sample, as well as following discontinuous gradient centrifugation and after incubation in an hypo-osmotic solution. CONCLUSION: We report a method to study in a simple single-step manner the meiotic segregation of double Robertsonian translocations in spermatozoa. Further, our results suggest that reproductive prognosis of affected individuals may be markedly improved by HOST-based sperm selection (HBSS).

3.
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.

4.
Artigo em Inglês | MEDLINE | ID: mdl-30362053

RESUMO

PURPOSE: To explore the three-dimensional (3D) organization of sperm genome in DPY19L2-deficient globozoospermic patients speculating a link between DPY19L2 and genome organization of sperm nucleus. METHODS: This is a study of chromatin organization in DPY19L2-deficient globozoospermic patients and healthy donors using three-dimensional fluorescence in situ hybridization (3D-FISH) combined with confocal laser scanning microscopy followed by 3D image analysis. The 3D structures of sperm nuclei, chromocenter, telomeric regions and chromosome territories (CTs), were reconstructed using IMARIS software, and the relative radial position for each individual signal was calculated. Statistical analysis used a non-parametric Mann-Whitney test was appropriate with significance at p < 0.05. RESULTS: DPY19L2-deficient globozoospermic patients display impaired sperm chromocenter organization resulting in an increased number of chromocenters (5.4 vs 3.5; p < 0.0001). Moreover, radial positions of telomeres are modified with a more central position in globozoospermic nuclei. 3D-FISH analysis of five chromosome territories (CTs) (X, Y, 7, 17, 18) showed that DPY19L2-deficient globozoospermic sperm nuclei display altered spatial organization of CT X, CT 7 and CT 18. CONCLUSIONS: Our findings strengthen the hypothesis that DPY19L2 might be considered as a LINC-like protein having a crucial role in the organization of nuclear chromatin in sperm nucleus through its interaction with nuclear lamina. Our results might also explain defective embryonic development after intracytoplasmic sperm injection (ICSI) performed with DPY19L2-deficient globozoospermic sperm.

5.
J Med Genet ; 2018 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-30287593

RESUMO

BACKGROUND: The clinical significance of 16p13.11 duplications remains controversial while frequently detected in patients with developmental delay (DD), intellectual deficiency (ID) or autism spectrum disorder (ASD). Previously reported patients were not or poorly characterised. The absence of consensual recommendations leads to interpretation discrepancy and makes genetic counselling challenging. This study aims to decipher the genotype-phenotype correlations to improve genetic counselling and patients' medical care. METHODS: We retrospectively analysed data from 16 013 patients referred to 12 genetic centers for DD, ID or ASD, and who had a chromosomal microarray analysis. The referring geneticists of patients for whom a 16p13.11 duplication was detected were asked to complete a questionnaire for detailed clinical and genetic data for the patients and their parents. RESULTS: Clinical features are mainly speech delay and learning disabilities followed by ASD. A significant risk of cardiovascular disease was noted. About 90% of the patients inherited the duplication from a parent. At least one out of four parents carrying the duplication displayed a similar phenotype to the propositus. Genotype-phenotype correlations show no impact of the size of the duplicated segment on the severity of the phenotype. However, NDE1 and miR-484 seem to have an essential role in the neurocognitive phenotype. CONCLUSION: Our study shows that 16p13.11 microduplications are likely pathogenic when detected in the context of DD/ID/ASD and supports an essential role of NDE1 and miR-484 in the neurocognitive phenotype. Moreover, it suggests the need for cardiac evaluation and follow-up and a large study to evaluate the aortic disease risk.

6.
Artigo em Inglês | MEDLINE | ID: mdl-30191619

RESUMO

OBJECTIVES: To validate and evaluate an integrated protocol for non-invasive prenatal genetic screening (NIPS) for common fetal aneuploidies in a clinical setting, using the semiconductor sequencing technology, Ion Proton. METHODS: This prospective cohort study included 2505 pregnant women from seven academic genetic laboratories (695 high risk pregnancies in a validation study and 1810 pregnancies with a risk higher than 1/250 without ultrasound anomalies, in a real NIPS clinical setting). Cell free DNA from plasma samples was sequenced using Ion Proton sequencer, and sequencing data were analyzed using the open-access software WISECONDOR. Performance metrics for detection of trisomies 21, 18 and 13, were calculated based on either fetal karyotype result or clinical data collected at birth. We also evaluated the failure rate and compared three methods of fetal fraction quantification (RASSF1A assay, DEFRAG and SANEFALCON software). RESULTS: Sensitivities and specificities were: 98.3% (95%CI: 93.5 - 99.7) and 99.9% (95%CI: 99.4 - 100) for T21, 96.7% (95%CI: 80.9 - 99.8) and 100% (95%CI: 99.6 - 100) for T18, 94.1% (95%CI: 69.2 - 99.7) and 100% (95%CI: 99.6 - 100) for T13. Our failure rate was 1.2% at first and as low as 0.6% after re-testing some of the failed samples. Fetal fraction estimation by RASSF1A assay was consistent with DEFRAG results, both of which are adequate for routine diagnosis. CONCLUSIONS: We describe one of the largest studies evaluating the Ion Proton based NIPS and the first clinical study reporting pregnancy outcome in a large set of patients. We demonstrate that this platform is highly efficient in detecting the three most common trisomies. Our protocol is robust and can be easily implemented in any medical genetics laboratory. This article is protected by copyright. All rights reserved.

7.
Am J Med Genet A ; 173(8): 2081-2087, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28573701

RESUMO

Interstitial 2p15p16.1 microdeletion is a rare chromosomal syndrome previously reported in 33 patients. It is characterized by intellectual disability, developmental delay, autism spectrum disorders, microcephaly, short stature, dysmorphic features, and multiple congenital organ defects. It is defined as a contiguous gene syndrome and two critical regions have been proposed at 2p15 and 2p16.1 loci. Nevertheless, patients with deletion of both critical regions shared similar features of the phenotype and the correlation genotype-phenotype is still unclear. We review all published cases and describe three additional patients, to define the phenotype-genotype correlation more precisely. We reported on two patients including the first prenatal case described so far, carrying a 2p15 deletion affecting two genes: XPO1 and part of USP34. Both patients shared similar features including facial dysmorphism and cerebral abnormalities. We considered the genes involved in the deleted segment to further understand the abnormal phenotype. The third case we described here was a 4-year-old boy with a heterozygous de novo 427 kb deletion encompassing BCL11A and PAPOLG at 2p16.1. He displayed speech delay, autistic traits, and motor stereotypies associated with brain structure abnormalities. We discuss the contribution of the genes included in the deletion to the abnormal phenotype. Our three new patients compared to previous cases, highlighted that despite two critical regions, both distal deletion at 2p16.1 and proximal deletion at 2p15 are associated with phenotypes that are very close to each other. Finally, we also discuss the genetic counseling of this microdeletion syndrome particularly in the course of prenatal diagnosis.


Assuntos
Anormalidades Múltiplas/genética , Deleção Cromossômica , Deficiências do Desenvolvimento/genética , Microcefalia/genética , Anormalidades Múltiplas/diagnóstico por imagem , Anormalidades Múltiplas/fisiopatologia , Encéfalo/diagnóstico por imagem , Encéfalo/fisiopatologia , Proteínas de Transporte/genética , Pré-Escolar , Cromossomos Humanos Par 2/genética , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/fisiopatologia , Feminino , Humanos , Lactente , Carioferinas/genética , Imagem por Ressonância Magnética , Masculino , Microcefalia/diagnóstico por imagem , Microcefalia/fisiopatologia , Proteínas Nucleares/genética , Fenótipo , Receptores Citoplasmáticos e Nucleares/genética , Proteases Específicas de Ubiquitina/genética
8.
PLoS One ; 11(5): e0155009, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27167625

RESUMO

OBJECTIVE: NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. METHOD: After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. RESULTS: The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. CONCLUSION: Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.


Assuntos
Síndrome de Down/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Diagnóstico Pré-Natal/métodos , Cromossomos Humanos Par 21/genética , DNA/sangue , Sondas de DNA/metabolismo , Síndrome de Down/sangue , Síndrome de Down/genética , Humanos , Curva ROC , Reprodutibilidade dos Testes
9.
Am J Med Genet A ; 170(7): 1912-7, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27119754

RESUMO

Chromosomal microarray analysis has become a powerful diagnostic tool in the investigation of patients with intellectual disability leading to the discovery of dosage sensitive genes implicated in the manifestation of various genomic disorders. Interstitial deletions of the short arm of chromosome 10 represent rare genetic abnormalities, especially those encompassing the chromosomal region 10p11-p12. To date, only 10 postnatal cases with microdeletion of this region have been described, and all patients shared a common phenotype, including intellectual disability, abnormal behavior, distinct dysmorphic features, visual impairment, and cardiac malformations. WAC was suggested to be the main candidate gene for intellectual disability associated with 10 p11-p12 deletion syndrome. Here, we describe a new case of de novo 10p11.23-p12.1 microdeletion in a patient with intellectual disability, abnormal behavior, and distinct dysmorphic features. Our observation allows us to redefine the smallest region of overlap among patients reported so far, with a size of 80 Kb and which contains only the WAC gene. These findings strengthen the hypothesis that haploinsufficency of WAC gene might be likely responsible for intellectual disability and behavior disorders. Our data also led us to propose a clinical pathway for patients with this recognizable genetic syndrome depending on the facial dysmorphisms. © 2016 Wiley Periodicals, Inc.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Predisposição Genética para Doença , Deficiência Intelectual/genética , Transtornos Mentais/genética , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 10/genética , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/fisiopatologia , Masculino , Transtornos Mentais/fisiopatologia , Mutação
12.
Eur J Hum Genet ; 23(8): 1010-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25351778

RESUMO

6q16 deletions have been described in patients with a Prader-Willi-like (PWS-like) phenotype. Recent studies have shown that certain rare single-minded 1 (SIM1) loss-of-function variants were associated with a high intra-familial risk for obesity with or without features of PWS-like syndrome. Although SIM1 seems to have a key role in the phenotype of patients carrying 6q16 deletions, some data support a contribution of other genes, such as GRIK2, to explain associated behavioural problems. We describe 15 new patients in whom de novo 6q16 deletions were characterised by comparative genomic hybridisation or single-nucleotide polymorphism (SNP) array analysis, including the first patient with fetopathological data. This fetus showed dysmorphic facial features, cerebellar and cerebral migration defects with neuronal heterotopias, and fusion of brain nuclei. The size of the deletion in the 14 living patients ranged from 1.73 to 7.84 Mb, and the fetus had the largest deletion (14 Mb). Genotype-phenotype correlations confirmed the major role for SIM1 haploinsufficiency in obesity and the PWS-like phenotype. Nevertheless, only 8 of 13 patients with SIM1 deletion exhibited obesity, in agreement with incomplete penetrance of SIM1 haploinsufficiency. This study in the largest series reported to date confirms that the PWS-like phenotype is strongly linked to 6q16.2q16.3 deletions and varies considerably in its clinical expression. The possible involvement of other genes in the 6q16.2q16.3-deletion phenotype is discussed.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Obesidade/genética , Penetrância , Síndrome de Prader-Willi/genética , Proteínas Repressoras/genética , Feto Abortado , Adolescente , Adulto , Criança , Pré-Escolar , Cromossomos Humanos Par 6/genética , Hibridização Genômica Comparativa , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Obesidade/complicações , Obesidade/patologia , Polimorfismo de Nucleotídeo Único , Síndrome de Prader-Willi/complicações , Síndrome de Prader-Willi/patologia , Gravidez
13.
Mol Cytogenet ; 7(1): 59, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25320640

RESUMO

BACKGROUND: Roberts syndrome (RBS) is a rare autosomal recessive disorder mainly characterized by growth retardation, limb defects and craniofacial anomalies. Characteristic cytogenetic findings are "railroad track" appearance of chromatids and premature centromere separation in metaphase spreads. Mutations in the ESCO2 (establishment of cohesion 1 homolog 2) gene located in 8p21.1 have been found in several families. ESCO2, a member of the cohesion establishing complex, has a role in the effective cohesion between sister chromatids. In order to analyze sister chromatids topography during interphase, we performed 3D-FISH using pericentromeric heterochromatin probes of chromosomes 1, 4, 9 and 16, on preserved nuclei from a fetus with RBS carrying compound heterozygous null mutations in the ESCO2 gene. RESULTS: Along with the first observation of an abnormal separation between sister chromatids in heterochromatic regions, we observed a statistically significant change in the intranuclear localization of pericentromeric heterochromatin of chromosome 1 in cells of the fetus compared to normal cells, demonstrating for the first time a modification in the spatial arrangement of chromosome domains during interphase. CONCLUSION: We hypothesize that the disorganization of nuclear architecture may result in multiple gene deregulations, either through disruption of DNA cis interaction -such as modification of chromatin loop formation and gene insulation - mediated by cohesin complex, or by relocation of chromosome territories. These changes may modify interactions between the chromatin and the proteins associated with the inner nuclear membrane or the pore complexes. This model offers a link between the molecular defect in cohesion and the complex phenotypic anomalies observed in RBS.

14.
Am J Clin Pathol ; 142(2): 248-53, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25015868

RESUMO

OBJECTIVES: We report here the unusual association of Silver-Russell syndrome (SRS) and cerebellar dysplasia with trisomy 7 mosaicism and maternal uniparental disomy of chromosome 7 [UPD(7)m]. METHODS: Low-level trisomy 7 mosaicism was diagnosed prenatally on amniocytes, and UPD(7)m was confirmed after birth. RESULTS: Medical examination at birth showed dysmorphic facial features of SRS. Cytogenetic analysis on several tissues and cells confirmed mosaic trisomy 7. Unusual severe psychomotor retardation, hypotonia, and choreoathetoid movement were noted at 6 months. Brain magnetic resonance imaging showed both cerebellar hypoplasia and dysplasia. CONCLUSIONS: This unusual association of SRS and dysplasia of the cerebellum might be related to the presence of the trisomy 7 mosaicism on the cerebellum. Our observation strengthens the hypothesis that the phenotype observed in patients with SRS with UPD(7)m might also result from an undetected low level of trisomy 7 mosaicism that could best be revealed by performing cytogenetic investigations.


Assuntos
Encéfalo/patologia , Síndrome de Silver-Russell/genética , Trissomia/genética , Dissomia Uniparental/genética , Adulto , Cerebelo/anormalidades , Cromossomos Humanos Par 7/genética , Análise Citogenética , Deficiências do Desenvolvimento/diagnóstico , Feminino , Humanos , Mosaicismo , Malformações do Sistema Nervoso/diagnóstico , Síndrome de Silver-Russell/complicações , Síndrome de Silver-Russell/diagnóstico
15.
Am J Med Genet A ; 164A(8): 2025-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24715584

RESUMO

Mutations of the CDKL5 gene cause early epileptic encephalopathy. Patients manifest refractory epilepsy, beginning before the age of 3 months, which is associated with severe psychomotor delay and features that overlap with Rett syndrome. We report here a patient with mosaicism for CDKL5 exonic deletion, with the presence of two mutant alleles. The affected 4-year-old girl presented with infantile spasms, beginning at the age of 9 months, but subsequent progression of the disease was consistent with the classical CDKL5-related phenotype. A deletion of exons 17 and 18 was suspected on the basis of Multiplex Ligation Probe Amplification analysis, but unexpected results for cDNA analysis, which showed the presence of an abnormal transcript with the deletion of exon 18 only, led us to suspect that two distinct events might have occurred. We used custom array-CGH to determine the size and breakpoints of these deletions. Exon 18 was deleted from one of the abnormal alleles, and exon 17 was deleted from the other. A Fork Stalling and Template Switching (FoSTeS) mechanism was proposed to explain the two events, given the presence of regions of microhomology at the breakpoints. We propose here an original involvement of the FoSTeS mechanism to explain the co-occurrence of these two events in the CDKL5 gene in a single patient. This patient highlights the difficulties involved in the detection of such abnormalities, particularly when they occur in a mosaic state and involve two distinct mutational events in a single gene.


Assuntos
Alelos , Deleção de Genes , Mosaicismo , Mutação , Proteínas Serina-Treonina Quinases/genética , Pré-Escolar , Pontos de Quebra do Cromossomo , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Éxons , Feminino , Duplicação Gênica , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Síndrome de Rett/diagnóstico , Síndrome de Rett/genética , Espasmos Infantis/diagnóstico , Espasmos Infantis/genética , Inativação do Cromossomo X
16.
Am J Med Genet A ; 158A(3): 617-21, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22302515

RESUMO

We report on a fetus with an isolated short femur detected by ultrasound and a de novo interstitial deletion of chromosome 15. The deletion was diagnosed prenatally by karyotype and further mapped by fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array-CGH) to bands 15q15.3 to 15q21.3 with a size of 11.11 Mb. Fetal autopsy showed characteristic minor anomalies, urinary abnormalities, and delayed bone maturation, but neither craniosynostosis, nor congenital heart defects as observed in previously reported cases. Despite the existence of ultrasound abnormalities, all five cases reported so far were diagnosed after birth. This is the first case of an interstitial deletion involving chromosomal band 15q15.3-q21.3 diagnosed prenatally and characterized at the molecular level. Our observation suggests the absence of imprinted genes in the area of 15q15-q22 and strengthens the hypothesis that a critical region for craniosynostosis may be mapped outside the deleted region in the present patient.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 15 , Fêmur/anormalidades , Diagnóstico Pré-Natal , Adulto , Hibridização Genômica Comparativa , Evolução Fatal , Feminino , Fêmur/embriologia , Humanos , Hibridização in Situ Fluorescente , Gravidez
17.
Psychiatry Res ; 197(3): 356-7, 2012 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-22365273

RESUMO

22q11.2 deletion syndrome, the most common microdeletion syndrome, exhibits a broad range of phenotypes, implying a cumbersome diagnosis due to atypical or paucisymptomatic presentations. We present two atypical cases of 22q11.2 deletion syndrome and suggest a preferential occurrence of the breakpoints in regions poor in repetitive elements of SINE/Alu family.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Hibridização Genômica Comparativa/métodos , Síndrome de DiGeorge/genética , Genoma Humano/genética , Adolescente , Adulto , Variações do Número de Cópias de DNA/genética , Síndrome de DiGeorge/diagnóstico , Feminino , Humanos , Masculino , Fenótipo , Sequências Repetitivas de Ácido Nucleico/genética
18.
Proc Natl Acad Sci U S A ; 108(47): 18995-9000, 2011 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-22065791

RESUMO

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10-secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti-IFN-α Ab immunotherapy in lupus patients.


Assuntos
Diferenciação Celular/imunologia , Ativação do Complemento/imunologia , Imunoterapia/métodos , Interferon-alfa/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Proteína Cofatora de Membrana/metabolismo , Linfócitos T Reguladores/imunologia , Anticorpos Monoclonais , Complemento C3b/imunologia , Primers do DNA/genética , Citometria de Fluxo , Humanos , Interferon-alfa/imunologia , Interleucina-10/imunologia , Leucócitos Mononucleares , Modelos Lineares , Proteína Cofatora de Membrana/imunologia , Reação em Cadeia da Polimerase em Tempo Real
20.
Epigenetics ; 6(8): 1035-46, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21775817

RESUMO

Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.


Assuntos
Neoplasias da Mama/patologia , Ilhas de CpG/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Proteína Relacionada ao Hormônio Paratireóideo/genética , Regiões Promotoras Genéticas/genética , Região 5'-Flanqueadora/genética , Azacitidina/farmacologia , Células Cultivadas , Ilhas de CpG/efeitos dos fármacos , Epigênese Genética , Feminino , Histonas/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Invasividade Neoplásica , Regiões Promotoras Genéticas/efeitos dos fármacos
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