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1.
Am J Hum Genet ; 107(3): 514-526, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32791035

RESUMO

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.


Assuntos
Anormalidades Múltiplas/genética , Astenozoospermia/genética , Axonema/genética , Infertilidade Masculina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Anormalidades Múltiplas/patologia , Alelos , Animais , Astenozoospermia/fisiopatologia , Axonema/patologia , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/genética , Homozigoto , Humanos , Infertilidade Masculina/patologia , Mutação com Perda de Função/genética , Perda de Heterozigosidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Mitocôndrias/genética , Cauda do Espermatozoide/metabolismo , Cauda do Espermatozoide/patologia , Testículo/metabolismo , Testículo/patologia , Sequenciamento Completo do Exoma
2.
Nature ; 584(7819): 136-141, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32581363

RESUMO

Clonally expanded blood cells that contain somatic mutations (clonal haematopoiesis) are commonly acquired with age and increase the risk of blood cancer1-9. The blood clones identified so far contain diverse large-scale mosaic chromosomal alterations (deletions, duplications and copy-neutral loss of heterozygosity (CN-LOH)) on all chromosomes1,2,5,6,9, but the sources of selective advantage that drive the expansion of most clones remain unknown. Here, to identify genes, mutations and biological processes that give selective advantage to mutant clones, we analysed genotyping data from the blood-derived DNA of 482,789 participants from the UK Biobank10. We identified 19,632 autosomal mosaic chromosomal alterations and analysed these for relationships to inherited genetic variation. We found 52 inherited, rare, large-effect coding or splice variants in 7 genes that were associated with greatly increased vulnerability to clonal haematopoiesis with specific acquired CN-LOH mutations. Acquired mutations systematically replaced the inherited risk alleles (at MPL) or duplicated them to the homologous chromosome (at FH, NBN, MRE11, ATM, SH2B3 and TM2D3). Three of the genes (MRE11, NBN and ATM) encode components of the MRN-ATM pathway, which limits cell division after DNA damage and telomere attrition11-13; another two (MPL and SH2B3) encode proteins that regulate the self-renewal of stem cells14-16. In addition, we found that CN-LOH mutations across the genome tended to cause chromosomal segments with alleles that promote the expansion of haematopoietic cells to replace their homologous (allelic) counterparts, increasing polygenic drive for blood-cell proliferation traits. Readily acquired mutations that replace chromosomal segments with their homologous counterparts seem to interact with pervasive inherited variation to create a challenge for lifelong cytopoiesis.


Assuntos
Evolução Clonal/genética , Células Clonais/metabolismo , Hematopoese/genética , Herança Multifatorial/genética , Adulto , Idoso , Alelos , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/patologia , Divisão Celular/genética , Aberrações Cromossômicas , Células Clonais/citologia , Células Clonais/patologia , Feminino , Predisposição Genética para Doença , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Humanos , Perda de Heterozigosidade/genética , Masculino , Pessoa de Meia-Idade , Mosaicismo , Reino Unido
3.
Cytogenet Genome Res ; 160(6): 309-315, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32599602

RESUMO

Warburg micro syndrome (WARBM) is a rare autosomal recessive disorder characterized by microcephaly, cortical dysplasia, intellectual disability, ocular abnormalities, spastic diplegia, and microgenitalia. WARBM has 4 subtypes arising from pathogenic variants in 4 genes (RAB18, RAB3GAP1, RAB3GAP2, and TBC1D20). Here, we report on a patient with a homozygous pathogenic c.665delC (p.Pro222HisfsTer30) variant in the RAB3GAP1 gene identified by whole-exome sequencing (WES) analyses. Only his father was a heterozygous carrier, and homozygosity mapping analysis of the WES data revealed large loss-of-heterozygosity regions in both arms of chromosome 2, interpreted as uniparental isodisomy. This uniparental disomy pattern could be due to paternal meiosis I nondisjunction because of the preserved heterozygosity in the pericentromeric region. This report provides novel insights, including a rare form of UPD, usage of homozygosity mapping analysis for the evaluation of isodisomy, and the first reported case of WARBM1 as a result of uniparental isodisomy.


Assuntos
Anormalidades Múltiplas/genética , Catarata/congênito , Cromossomos Humanos Par 2/genética , Córnea/anormalidades , Homozigoto , Hipogonadismo/genética , Deficiência Intelectual/genética , Microcefalia/genética , Atrofia Óptica/genética , Dissomia Uniparental/genética , Sequenciamento Completo do Exoma , Adolescente , Adulto , Catarata/genética , Feminino , Humanos , Lactente , Perda de Heterozigosidade/genética , Masculino , Pais , Polimorfismo de Nucleotídeo Único/genética , Proteínas rab3 de Ligação ao GTP/genética
4.
Nat Commun ; 11(1): 2517, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32433464

RESUMO

Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities.


Assuntos
Genes Essenciais , Perda de Heterozigosidade , Neoplasias/genética , Alelos , Proliferação de Células , DNA Primase/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Humanos , Modelos Genéticos , Neoplasias/fisiopatologia , Proteínas de Ligação a RNA/genética
5.
Nat Commun ; 11(1): 2333, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393735

RESUMO

Missense-type mutant p53 plays a tumor-promoting role through gain-of-function (GOF) mechanism. In addition, the loss of wild-type TP53 through loss of heterozygosity (LOH) is widely found in cancer cells. However, malignant progression induced by cooperation of TP53 GOF mutation and LOH remains poorly understood. Here, we show that mouse intestinal tumors carrying Trp53 GOF mutation with LOH (AKTPM/LOH) are enriched in metastatic lesions when heterozygous Trp53 mutant cells (AKTP+/M) are transplanted. We show that Trp53 LOH is required for dormant cell survival and clonal expansion of cancer cells. Moreover, AKTPM/LOH cells show an increased in vivo tumor-initiating ability compared with AKTPNull and AKTP+/M cells. RNAseq analyses reveal that inflammatory and growth factor/MAPK pathways are specifically activated in AKTPM/LOH cells, while the stem cell signature is upregulated in both AKTPM/LOH and AKTPNull cells. These results indicate that TP53/Trp53 LOH promotes TP53/Trp53 GOF mutation-driven metastasis through the activation of distinct pathway combination.


Assuntos
Carcinogênese/genética , Carcinogênese/patologia , Neoplasias Hepáticas/secundário , Mutação/genética , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Células Clonais , Análise por Conglomerados , Feminino , Mutação com Ganho de Função , Humanos , Inflamação/patologia , Neoplasias Intestinais/patologia , Perda de Heterozigosidade , Sistema de Sinalização das MAP Quinases , Camundongos , Organoides/patologia
6.
PLoS One ; 15(4): e0231809, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32310990

RESUMO

Genetic factors are often overlooked in conservation planning, despite their importance in small isolated populations. We used mitochondrial and microsatellite markers to investigate population genetics of the adder (Vipera berus) in southern Britain, where numbers are declining. We found no evidence for loss of heterozygosity in any of the populations studied. Genetic diversity was comparable across sites, in line with published levels for mainland Europe. However, further analysis revealed a striking level of relatedness. Genetic networks constructed from inferred first degree relationships suggested a high proportion of individuals to be related at a level equivalent to that of half-siblings, with rare inferred full-sib dyads. These patterns of relatedness can be attributed to the high philopatry and low vagility of adders, which creates high local relatedness, in combination with the polyandrous breeding system in the adder, which may offset the risk of inbreeding in closed populations. We suggest that reliance on standard genetic indicators of inbreeding and diversity may underestimate demographic and genetic factors that make adder populations vulnerable to extirpation. We stress the importance of an integrated genetic and demographic approach in the conservation of adders, and other taxa of similar ecology.


Assuntos
Endogamia , Viperidae/genética , Animais , Feminino , Variação Genética , Genética Populacional , Perda de Heterozigosidade , Masculino , Repetições de Microssatélites , Mitocôndrias/genética , Reino Unido , Viperidae/fisiologia
8.
Nat Genet ; 52(3): 283-293, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32139907

RESUMO

Whole-genome doubling (WGD) is a prevalent event in cancer, involving a doubling of the entire chromosome complement. However, despite its prevalence and prognostic relevance, the evolutionary selection pressures for WGD in cancer have not been investigated. Here, we combine evolutionary simulations with an analysis of cancer sequencing data to explore WGD during cancer evolution. Simulations suggest that WGD can be selected to mitigate the irreversible, ratchet-like, accumulation of deleterious somatic alterations, provided that they occur at a sufficiently high rate. Consistent with this, we observe an enrichment for WGD in tumor types with extensive loss of heterozygosity, including lung squamous cell carcinoma and triple-negative breast cancers, and we find evidence for negative selection against homozygous loss of essential genes before, but not after, WGD. Finally, we demonstrate that loss of heterozygosity and temporal dissection of mutations can be exploited to identify novel tumor suppressor genes and to obtain a deeper characterization of known cancer genes.


Assuntos
Adenocarcinoma de Pulmão/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Duplicação Gênica , Genoma Humano/genética , Neoplasias Pulmonares/genética , Proteínas Supressoras de Tumor/genética , Estudos de Coortes , Simulação por Computador , Variações do Número de Cópias de DNA , Evolução Molecular , Humanos , Estudos Longitudinais , Perda de Heterozigosidade , Mutação , Estudos Prospectivos
9.
Nat Commun ; 11(1): 1308, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161261

RESUMO

Cancer chemotherapy targeting frequent loss of heterozygosity events is an attractive concept, since tumor cells may lack enzymatic activities present in normal constitutional cells. To find exploitable targets, we map prevalent genetic polymorphisms to protein structures and identify 45 nsSNVs (non-synonymous small nucleotide variations) near the catalytic sites of 17 enzymes frequently lost in cancer. For proof of concept, we select the gastrointestinal drug metabolic enzyme NAT2 at 8p22, which is frequently lost in colorectal cancers and has a common variant with 10-fold reduced activity. Small molecule screening results in a cytotoxic kinase inhibitor that impairs growth of cells with slow NAT2 and decreases the growth of tumors with slow NAT2 by half as compared to those with wild-type NAT2. Most of the patient-derived CRC cells expressing slow NAT2 also show sensitivity to 6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine (APA) treatment. These findings indicate that the therapeutic index of anti-cancer drugs can be altered by bystander mutations affecting drug metabolic genes.


Assuntos
Antineoplásicos/farmacologia , Arilamina N-Acetiltransferase/genética , Neoplasias Colorretais/tratamento farmacológico , Perda de Heterozigosidade , Inibidores de Proteínas Quinases/farmacologia , Alelos , Animais , Antineoplásicos/uso terapêutico , Arilamina N-Acetiltransferase/metabolismo , Efeito Espectador/genética , Estudos de Casos e Controles , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Nus , Polimorfismo Genético , Inibidores de Proteínas Quinases/uso terapêutico , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
BMC Cancer ; 20(1): 126, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059662

RESUMO

BACKGROUND: The loss of a single copy of adenomatous polyposis coli (Apc) in leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1)-expressing colonic progenitor cells induces rapid growth of adenomas in mice with high penetrance and multiplicity. The tumors lack functional APC, and a genetic loss of heterozygosity of Apc was previously observed. METHODS: To identify genomic features of early tumorigenesis, and to profile intertumoral genetic heterogeneity, tumor exome DNA (n = 9 tumors) and mRNA (n = 5 tumors) sequences were compared with matched nontumoral colon tissue. Putative somatic mutations were called after stringent variant filtering. Somatic signatures of mutational processes were determined and splicing patterns were observed. RESULTS: The adenomas were found to be genetically heterogeneous and unexpectedly hypermutated, displaying a strong bias toward G:C > A:T mutations. A genetic loss of heterozygosity of Apc was not observed, however, an epigenetic loss of heterozygosity was apparent in the tumor transcriptomes. Complex splicing patterns characterized by a loss of intron retention were observed uniformly across tumors. CONCLUSION: This study demonstrates that early tumors originating from intestinal stem cells with reduced Lrig1 and Apc expression are highly mutated and genetically heterogeneous, with an inflammation-associated mutational signature and complex splicing patterns that are uniform across tumors.


Assuntos
Neoplasias do Colo/genética , Epigênese Genética , Genes APC , Perda de Heterozigosidade , Glicoproteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Animais , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Exoma , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Transcriptoma
11.
BMC Cancer ; 20(1): 84, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005109

RESUMO

BACKGROUND: Cancer subtyping has mainly relied on pathological and molecular means. Massively parallel sequencing-enabled subtyping requires genomic markers to be developed based on global features rather than individual mutations for effective implementation. METHODS: In the present study, the whole genome sequences (WGS) of 110 liver cancers of Japanese patients published with different pathologies were analyzed with respect to their single nucleotide variations (SNVs) comprising both gain-of-heterozygosity (GOH) and loss-of-heterozygosity (LOH) mutations, the signatures of combined GOH and LOH mutations, along with recurrent copy number variations (CNVs). RESULTS: The results, obtained based on the WGS sequences as well as the Exome subset within the WGSs that covered ~ 2.0% of the WGS and the AluScan-subset within the WGSs that were amplifiable by Alu element-consensus primers and covered ~ 2.1% of the WGS, indicated that the WGS samples could be employed with the mutational parameters of SNV load, LOH%, the Signature α%, and survival-associated recurrent CNVs (srCNVs) as genomic markers for subtyping to stratify liver cancer patients prognostically into the long and short survival subgroups. The usage of the AluScan-subset data, which could be implemented with sub-micrograms of DNA samples and vastly reduced sequencing analysis task, outperformed the usage of WGS data when LOH% was employed as stratifying criterion. CONCLUSIONS: Thus genomic subtyping performed with novel genomic markers identified in this study was effective in predicting patient-survival duration, with cohorts of hepatocellular carcinomas alone and those including intrahepatic cholangiocarcinomas. Such relatively heterogeneity-insensitive genomic subtyping merits further studies with a broader spectrum of cancers.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Elementos Alu , Variações do Número de Cópias de DNA , Humanos , Japão , Perda de Heterozigosidade , Mutação , Polimorfismo de Nucleotídeo Único , Prognóstico , Análise de Sobrevida , Sequenciamento Completo do Genoma
12.
Cancer Genet ; 242: 8-14, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32058318

RESUMO

Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy with the majority of patients being classified as B-cell lineage (B-ALL). The sub-classification of B-ALL is based on genomic architecture. Recent studies have demonstrated the capability of SNP-microarrays to detect genomic changes in B-ALL which cannot be observed by conventional cytogenetic methods. In current clinical trials, B-ALL patients at high risk of relapse are mainly identified by adverse cancer genomics and/or poor response to early therapy. To test the hypothesis that inclusion of SNP-microarrays in frontline diagnostics could more efficiently and accurately identify adverse genomic factors than conventional techniques, we evaluated the Australian high-risk B-ALL cohort enrolled on AIEOP-BFM ALL 2009 study (n = 33). SNP-microarray analysis identified additional aberrations in 97% of patients (32/33) compared to conventional techniques. This changed the genomic risk category of 24% (8/33) of patients. Additionally, 27% (9/33) of patients exhibited a 'hyperdiploid' genome, which is generally associated with a good genomic risk and favourable outcomes. An enrichment of IKZF1 deletions was observed with one third of the cohort affected. Our findings suggest the current classification system could be improved and highlights the need to use more sensitive techniques such as SNP-microarray for cytogenomic risk stratification in B-ALL.


Assuntos
Fator de Transcrição Ikaros/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Austrália , Criança , Pré-Escolar , Aberrações Cromossômicas , Bandeamento Cromossômico , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feminino , Proteínas de Fusão bcr-abl/genética , Deleção de Genes , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-Nascido , Perda de Heterozigosidade , Masculino , Neoplasia Residual , Proteínas de Fusão Oncogênica/genética , Poliploidia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/classificação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Estudos Prospectivos , Medição de Risco , Deleção de Sequência
13.
Mol Carcinog ; 59(4): 412-424, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32039517

RESUMO

Somatic copy number alterations (SCNAs) are important biological characteristics that can identify genome-wide alterations in renal cell carcinoma (RCC). Recent studies have shown that SCNAs have potential value for determining the prognosis of RCC. We examined SCNAs using the Affymetrix platform to analyze samples from 59 patients with clear cell RCCs (ccRCCs) including first cohort (30 cases) and second cohort (validation cohort, 29 cases). We stratified SCNAs in the ccRCCs using a hierarchical cluster analysis based on SCNA types, including gain, loss of heterozygosity (LOH), copy neutral LOH, mosaic, and mixed types. In this way, the examined two cohorts were categorized into two subgroups (1 and 2). Although the frequency of mixed type was higher in subgroup 1 than in subgroup 2 in the two cohorts, the association did not reach statistical significance. There was a significant difference in the frequency of metachronous metastasis between subgroups 1 and 2 (subgroup 2 > 1). In addition, subgroup 2 was retained in multivariate analysis of both cohorts. We examined whether there were specific alleles differing between subgroups 1 and 2 in both cohorts. We found that there was indeed a statistically significant difference in the 3p mixed types. Among the 3p mixed type, we found that 3p24.3 mixed type was inversely correlated with the presence of metachronous metastasis in ccRCC. The association was also retained in multivariate analysis in second cohort. We suggest that the 3p24.3 mixed type may be a novel marker to predict a favorable prognosis in ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Variações do Número de Cópias de DNA , Neoplasias Renais/genética , Perda de Heterozigosidade , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/patologia , Análise por Conglomerados , Estudos de Coortes , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Polimorfismo de Nucleotídeo Único , Prognóstico
16.
Genes Cells ; 25(2): 124-138, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31917895

RESUMO

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.


Assuntos
Perda de Heterozigosidade , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Oryzias/genética , Animais , Animais Geneticamente Modificados , Carcinogênese , Linhagem Celular , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA , Feminino , Regulação da Expressão Gênica , Fígado/patologia , Masculino , Mutagênese , Mutação , Proteínas Recombinantes , Transcriptoma
17.
Methods Mol Biol ; 2102: 251-270, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989560

RESUMO

The mouse lymphoma assay (MLA), a forward mutation assay using the Tk+/--3.7.2C clone of the L5178Y mouse lymphoma cell line and the Thymidine kinase (Tk) gene, has been widely used as an in vitro genetic toxicity assay for more than four decades. The MLA can evaluate the ability of mutagens to induce a wide range of genetic events including both gene mutations and chromosomal mutations and has been recommended as one component of several genotoxicity test batteries. Tk-deficient mutants often exhibit chromosomal abnormalities involving the distal end of chromosome 11 where the Tk gene is located, in mice, and the type of chromosome alteration can be analyzed using a loss of heterozygosity (LOH) approach. LOH has been considered an important event in human tumorigenesis and can result from any of the following several mechanisms: large deletions, mitotic recombination, and chromosome loss. In this chapter, the authors describe the procedures for the detection of LOH in the Tk mutants from the MLA, and apply LOH analysis for understanding the types of genetic damage that is induced by individual chemicals.


Assuntos
Cromossomos Humanos Par 11/genética , Perda de Heterozigosidade , Linfoma/genética , Testes de Mutagenicidade/métodos , Timidina Quinase/genética , Alelos , Animais , Aberrações Cromossômicas , DNA/isolamento & purificação , Heterozigoto , Humanos , Camundongos , Repetições de Microssatélites/genética , Mutação , Reação em Cadeia da Polimerase , Timidina Quinase/metabolismo , Fluxo de Trabalho
18.
Methods Mol Biol ; 2102: 333-348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989565

RESUMO

Determining mutant frequencies in endogenous reporter genes is a tool for identifying potentially genotoxic environmental agents, and discovering phenotypes prone to genomic instability and diseases, such as cancer. Here, we describe a high-throughput method for identifying mouse spleen lymphocytes with mutations in the endogenous X-linked hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the endogenous autosomal thymidine kinase (Tk) gene. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of HPRT- and TK-deficient cells. The same procedure can be utilized for quantifying Hprt mutations in most strains of mice (and, with minor changes, in other mammalian species), while mutations in the Tk gene can be determined only in transgenic mice that are heterozygous for inactivation of this gene. Expanded mutant clones can be further analyzed to classify the types of mutations in the Tk gene (small intragenic mutations vs. large chromosomal mutations) and to determine the nature of intragenic mutation at both the Hprt and Tk genes.


Assuntos
Análise Mutacional de DNA/métodos , Ensaios de Triagem em Larga Escala/métodos , Hipoxantina Fosforribosiltransferase/genética , Linfócitos/metabolismo , Timidina Quinase/genética , Animais , Células Cultivadas , Células Clonais/metabolismo , Genes Reporter , Hipoxantina Fosforribosiltransferase/metabolismo , Perda de Heterozigosidade , Camundongos , Camundongos Transgênicos , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina Quinase/metabolismo , Fluxo de Trabalho
19.
Nat Protoc ; 15(2): 266-315, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907453

RESUMO

Mouse models of human cancer have transformed our ability to link genetics, molecular mechanisms and phenotypes. Both reverse and forward genetics in mice are currently gaining momentum through advances in next-generation sequencing (NGS). Methodologies to analyze sequencing data were, however, developed for humans and hence do not account for species-specific differences in genome structures and experimental setups. Here, we describe standardized computational pipelines specifically tailored to the analysis of mouse genomic data. We present novel tools and workflows for the detection of different alteration types, including single-nucleotide variants (SNVs), small insertions and deletions (indels), copy-number variations (CNVs), loss of heterozygosity (LOH) and complex rearrangements, such as in chromothripsis. Workflows have been extensively validated and cross-compared using multiple methodologies. We also give step-by-step guidance on the execution of individual analysis types, provide advice on data interpretation and make the complete code available online. The protocol takes 2-7 d, depending on the desired analyses.


Assuntos
Genômica/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , Animais , Mutação INDEL , Perda de Heterozigosidade , Camundongos , Polimorfismo de Nucleotídeo Único , Fluxo de Trabalho
20.
PLoS One ; 15(1): e0227250, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910244

RESUMO

Oomycete plant pathogens are difficult to control and routine genetic research is challenging. A major problem is instability of isolates. Here we characterize >600 field and single zoospore isolates of Phytophthora capsici for inheritance of mating type, sensitivity to mefenoxam, chromosome copy number and heterozygous allele frequencies. The A2 mating type was highly unstable with 26% of 241 A2 isolates remaining A2. The A1 mating type was stable. Isolates intermediately resistant to mefenoxam produced fully resistant single-spore progeny. Sensitive isolates remained fully sensitive. Genome re-sequencing of single zoospore isolates revealed extreme aneuploidy; a phenomenon dubbed Dynamic Extreme Aneuploidy (DEA). DEA is characterized by the asexual inheritance of diverse intra-genomic combinations of chromosomal ploidy ranging from 2N to 3N and heterozygous allele frequencies that do not strictly correspond to ploidy. Isolates sectoring on agar media showed dramatically altered heterozygous allele frequencies. DEA can explain the rapid increase of advantageous alleles (e.g. drug resistance), mating type switches and copy neutral loss of heterozygosity (LOH). Although the mechanisms driving DEA are unknown, it can play an important role in adaptation and evolution and seriously hinders all aspects of P. capsici research.


Assuntos
Aneuploidia , Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Reprodução Assexuada/genética , Verduras/microbiologia , Alelos , Evolução Biológica , Mapeamento Cromossômico , Cucumis sativus/microbiologia , Variação Genética , Genótipo , Perda de Heterozigosidade , Phytophthora/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Análise de Célula Única , Esporos/genética
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