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1.
Genes (Basel) ; 12(1)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445578

RESUMO

Gilles de la Tourette syndrome (GTS) is a complex neurodevelopmental disorder characterized by motor and vocal tics. Most of the GTS individuals have comorbid diagnoses, of which obsessive-compulsive disorder (OCD) and attention deficit-hyperactivity disorder (ADHD) are the most common. Several neurotransmitter systems have been implicated in disease pathogenesis, and amongst these, the dopaminergic and the serotonergic pathways are the most widely studied. In this study, we aimed to investigate whether the serotonin transporter (SERT) gene (SLC6A4) was differentially expressed among GTS individuals compared to healthy controls, and whether DNA variants (the SERT-linked polymorphic region 5-HTTLPR, together with the associated rs25531 and rs25532 variants, and the rare Ile425Val variant) or promoter methylation of SLC6A4 were associated with gene expression levels or with the presence of OCD as comorbidity. We observed that SLC6A4 expression is upregulated in GTS individuals compared to controls. Although no specific genotype, allele or haplotype was overrepresented in GTS individuals compared to controls, we observed that the LAC/LAC genotype of the 5-HTTLPR/rs25531/rs25532 three-locus haplotype was associated with higher SLC6A4 mRNA expression levels in GTS individuals, but not in the control group.

2.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
3.
Front Neurol ; 11: 163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32265818

RESUMO

Background: Gilles de la Tourette syndrome (GTS) is a neurodevelopmental condition characterized by motor and vocal tics. The underlying etiology remains largely unknown, and GTS is considered as a complex multifactorial disorder associated with effects of several genes in combination with environmental factors. The inner mitochondrial membrane peptidase, subunit 2 (IMMP2L) has been suggested as one of the susceptibility genes for GTS, and IMMP2L-deficient mouse and human cells show increased levels of mitochondrial oxidative stress and altered cell fate programming. Hence, a potential involvement of IMMP2L-induced mitochondrial dysfunction in GTS pathology is yet to be elucidated. To address this, we investigated mitochondrial function in a group of GTS patients with intragenic IMMP2L deletions and compared with GTS without IMMP2L deletions and healthy controls. Methods: Mitochondrial function in fibroblasts from GTS patients and non-GTS parents (with and without IMMP2L deletions) compared to healthy controls were evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, mitochondrial mass, and mitochondrial respiration. In addition, we evaluated apoptosis and senescence. Results: None of the mitochondrial parameters assessed in this study were significantly distinctive when comparing GTS patients with and without IMMP2L deletions against healthy controls or parents with or without IMMP2L deletions, and we did not observe altered cell programming. Conclusion: This study suggests that IMMP2L deletions do not lead to a substantial general mitochondrial dysfunction in GTS fibroblasts. Assessing a large cohort of controls and patients of similar age and gender would possibly reveal small differences in mitochondrial function. However, it is possible that IMMP2L variants affect mitochondrial function during specific instances of stress stimuli or in brain regions suggested to be affected in GTS.

4.
Proc Natl Acad Sci U S A ; 115(51): 13003-13008, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30509972

RESUMO

The instability of chromosome fragile sites is implicated as a causative factor in several human diseases, including cancer [for common fragile sites (CFSs)] and neurological disorders [for rare fragile sites (RFSs)]. Previous studies have indicated that problems arising during DNA replication are the underlying source of this instability. Although the role of replication stress in promoting instability at CFSs is well documented, much less is known about how the fragility of RFSs arises. Many RFSs, as exemplified by expansion of a CGG trinucleotide repeat sequence in the fragile X syndrome-associated FRAXA locus, exhibit fragility in response to folate deficiency or other forms of "folate stress." We hypothesized that such folate stress, through disturbing the replication program within the pathologically expanded repeats within FRAXA, would lead to mitotic abnormalities that exacerbate locus instability. Here, we show that folate stress leads to a dramatic increase in missegregation of FRAXA coupled with the formation of single-stranded DNA bridges in anaphase and micronuclei that contain the FRAXA locus. Moreover, chromosome X aneuploidy is seen when these cells are exposed to folate deficiency for an extended period. We propose that problematic FRAXA replication during interphase leads to a failure to disjoin the sister chromatids during anaphase. This generates further instability not only at FRAXA itself but also of chromosome X. These data have wider implications for the effects of folate deficiency on chromosome instability in human cells.


Assuntos
Sítios Frágeis do Cromossomo , Cromossomos Humanos X , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Linfócitos/patologia , Mitose , Estresse Fisiológico , Células Cultivadas , Replicação do DNA , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Linfócitos/metabolismo , Masculino , Expansão das Repetições de Trinucleotídeos
5.
Methods Mol Biol ; 1672: 471-482, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29043642

RESUMO

A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all individuals) or rare (RFSs; present in only a few individuals). Interest in fragile sites has remained high since their discovery in 1965, because of their association with human disease. CFSs are recognized as drivers of oncogene activation and genome instability in cancer cells, while some RFSs are associated with neurodegenerative diseases. This review summaries our current understanding of the nature and causes of fragile site "expression", including the recently characterized phenomenon of telomere fragility. In particular, we focus on a description of the methodologies and technologies for detection and analysis of chromosome fragile sites.


Assuntos
Sítios Frágeis do Cromossomo , Instabilidade Genômica , Hibridização in Situ Fluorescente , Animais , Replicação do DNA , Expressão Gênica , Loci Gênicos , Humanos , Hibridização in Situ Fluorescente/métodos
6.
Nature ; 528(7581): 286-90, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26633632

RESUMO

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.


Assuntos
Carcinogênese/genética , Reparo do DNA/fisiologia , Replicação do DNA , Endodesoxirribonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Mitose/genética , Estresse Fisiológico/genética , Linhagem Celular Tumoral , Instabilidade Cromossômica , Sítios Frágeis do Cromossomo , Segregação de Cromossomos , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endonucleases/metabolismo , Células HCT116 , Células HT29 , Células HeLa , Humanos , Modelos Biológicos , Não Disjunção Genética/genética
7.
Nat Commun ; 6: 8962, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26643143

RESUMO

PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH(-/-) cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro. Consistent with this, a human PICH(-/-) cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas Aviárias/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Segregação de Cromossomos/genética , DNA Helicases/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitose/genética , Animais , Proteínas Aviárias/metabolismo , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Galinhas , Instabilidade Cromossômica/genética , DNA Helicases/metabolismo , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Humanos , Linfócitos/metabolismo
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