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1.
Am J Hum Genet ; 108(9): 1611-1630, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34343493

RESUMO

Genome-wide association studies (GWASs) have identified a melanoma-associated locus on chromosome band 7p21.1 with rs117132860 as the lead SNP and a secondary independent signal marked by rs73069846. rs117132860 is also associated with tanning ability and cutaneous squamous cell carcinoma (cSCC). Because ultraviolet radiation (UVR) is a key environmental exposure for all three traits, we investigated the mechanisms by which this locus contributes to melanoma risk, focusing on cellular response to UVR. Fine-mapping of melanoma GWASs identified four independent sets of candidate causal variants. A GWAS region-focused Capture-C study of primary melanocytes identified physical interactions between two causal sets and the promoter of the aryl hydrocarbon receptor (AHR). Subsequent chromatin state annotation, eQTL, and luciferase assays identified rs117132860 as a functional variant and reinforced AHR as a likely causal gene. Because AHR plays critical roles in cellular response to dioxin and UVR, we explored links between this SNP and AHR expression after both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and ultraviolet B (UVB) exposure. Allele-specific AHR binding to rs117132860-G was enhanced following both, consistent with predicted weakened AHR binding to the risk/poor-tanning rs117132860-A allele, and allele-preferential AHR expression driven from the protective rs117132860-G allele was observed following UVB exposure. Small deletions surrounding rs117132860 introduced via CRISPR abrogates AHR binding, reduces melanocyte cell growth, and prolongs growth arrest following UVB exposure. These data suggest AHR is a melanoma susceptibility gene at the 7p21.1 risk locus and rs117132860 is a functional variant within a UVB-responsive element, leading to allelic AHR expression and altering melanocyte growth phenotypes upon exposure.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 7 , Loci Gênicos , Melanócitos/metabolismo , Melanoma/genética , Receptores de Hidrocarboneto Arílico/genética , Neoplasias Cutâneas/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Cromatina/química , Cromatina/metabolismo , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Melanócitos/efeitos da radiação , Melanoma/metabolismo , Melanoma/patologia , Dibenzodioxinas Policloradas/toxicidade , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Banho de Sol , Raios Ultravioleta/efeitos adversos
2.
Am J Hum Genet ; 108(9): 1590-1610, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34390653

RESUMO

Our study investigated the underlying mechanism for the 14q24 renal cell carcinoma (RCC) susceptibility risk locus identified by a genome-wide association study (GWAS). The sentinel single-nucleotide polymorphism (SNP), rs4903064, at 14q24 confers an allele-specific effect on expression of the double PHD fingers 3 (DPF3) of the BAF SWI/SNF complex as assessed by massively parallel reporter assay, confirmatory luciferase assays, and eQTL analyses. Overexpression of DPF3 in renal cell lines increases growth rates and alters chromatin accessibility and gene expression, leading to inhibition of apoptosis and activation of oncogenic pathways. siRNA interference of multiple DPF3-deregulated genes reduces growth. Our results indicate that germline variation in DPF3, a component of the BAF complex, part of the SWI/SNF complexes, can lead to reduced apoptosis and activation of the STAT3 pathway, both critical in RCC carcinogenesis. In addition, we show that altered DPF3 expression in the 14q24 RCC locus could influence the effectiveness of immunotherapy treatment for RCC by regulating tumor cytokine secretion and immune cell activation.


Assuntos
Carcinoma de Células Renais/genética , Cromossomos Humanos Par 14 , Proteínas de Ligação a DNA/genética , Loci Gênicos , Neoplasias Renais/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética , Carcinogênese/genética , Carcinogênese/imunologia , Carcinogênese/patologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Cromatina/química , Cromatina/imunologia , Montagem e Desmontagem da Cromatina/imunologia , Citocinas/genética , Citocinas/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoterapia/métodos , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Polimorfismo de Nucleotídeo Único , Fator de Transcrição STAT3/imunologia , Linfócitos T Citotóxicos , Fatores de Transcrição/imunologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-34162668

RESUMO

IKZF1 encodes Ikaros, a zinc finger-containing transcription factor crucial to the development of the hematopoietic system. Germline pathogenic variants in IKZF1 have been reported in patients with acute lymphocytic leukemia and immunodeficiency syndromes. Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by erythroid hypoplasia, associated with a spectrum of congenital anomalies and an elevated risk of certain cancers. DBA is usually caused by heterozygous pathogenic variants in genes that function in ribosomal biogenesis; however, in many cases the genetic etiology is unknown. We identified a germline IKZF1 variant, rs757907717 C > T, in identical twins with DBA-like features and autoimmune gastrointestinal disease. rs757907717 C > T results in a p.R381C amino acid change in the IKZF1 Ik-x isoform (p.R423C on isoform Ik-1), which we show is associated with altered global gene expression and perturbation of transcriptional networks involved in hematopoietic system development. These data suggest that this missense substitution caused a DBA-like syndrome in this family because of alterations in hematopoiesis, including dysregulation of networks essential for normal erythropoiesis and the immune system.

4.
J Hum Genet ; 64(6): 545-550, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30850729

RESUMO

Mosaic protein truncating variants (PTVs) in the phosphatase, Mg2+/Mn2+dependent 1D (PPM1D) gene in blood-derived DNA have been associated with increased risk of breast cancer. We analyzed PPM1D PTVs in blood from 3817 breast cancer cases and 3058 controls by deep sequencing of a previously defined region in exon 6 of PPM1D. We identified 50 of 6875 (0.73%) participants having a mosaic PPM1D PTV. We observed a higher frequency of mosaic PPM1D PTVs with increasing age (Ptrend = 2.9 × 10-6). We did not observe an overall association between PPM1D PTVs and increased breast cancer risk (OR = 1.51, 95% CI = 0.84-2.71). Evidence for an association was observed in a subset of cases with DNA collected 1-year or more before breast cancer diagnosis (OR = 3.44, 95% CI = 1.62-7.30, P-value = 0.001); however, no significant association was observed for the larger series of cases with DNA collected post diagnosis (OR = 1.01, 95% CI = 0.51-2.01, P-value = 0.98). Our study indicates that the PPM1D PTVs are present at higher rates than previously reported and the frequency of PPM1D PTVs increases with age. We observed limited evidence for an association between mosaic PPM1D PTVs and breast cancer risk, suggesting mosaic PPM1D PTVs in the blood likely do not influence risk of breast cancer.


Assuntos
Envelhecimento/genética , Neoplasias da Mama/genética , Predisposição Genética para Doença , Proteína Fosfatase 2C/genética , Idoso , Envelhecimento/patologia , Neoplasias da Mama/patologia , Éxons , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Mutação , Fatores de Risco
5.
Hum Mol Genet ; 26(22): 4388-4394, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28973384

RESUMO

Recent studies have reported a higher than anticipated frequency of large clonal autosomal mosaic events >2 Mb in size in the aging population. Mosaic events are detected from analyses of intensity parameters of linear stretches with deviations in heterozygous probes of single nucleotide polymorphism microarrays. The non-random distribution of detected mosaic events throughout the genome suggests common mechanisms could influence the formation of mosaic events. Here we use publicly available data tracks from the University of California Santa Cruz Genome Browser to investigate the genomic characteristics of the regions at the terminal ends of two frequent types of large structural mosaic events: telomeric neutral events and interstitial losses. We observed breakpoints are more likely to occur in regions enriched for open chromatin, increased gene density, elevated meiotic recombination rates and in the proximity of repetitive elements. These observations suggest that detected mosaic event breakpoints are preferentially recovered in genomic regions that are observed to be active and thus more accessible to environmental exposures and events related to gene transcription. We propose that errors in DNA repair pathways, such as non-homologous end joining and homologous recombination, may be important cellular mechanisms that lead to the formation of large structural mosaic events such as interstitial losses and copy neutral events that include telomeres. Further studies using next generation sequencing technologies should be instrumental in mapping the specific junctions of mosaic events to the nucleotide and provide insights into the molecular mechanisms responsible for clonal somatic structural events.


Assuntos
Quebra Cromossômica , Cromossomos Humanos , Cromatina , Quebras de DNA , Variações do Número de Cópias de DNA , Bases de Dados de Ácidos Nucleicos , Genoma Humano , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mosaicismo , Polimorfismo de Nucleotídeo Único , Recombinação Genética
6.
Cancer Res ; 77(13): 3666-3671, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28446466

RESUMO

Cancer treatments composed of immune checkpoint inhibitors and oncogene-targeted drugs might improve cancer management, but there has been little investigation of their combined potential as yet. To estimate the fraction of cancer cases that might benefit from such combination therapy, we conducted an exploratory study of cancer genomic datasets to determine the proportion with somatic mutation profiles amenable to either immunotherapy or targeted therapy. We surveyed 13,349 genomic profiles from public databases for cases with specific mutations targeted by current agents or a burden of exome-wide nonsynonymous mutations (NsM) that exceed a proposed threshold for response to checkpoint inhibitors. Overall, 8.9% of cases displayed profiles that could benefit from combination therapy, which corresponded to approximately 11.2% of U.S. annual incident cancer cases. Frequently targetable mutations were in PIK3CA, BRAF, NF1, NRAS, and PTEN We also noted a high burden of NsM in cases with targetable mutations in SMO, DDR2, FGFR1, PTCH1, FGFR2, and MET Our results indicate that a significant proportion of solid tumor patients are eligible for immuno-targeted combination therapy, and they suggest prioritizing specific cancers for trials of certain targeted and checkpoint inhibitor drugs. Cancer Res; 77(13); 3666-71. ©2017 AACR.


Assuntos
Imunoterapia/métodos , Neoplasias/terapia , Terapia Combinada , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/imunologia
7.
Nat Commun ; 7: 12098, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27384883

RESUMO

Genome-wide association studies have identified multiple renal cell carcinoma (RCC) susceptibility loci. Here, we use regional imputation and bioinformatics analysis of the 12p12.1 locus to identify the single-nucleotide polymorphism (SNP) rs7132434 as a potential functional variant. Luciferase assays demonstrate allele-specific regulatory activity and, together with data from electromobility shift assays, suggest allele-specific differences at rs7132434 for AP-1 transcription factor binding. In an analysis of The Cancer Genome Atlas data, SNPs highly correlated with rs7132434 show allele-specific differences in BHLHE41 expression (trend P value=6.3 × 10(-7)). Cells overexpressing BHLHE41 produce larger mouse xenograft tumours, while RNA-seq analysis reveals that constitutively increased BHLHE41 induces expression of IL-11. We conclude that the RCC risk allele at 12p12.1 maps to rs7132434, a functional variant in an enhancer that upregulates BHLHE41 expression which, in turn, induces IL-11, a member of the IL-6 cytokine family.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma de Células Renais/genética , Cromossomos Humanos Par 12/química , Loci Gênicos , Predisposição Genética para Doença , Interleucina-11/genética , Neoplasias Renais/genética , Alelos , Animais , Atlas como Assunto , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 12/metabolismo , Biologia Computacional , Humanos , Interleucina-11/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Transplante de Neoplasias , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
8.
Cancer Res ; 76(13): 3767-72, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27197178

RESUMO

Immune checkpoint inhibitor treatment represents a promising approach toward treating cancer and has been shown to be effective in a subset of melanoma, non-small cell lung cancer (NSCLC), and kidney cancers. Recent studies have suggested that the number of nonsynonymous mutations (NsM) can be used to select melanoma and NSCLC patients most likely to benefit from checkpoint inhibitor treatment. It is hypothesized that a higher burden of NsM generates novel epitopes and gene products, detected by the immune system as foreign. We conducted an assessment of NsM across 7,757 tumor samples drawn from 26 cancers sequenced in the Cancer Genome Atlas (TCGA) Project to estimate the subset of cancers (both types and fractions thereof) that fit the profile suggested for melanoma and NSCLC. An additional independent set of 613 tumors drawn from 5 cancers were analyzed for replication. An analysis of the receiver operating characteristic curves of published data on checkpoint inhibitor response in melanoma and NSCLC data estimates a cutoff of 192 NsM with 74% sensitivity and 59.3% specificity to discriminate potential clinical benefit. Across the 7,757 samples of TCGA, 16.2% displayed an NsM count that exceeded the threshold of 192. It is notable that more than 30% of bladder, colon, gastric, and endometrial cancers have NsM counts above 192, which was also confirmed in melanoma and NSCLC. Our data could inform the prioritization of tumor types (and subtypes) for possible clinical trials to investigate further indications for effective use of immune checkpoint inhibitors, particularly in adult cancers. Cancer Res; 76(13); 3767-72. ©2016 AACR.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/genética , Mutação/genética , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Biomarcadores Tumorais/genética , Humanos , Estadiamento de Neoplasias , Neoplasias/genética , Neoplasias/imunologia , Prognóstico , Células Tumorais Cultivadas
9.
Am J Hum Genet ; 96(3): 487-97, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25748358

RESUMO

Analyses of genome-wide association study (GWAS) data have revealed that detectable genetic mosaicism involving large (>2 Mb) structural autosomal alterations occurs in a fraction of individuals. We present results for a set of 24,849 genotyped individuals (total GWAS set II [TGSII]) in whom 341 large autosomal abnormalities were observed in 168 (0.68%) individuals. Merging data from the new TGSII set with data from two prior reports (the Gene-Environment Association Studies and the total GWAS set I) generated a large dataset of 127,179 individuals; we then conducted a meta-analysis to investigate the patterns of detectable autosomal mosaicism (n = 1,315 events in 925 [0.73%] individuals). Restricting to events >2 Mb in size, we observed an increase in event frequency as event size decreased. The combined results underscore that the rate of detectable mosaicism increases with age (p value = 5.5 × 10(-31)) and is higher in men (p value = 0.002) but lower in participants of African ancestry (p value = 0.003). In a subset of 47 individuals from whom serial samples were collected up to 6 years apart, complex changes were noted over time and showed an overall increase in the proportion of mosaic cells as age increased. Our large combined sample allowed for a unique ability to characterize detectable genetic mosaicism involving large structural events and strengthens the emerging evidence of non-random erosion of the genome in the aging population.


Assuntos
Aberrações Cromossômicas , Genoma Humano , Mosaicismo , Idoso , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/genética
10.
Breast Cancer (Auckl) ; 8: 135-44, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25368520

RESUMO

Common genetic variants mapping to two distinct regions of RAD51B, a paralog of RAD51, have been associated with breast cancer risk in genome-wide association studies (GWAS). RAD51B is a plausible candidate gene because of its established role in the homologous recombination (HR) process. How germline genetic variation in RAD51B confers susceptibility to breast cancer is not well understood. Here, we investigate the molecular function of RAD51B in breast cancer cell lines by knocking down RAD51B expression by small interfering RNA and treating cells with DNA-damaging agents, namely cisplatin, hydroxyurea, or methyl-methanesulfonate. Our results show that RAD51B-depleted breast cancer cells have increased sensitivity to DNA damage, reduced efficiency of HR, and altered cell cycle checkpoint responses. The influence of RAD51B on the cell cycle checkpoint is independent of its role in HR and further studies are required to determine whether these functions can explain the RAD51B breast cancer susceptibility alleles.

11.
Blood ; 124(1): 24-32, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24829207

RESUMO

Diamond-Blackfan anemia (DBA) is a cancer-prone inherited bone marrow failure syndrome. Approximately half of DBA patients have a germ-line mutation in a ribosomal protein gene. We used whole-exome sequencing to identify disease-causing genes in 2 large DBA families. After filtering, 1 nonsynonymous mutation (p.I31F) in the ribosomal protein S29 (RPS29[AUQ1]) gene was present in all 5 DBA-affected individuals and the obligate carrier, and absent from the unaffected noncarrier parent in 1 DBA family. A second DBA family was found to have a different nonsynonymous mutation (p.I50T) in RPS29. Both mutations are amino acid substitutions in exon 2 predicted to be deleterious and resulted in haploinsufficiency of RPS29 expression compared with wild-type RPS29 expression from an unaffected control. The DBA proband with the p.I31F RPS29 mutation had a pre-ribosomal RNA (rRNA) processing defect compared with the healthy control. We demonstrated that both RPS29 mutations failed to rescue the defective erythropoiesis in the rps29(-/-) mutant zebra fish DBA model. RPS29 is a component of the small 40S ribosomal subunit and essential for rRNA processing and ribosome biogenesis. We uncovered a novel DBA causative gene, RPS29, and showed that germ-line mutations in RPS29 can cause a defective erythropoiesis phenotype using a zebra fish model.


Assuntos
Anemia de Diamond-Blackfan/genética , Mutação , Proteínas Ribossômicas/genética , Idade de Início , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma/genética , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra
12.
Mol Cell ; 46(1): 43-53, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22500736

RESUMO

The BLM helicase has been shown to maintain genome stability by preventing accumulation of aberrant recombination intermediates. We show here that the Saccharomyces cerevisiae BLM ortholog, Sgs1, plays an integral role in normal meiotic recombination, beyond its documented activity limiting aberrant recombination intermediates. In wild-type meiosis, temporally and mechanistically distinct pathways produce crossover and noncrossover recombinants. Crossovers form late in meiosis I prophase, by polo kinase-triggered resolution of Holliday junction (HJ) intermediates. Noncrossovers form earlier, via processes that do not involve stable HJ intermediates. In contrast, sgs1 mutants abolish early noncrossover formation. Instead, both noncrossovers and crossovers form by late HJ intermediate resolution, using an alternate pathway requiring the overlapping activities of Mus81-Mms4, Yen1, and Slx1-Slx4, nucleases with minor roles in wild-type meiosis. We conclude that Sgs1 is a primary regulator of recombination pathway choice during meiosis and suggest a similar function in the mitotic cell cycle.


Assuntos
Troca Genética/fisiologia , DNA Cruciforme/metabolismo , DNA Fúngico/metabolismo , Prófase Meiótica I/fisiologia , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , DNA Cruciforme/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Mutação , RecQ Helicases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Fam Cancer ; 10(3): 545-8, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21614589

RESUMO

The presence of pancreatic cancer (PC) in melanoma-prone families has been consistently associated with an increased frequency of CDKN2A mutations, the major high-risk susceptibility gene identified for melanoma. However, the precise relationship between CDKN2A, melanoma and PC remains unknown. We evaluated a recently identified PC susceptibility gene PALB2 using both sequencing and tagging to determine whether PALB2 might explain part of the relationship between CDKN2A, melanoma, and PC. No disease-related mutations were identified from sequencing PALB2 in multiple pancreatic cancer patients or other mutation carrier relatives of PC patients from the eight melanoma-prone families with CDKN2A mutations and PC. In addition, no significant associations were observed between 11 PALB2 tagging SNPs and melanoma risk in 23 melanoma-prone families with CDKN2A mutations or the subset of 11 families with PC or PC-related CDKN2A mutations. The results suggested that PALB2 does not explain the relationship between CDKN2A, melanoma, and pancreatic cancer in these melanoma-prone families.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Melanoma/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/etiologia , Neoplasias Cutâneas/genética , Proteínas Supressoras de Tumor/genética , Estudos de Casos e Controles , DNA/genética , Família , Proteína do Grupo de Complementação N da Anemia de Fanconi , Feminino , Humanos , Masculino , Melanoma/complicações , Reação em Cadeia da Polimerase , Prognóstico , Neoplasias Cutâneas/complicações
14.
J Med Genet ; 48(4): 285-8, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21209122

RESUMO

BACKGROUND: Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterised by dystrophic nails, abnormal skin pigmentation and oral leukoplakia. Patients are at very high risk of cancer and other medical problems. They have exceedingly short telomeres for their age and approximately 60% have a germline mutation in a gene important in telomere biology (DKC1, TERC, TERT, TINF2, NOP10, or NHP2). The shelterin complex consists of six proteins encoded by TINF2, ACD, POT1, TERF1, TERF2 and TERF2IP, which are essential for telomeric stability. TINF2 mutations are present in 11-25% of patients with DC. METHODS: Bi-directional sequence analysis was conducted of all exons, intron-exon boundaries and the proximal promoter of the other five shelterin genes to determine whether mutations in these genes were associated with DC. Sixteen mutation-negative patients, nine with DC and seven patients with short telomeres and bone marrow failure, were evaluated. RESULTS: Two variants were identified, ACD Ex1+189 G→A and TERF1 Ex9+59 G→A, which were each present in one patient and a healthy parent but absent in 364 controls. Three other variants were rare (<1%) but present in both patients and controls. DISCUSSION: These data suggest that except for TINF2, mutations in shelterin genes are not a common cause of DC.


Assuntos
Disceratose Congênita/genética , Proteínas de Ligação a Telômeros/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Telômero/genética
15.
Genes Dev ; 25(1): 11-6, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21205863

RESUMO

Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.


Assuntos
Disceratose Congênita/enzimologia , Disceratose Congênita/genética , Mutação/genética , Transporte Proteico/fisiologia , Telomerase/metabolismo , Sequência de Aminoácidos , Animais , Disceratose Congênita/fisiopatologia , Humanos , Modelos Moleculares , Chaperonas Moleculares , Linhagem , Transporte Proteico/genética , Alinhamento de Sequência , Telomerase/genética
16.
Methods Mol Biol ; 557: 209-34, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19799185

RESUMO

Joint Molecule (JM) recombination intermediates result from DNA strand-exchange between homologous chromosomes. Physical monitoring of JM formation in budding yeast has provided a wealth of information about the timing and mechanism of meiotic recombination. These assays are especially informative when applied to the analysis of mutants for which genetic analysis of recombination is impossible, i.e. mutants that die during meiosis. This chapter describes three distinct methods to stabilize JMs against thermally driven dissolution as well as electrophoretic approaches to resolve and detect JMs at two well-characterized recombination hotspots.


Assuntos
Instabilidade Cromossômica/fisiologia , Meiose/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Algoritmos , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Ágar/métodos , Modelos Biológicos , Mapeamento por Restrição/métodos , Saccharomyces cerevisiae/química
17.
Mol Cell ; 31(3): 313-23, 2008 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-18691964

RESUMO

Budding yeast lacking the Sgs1 helicase and the Mus81/Mms4 endonuclease are inviable, and indirect studies implicate homologous recombination gone awry as the cause of death. We show that mutants lacking both enzymes have profound defects in meiotic recombination intermediate metabolism and crossover (CO) formation. Recombination intermediates (joint molecules, JMs) accumulate in these cells, many with structures that are infrequent in wild-type cells. These JMs persist, preventing nuclear division. Using an inducible expression system, we restored Mus81 or Sgs1 to sgs1 mus81 cells at a time when JMs are forming. Mus81 expression did not prevent JM formation but did restore JM resolution, CO formation, and nuclear division. In contrast, Sgs1 expression reduced the extent of JM accumulation. These results indicate that Sgs1 and Mus81/Mms4 collaborate to direct meiotic recombination toward interhomolog interactions that promote proper chromosome segregation, and also indicate that Mus81/Mms4 promotes JM resolution in vivo.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Meiose , RecQ Helicases/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Transativadores/metabolismo , Núcleo Celular/enzimologia , DNA Cruciforme , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Mutação/genética , Estágio Paquíteno , Regiões Promotoras Genéticas/genética , RecQ Helicases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética
18.
PLoS Genet ; 2(9): e155, 2006 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-17002499

RESUMO

Sgs1, the budding yeast homolog of the mammalian BLM helicase, has been implicated in preventing excess recombination during both vegetative growth and meiosis. Most meiotic crossover (CO) recombination requires full function of a set of yeast proteins (Zip1, Zip2, Zip3, Zip4/Spo22, Mer3, Msh4, and Msh5, termed the SIC or ZMM proteins) that are also required for homologous chromosome synapsis. We report here genetic and molecular assays showing that sgs1 single mutants display relatively modest increases in CO recombination (less than 1.6-fold relative to wild-type). In contrast, a much greater CO increase is seen when an sgs1 mutation is introduced into the CO- and synapsis-deficient zip1, zip2, zip3, mer3, or msh4 mutants (2- to 8-fold increase). Furthermore, close juxtaposition of the axes of homologous chromosomes is restored. CO restoration in the mutants is not accompanied by significant changes in noncrossover (NCO) recombinant frequencies. These findings show that Sgs1 has potent meiotic anti-CO activity, which is normally antagonized by SIC/ZMM proteins. Our data reinforce previous proposals for an early separation of meiotic processes that form CO and NCO recombinants.


Assuntos
Pareamento Cromossômico/fisiologia , Cromossomos Fúngicos/metabolismo , Troca Genética , DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , DNA Helicases/deficiência , Dados de Sequência Molecular , Mutação/genética , RecQ Helicases , Saccharomyces cerevisiae/genética , Esporos Fúngicos/metabolismo
19.
Genetics ; 169(3): 1353-67, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15654098

RESUMO

To study the mechanism of meiotic recombination in Saccharomyces cerevisiae, we examined recombination in an interval where the majority of events are initiated at a single hotspot for DNA double-strand breaks (DSBs), with little or no expected contribution by outside initiation events. This interval contained infrequently corrected palindromic markers 300 bp to the left and 600 bp to the right of the DSB hotspot. Conversion of single markers occurred frequently, while conversion of both markers occurred rarely, and many of the tetrads in which both markers converted were the products of multiple events. These data indicate that most meiotic recombination intermediates are asymmetrically positioned around the initiating DSB, with a short (<300 bp) tract of heteroduplex DNA (hDNA) to one side and hDNA on the other side frequently extending 600 bp or more. One consequence of this asymmetry is the preferential concentration of crossovers in the vicinity of the initiating DSB.


Assuntos
Troca Genética/genética , Meiose/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Pareamento Incorreto de Bases/genética , Dano ao DNA/genética , DNA Fúngico/genética , Diploide , Eletroforese em Gel de Campo Pulsado , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Marcadores Genéticos , Modelos Genéticos
20.
Nat Cell Biol ; 5(5): 480-5, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12717442

RESUMO

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.


Assuntos
Proteínas de Ciclo Celular , Centrômero/genética , Segregação de Cromossomos/genética , Células Eucarióticas/metabolismo , Meiose/genética , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae , Leveduras/enzimologia , Leveduras/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Cultivadas , Cromossomos/genética , Replicação do DNA/genética , Imunofluorescência , Regulação Fúngica da Expressão Gênica/genética , Cinetocoros/metabolismo , Proteínas Quinases/deficiência , Proteínas Serina-Treonina Quinases , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
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