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2.
Neuro Oncol ; 2019 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-30722027

RESUMO

Background: Neurofibromatosis type 1 (NF1) is a tumor-predisposition disorder caused by germline mutations in NF1. NF1 patients have an 8-16% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST), a highly-aggressive soft-tissue sarcoma, often arising from pre-existing benign plexiform neurofibromas (PN) and atypical neurofibromas (ANF). ANF are distinct from both PN and MPNST, representing an intermediate step in malignant transformation. Methods: In the first comprehensive genomic analysis of ANF originating from multiple patients, we performed tumor/normal whole-exome sequencing (WES) of 16 ANFs. In addition, we conducted WES of three MPNSTs, copy-number meta-analysis of 26 ANFs and 28 MPNSTs, and whole transcriptome sequencing analysis of five ANFs and five MPNSTs. Results: We identified a low number of mutations (median 1, range 0-5) in the exomes of ANFs (only NF1 somatic mutations were recurrent), and frequent deletions of CDKN2A/B (69%) and SMARCA2 (42%). We determined that polycomb repressor complex 2 (PRC2) genes EED or SUZ12 were frequently mutated, deleted or downregulated in MPNSTs but not in ANFs. Our pilot gene expression study revealed upregulated NRAS, MDM2, CCND1/2/3 and CDK4/6 in ANFs and MPNSTs, and overexpression of EZH2 in MPNSTs only. Conclusions: The PN-ANF transition is primarily driven by the deletion of CDKN2A/B. Further progression from ANF to MPNST likely involves broad chromosomal rearrangements and frequent inactivation of the PRC2 genes, loss of the DNA repair genes, and copy-number increase of signal transduction, cell cycle and pluripotency self-renewal genes.

3.
Genome Med ; 10(1): 99, 2018 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-30583724

RESUMO

BACKGROUND: Prior research has established that the prevalence of pathogenic/likely pathogenic (P/LP) variants across all of the American College of Medical Genetics (ACMG) Secondary Findings (SF) genes is approximately 0.8-5%. We investigated the prevalence of P/LP variants in the 24 ACMG SF v2.0 cancer genes in a family-based cancer research cohort (n = 1173) and in cancer-free ethnicity-matched controls (n = 982). METHODS: We used InterVar to classify variants and subsequently conducted a manual review to further examine variants of unknown significance (VUS). RESULTS: In the 24 genes on the ACMG SF v2.0 list associated with a cancer phenotype, we observed 8 P/LP unique variants (8 individuals; 0.8%) in controls and 11 P/LP unique variants (14 individuals; 1.2%) in cases, a non-significant difference. We reviewed 115 VUS. The median estimated per-variant review time required was 30 min; the first variant within a gene took significantly (p = 0.0009) longer to review (median = 60 min) compared with subsequent variants (median = 30 min). The concordance rate was 83.3% for the variants examined by two reviewers. CONCLUSION: The 115 VUS required database and literature review, a time- and labor-intensive process hampered by the difficulty in interpreting conflicting P/LP determinations. By rigorously investigating the 24 ACMG SF v2.0 cancer genes, our work establishes a benchmark P/LP variant prevalence rate in a familial cancer cohort and controls.


Assuntos
Genes Neoplásicos/genética , Predisposição Genética para Doença , Mutação , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Idoso , Estudos de Coortes , Análise Mutacional de DNA , Grupos Étnicos , Feminino , Humanos , Masculino
4.
BMC Cancer ; 17(1): 127, 2017 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-28193203

RESUMO

BACKGROUND: Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined. CASE PRESENTATION: Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivation of NF2, confirming the central role of the gene in meningioma formation. The genome of the benign tumor closely resembled that of a normal diploid cell and had only one other deleterious mutation (EPHB3). In contrast, the chromosomal architecture of the grade II tumor was highly re-arranged, yet uniform among all analyzed fragments, implying that this large and fast growing tumor was composed of relatively few clones. Besides multiple gains and losses, the grade II meningioma harbored numerous chromosomal translocations. WES analysis of the atypical tumor identified deleterious mutations in two genes: ADAMTSL3 and CAPN5 in all fragments, indicating that the mutations were present in the cell undergoing fast clonal expansion CONCLUSIONS: This is the first WES study of NF2-associated meningiomas. Besides second NF2 copy inactivation, we found low somatic burden in both tumors and high level of genomic instability in the atypical meningioma. Genomic instability resulting in altered gene dosage and compromised structural integrity of multiple genes may be the primary reason of the high growth rate for the grade II tumor. Further study of ADAMTSL3 and CAPN5 may lead to elucidation of their molecular implications in meningioma pathogenesis.


Assuntos
Neoplasias dos Nervos Cranianos/genética , Genes da Neurofibromatose 2 , Genômica/métodos , Neoplasias Meníngeas/genética , Meningioma/genética , Mutação/genética , Adulto , Neoplasias dos Nervos Cranianos/patologia , Neoplasias dos Nervos Cranianos/cirurgia , Feminino , Genótipo , Humanos , Neoplasias Meníngeas/patologia , Neoplasias Meníngeas/cirurgia , Meningioma/patologia , Meningioma/cirurgia , Gradação de Tumores , Prognóstico
5.
Haematologica ; 101(7): 846-52, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26721895

RESUMO

Familial acute myeloid leukemia is rare and linked to germline mutations in RUNX1, GATA2 or CCAAT/enhancer binding protein-α (CEBPA). We re-evaluated a large family with acute myeloid leukemia originally seen at NIH in 1969. We used whole exome sequencing to study this family, and conducted in silico bioinformatics analysis, protein structural modeling and laboratory experiments to assess the impact of the identified CEBPA Q311P mutation. Unlike most previously identified germline mutations in CEBPA, which were N-terminal frameshift mutations, we identified a novel Q311P variant that was located in the C-terminal bZip domain of C/EBPα. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mu-tant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation, consistent with a loss-of-function mutation. From 45 years of follow up, we observed incomplete penetrance (46%) of CEBPA Q311P. This study of a large multi-generational pedigree reveals that a germline mutation in the C-terminal bZip domain can alter the ability of C/EBP-α to bind DNA and reduces transactivation, leading to acute myeloid leukemia.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/genética , Exoma , Mutação em Linhagem Germinativa , Leucemia Mieloide Aguda/genética , Domínios e Motivos de Interação entre Proteínas , Adolescente , Adulto , Alelos , Proteína alfa Estimuladora de Ligação a CCAAT/química , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Criança , Pré-Escolar , Família , Feminino , Seguimentos , Regulação Leucêmica da Expressão Gênica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Conformação Proteica , Multimerização Proteica , Adulto Jovem
6.
Hum Genet ; 134(7): 775-87, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25939664

RESUMO

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.


Assuntos
Mutação em Linhagem Germinativa , Leucemia Mielomonocítica Juvenil/genética , Mutação de Sentido Incorreto , Linhagem , Proteínas Proto-Oncogênicas c-cbl/genética , Ubiquitinação/genética , Adolescente , Adulto , Criança , Pré-Escolar , Exoma , Feminino , Seguimentos , Humanos , Lactente , Masculino , Modelos Moleculares , Penetrância , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-cbl/química
7.
PLoS Genet ; 10(10): e1004575, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25329635

RESUMO

Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Neurofibromatose 1/genética , Polimorfismo de Nucleotídeo Único , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Estudos de Coortes , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Variação Genética , Humanos , Masculino , Complexo Mediador/genética , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Neurofibromatose 1/patologia , Proteínas Nucleares/genética , Fenótipo , Proteínas/genética , ATPases Vacuolares Próton-Translocadoras/genética
8.
PLoS One ; 9(6): e98686, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24892279

RESUMO

Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings' history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ∼ 3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463-65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of ≤ 1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.


Assuntos
Eczema/diagnóstico , Eczema/genética , Transtornos do Crescimento/diagnóstico , Transtornos do Crescimento/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Microcefalia/diagnóstico , Microcefalia/genética , Adulto , Disceratose Congênita/diagnóstico , Disceratose Congênita/genética , Facies , Feminino , Mutação da Fase de Leitura/genética , Heterogeneidade Genética , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Telômero/genética
9.
Genes Chromosomes Cancer ; 51(5): 429-37, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22250039

RESUMO

Neurofibromatosis type 1 (NF1) is a common, autosomal dominant, tumor-predisposition syndrome that arises secondary to mutations in NF1. Glomus tumors are painful benign tumors that originate from the glomus body in the fingers and toes due to biallelic inactivation of NF1. We karyotyped cultures from four previously reported and one new glomus tumor and hybridized tumor (and matching germline) DNA on Illumina HumanOmni1-Quad SNP arrays (≈ 1 × 10(6) SNPs). Two tumors displayed evidence of copy-neutral loss of heterozygosity of chromosome arm 17q not observed in the germline sample, consistent with a mitotic recombination event. One of these two tumors, NF1-G12, featured extreme polyploidy (near-tetraploidy, near-hexaploidy, or near-septaploidy) across all chromosomes. In the remaining four tumors, there were few cytogenetic abnormalities observed, and copy-number analysis was consistent with diploidy in all chromosomes. This is the first study of glomus tumors cytogenetics, to our knowledge, and the first to report biallelic inactivation of NF1 secondary to mitotic recombination of chromosome arm 17q in multiple NF1-associated glomus tumors. We have observed mitotic recombination in 22% of molecularly characterized NF1-associated glomus tumors, suggesting that it is a not uncommon mechanism in the reduction to homozygosity of the NF1 germline mutation in these tumors. In tumor NF1-G12, we hypothesize that mitotic recombination also "unmasked" (reduced to homozygosity) a hypomorphic germline allele in a gene on chromosome arm 17q associated with chromosomal instability, resulting in the extreme polyploidy.


Assuntos
Cromossomos Humanos Par 17 , Genes da Neurofibromatose 1 , Tumor Glômico/genética , Perda de Heterozigosidade , Neurofibromatose 1/complicações , Recombinação Genética , Adulto , Células Cultivadas , Análise por Conglomerados , Variações do Número de Cópias de DNA , Tumor Glômico/complicações , Humanos , Cariotipagem , Masculino , Mitose , Neurofibromatose 1/genética , Poliploidia
10.
BMC Genomics ; 11: 194, 2010 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-20307317

RESUMO

BACKGROUND: Neurofibromatosis type 1 (NF1) is a common monogenic tumor-predisposition disorder that arises secondary to mutations in the tumor suppressor gene NF1. Haploinsufficiency of NF1 fosters a permissive tumorigenic environment through changes in signalling between cells, however the intracellular mechanisms for this tumor-promoting effect are less clear. Most primary human NF1+/- cells are a challenge to obtain, however lymphoblastoid cell lines (LCLs) have been collected from large NF1 kindreds. We hypothesized that the genetic effects of NF1-haploinsufficiency may be discerned by comparison of genome-wide transcriptional profiling in somatic, non-tumor cells (LCLs) from NF1-affected and -unaffected individuals. As a cross-species filter for heterogeneity, we compared the results from two human kindreds to whole-genome transcriptional profiling in spleen-derived B lymphocytes from age- and gender-matched Nf1+/- and wild-type mice, and used gene set enrichment analysis (GSEA), Onto-Express, Pathway-Express and MetaCore tools to identify genes perturbed in NF1-haploinsufficiency. RESULTS: We observed moderate expression of NF1 in human LCLs and of Nf1 in CD19+ mouse B lymphocytes. Using the t test to evaluate individual transcripts, we observed modest expression differences in the transcriptome in NF1-haploinsufficient LCLs and Nf1-haploinsuffiicient mouse B lymphocytes. However, GSEA, Onto-Express, Pathway-Express and MetaCore analyses identified genes that control cell cycle, DNA replication and repair, transcription and translation, and immune response as the most perturbed in NF1-haploinsufficient conditions in both human and mouse. CONCLUSIONS: Haploinsufficiency arises when loss of one allele of a gene is sufficient to give rise to disease. Haploinsufficiency has traditionally been viewed as a passive state. Our observations of perturbed, up-regulated cell cycle and DNA repair pathways may functionally contribute to NF1-haploinsufficiency as an "active state" that ultimately promotes the loss of the wild-type allele.


Assuntos
Ciclo Celular , Reparo do DNA , DNA/genética , Haplótipos , Neurofibromatose 1/genética , Adolescente , Adulto , Idoso , Alelos , Animais , Linhagem Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Neurofibromatose 1/imunologia , Neurofibromina 1/genética , Análise de Sequência com Séries de Oligonucleotídeos
11.
Cancer Res ; 69(18): 7393-401, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19738042

RESUMO

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1(-/-) glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3'-end of CRTAC1 and a deletion of the 5'-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1.


Assuntos
Tumor Glômico/genética , Neurofibromatose 1/genética , Actinas/biossíntese , Adolescente , Adulto , Criança , Hibridização Genômica Comparativa , Feminino , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Dosagem de Genes , Inativação Gênica , Genes da Neurofibromatose 1 , Tumor Glômico/metabolismo , Tumor Glômico/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , Neurofibromatose 1/metabolismo , Neurofibromatose 1/patologia , Reação em Cadeia da Polimerase , Receptores Androgênicos/metabolismo , Pele/citologia , Células Tumorais Cultivadas , Adulto Jovem , Proteínas ras/metabolismo
12.
Invest Ophthalmol Vis Sci ; 50(11): 5035-43, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19516012

RESUMO

PURPOSE: The presence of two or more Lisch nodules (melanocytic hamartomas of the iris) is one of seven diagnostic criteria for neurofibromatosis type 1 (NF1), a common monogenic disorder of dysregulated neurocutaneous growth. The hypothesis that Lisch nodules arise secondary to exposure to ultraviolet (UV) radiation from sunlight was investigated. METHODS: Lisch nodule burden was mapped and quantified in the irides of 77 adults with NF1. Lifetime sunlight (UV radiation) exposure was inventoried, NF1 neurocutaneous severity determined, and two NF1 mutations predictive of severity selectively genotyped. RESULTS: There was high interindividual variability in Lisch nodule burden. Lisch nodules were primarily located in the inferior hemifield (half) of the iris, regardless of its color (P = 3.0 x 10(-20)). Light irides harbored significantly more Lisch nodules than dark irides (P = 4.8 x 10(-5)). There was no statistically significant correlation of Lisch nodule burden to lifetime sunlight exposure "dose" or NF1 neurocutaneous severity. CONCLUSIONS: The difference in Lisch nodule burden between the superior and inferior iris hemifields is most likely due to the sunlight-shielding effects on the superior half by periocular structures. The difference in Lisch nodule burden between light and dark irides is probably due to the photoprotective effects of pigmentation. The genes underlying the control of iris color may thus be viewed as modifiers of severity of Lisch nodule burden in NF1. Given the role of UV radiation and, presumably, DNA damage in Lisch nodule pathogenesis, "benign tumor of the iris," not "hamartoma," may be a better descriptor.


Assuntos
Hamartoma/etiologia , Neoplasias da Íris/etiologia , Iris/efeitos da radiação , Neoplasias Induzidas por Radiação/etiologia , Neurofibromatose 1/etiologia , Neoplasias Cutâneas/etiologia , Adolescente , Adulto , Idoso , Cor de Olho , Feminino , Genes da Neurofibromatose 1/fisiologia , Genótipo , Hamartoma/genética , Hamartoma/patologia , Humanos , Iris/patologia , Neoplasias da Íris/genética , Neoplasias da Íris/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Induzidas por Radiação/genética , Neoplasias Induzidas por Radiação/patologia , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Polimorfismo de Nucleotídeo Único , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Luz Solar , Inquéritos e Questionários , Carga Tumoral , Raios Ultravioleta/efeitos adversos , Adulto Jovem
13.
J Biol Chem ; 277(4): 2702-8, 2002 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-11723123

RESUMO

Mini-chromosome maintenance (MCM) proteins were originally identified in yeast, and homologues have been identified in several other eukaryotic organisms, including mammals. These findings suggest that the mechanisms by which eukaryotic cells initiate and regulate DNA replication have been conserved throughout evolution. However, it is clear that many mammalian origins are much more complex than those of yeast. An example is the Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the spacer between the DHFR and 2BE2121 genes. This origin consists of a broad zone of potential sites scattered throughout the 55-kb spacer, with several subregions (e.g. ori-beta, ori-beta', and ori-gamma) being preferred. We show here that antibodies to human MCMs 2-7 recognize counterparts in extracts prepared from hamster cells; furthermore, co-immunoprecipitation data demonstrate the presence of an MCM2-3-5 subcomplex as observed in other species. To determine whether MCM proteins play a role in initiation and/or elongation in Chinese hamster cells, we have examined in vivo protein-DNA interactions between the MCMs and chromatin in the DHFR locus using a chromatin immunoprecipitation (ChIP) approach. In synchronized cultures, MCM complexes associate preferentially with DNA in the intergenic initiation zone early in S-phase during the time that replication initiates. However, significant amounts of MCMs were also detected over the two genes, in agreement with recent observations that the MCM complex co-purifies with RNA polymerase II. As cells progress through S-phase, the MCMs redistribute throughout the DHFR domain, suggesting a dynamic interaction with DNA. In asynchronous cultures, in which replication forks should be found at any position in the genome, MCM proteins were distributed relatively evenly throughout the DHFR locus. Altogether, these data are consistent with studies in yeast showing that MCM subunits localize to origins during initiation and then migrate outward with the replication forks. This constitutes the first evidence that mammalian MCM complexes perform a critical role during the initiation and elongation phases of replication at the DHFR origin in hamster cells.


Assuntos
Proteína 1 de Manutenção de Minicromossomo/fisiologia , Origem de Replicação , Tetra-Hidrofolato Desidrogenase/genética , Animais , Western Blotting , Células CHO , Linhagem Celular , Movimento Celular , Centrifugação com Gradiente de Concentração , Césio/farmacologia , Cloretos/farmacologia , Cricetinae , Fixadores/farmacologia , Formaldeído/farmacologia , Fase G1 , Humanos , Proteína 1 de Manutenção de Minicromossomo/química , Modelos Químicos , Modelos Genéticos , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Fase S , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/química , Fatores de Tempo
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