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1.
Artigo em Russo | MEDLINE | ID: mdl-31317896

RESUMO

AIM: Long continuous stretches of homozygosity (LCSH) are regularly detected in studies using molecular karyotyping (SNP array). Despite this type of variation being able to provide meaningful data on the parents' kinship, uniparental disomy and chromosome rearrangements, LCSH are rarely considered as a possible epigenetic cause of neurodevelopmental disorders. Despite their direct relationship to imprinting, LCSH in imprinted loci have not been considered in terms of pathogenicity. The present work is aimed at studying LCSH in chromosomal regions containing imprinted genes previously associated with disease in children with idiopathic intellectual disability, autism, congenital malformations and/or epilepsy. MATERIAL AND METHODS: Five hundred and four patients with autism spectrum disorders and intellectual disability were examined. RESULTS: LCSH affecting imprinted loci associated with various diseases were identified in 40 (7.9%) individuals. Chromosomal region 7q21.3 was affected in twenty three cases, 15q11.2 in twelve, 11p15.5 in five, 7q32.2 in four. Four patients had 2 LCSH affecting imprinted loci. Besides one LCSH in 7q31.33q32.3 (~4 Mbp) region, all LCSH were 1-1.6 Mbp. Clinically, these cases resembled the corresponding imprinting diseases (e.g. Silver-Russell, Beckwith-Wiedemann, Prader-Willi, Angelman syndromes). Parental kinship was identified in 8 cases (1.59%), which were not affected by LCSH at imprinted loci. CONCLUSION: The present study shows that LCSH affecting chromosomal regions 7q21.3, 7q32.2, 11p15.5 and 15p11.2 occur in about 7.9% of children with intellectual disability, autism, congenital malformations and/or epilepsy. Consequently, this type of epigenetic mutations is obviously common in a group of children with neurodevelopmental disorders. LCSH less than 2.5-10 Mbp are usually ignored in molecular karyotyping (SNP array) studies and, therefore, an important epigenetic cause of intellectual disability, autism or epilepsy with high probability remains without attention.


Assuntos
Síndrome de Angelman , Transtorno do Espectro Autista , Epigenômica , Deficiência Intelectual , Síndrome de Angelman/genética , Transtorno do Espectro Autista/genética , Criança , Humanos , Deficiência Intelectual/genética , Perda de Heterozigosidade
3.
J Cancer Res Clin Oncol ; 145(9): 2241-2250, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31342168

RESUMO

PURPOSE: The tripartite motif (TRIM)16 acts as a tumour suppressor in both squamous cell carcinoma (SCC) and melanoma. TRIM16 is known to be secreted by keratinocytes, but no studies have been reported yet to assess the relationship between TRIM16 keratinocyte expression and melanoma development. METHODS: To study the role of TRIM16 in skin cancer development, we developed a keratinocyte TRIM16-specific knockout mouse model, and used the classical two-stage skin carcinogenesis challenge method, to assess the loss of keratinocyte TRIM16 on both papilloma, SCC and melanoma development in the skin after topical carcinogen treatment. RESULTS: Heterozygous, but not homozygous, TRIM16 knockout mice exhibited an accelerated development of skin papillomas and melanomas, larger melanoma lesions and an increased potential for lymph node metastasis. CONCLUSION: This study provides the first evidence that keratinocyte loss of the putative melanoma tumour suppressor protein, TRIM16, enhances melanomagenesis. Our data also suggest that TRIM16 expression in keratinocytes is involved in cross talk between keratinocytes and melanocytes, and has a role in melanoma tumorigenesis.


Assuntos
Proteínas de Transporte/genética , Queratinócitos/metabolismo , Perda de Heterozigosidade/fisiologia , Linfonodos/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Neoplasias Cutâneas/genética , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Queratinócitos/patologia , Linfonodos/patologia , Metástase Linfática , Melanócitos/patologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Knockout , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
4.
Appl Microbiol Biotechnol ; 103(12): 4869-4880, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053912

RESUMO

The yeast Saccharomyces cerevisiae has been widely used as a model system for studying the physiological and pharmacological action of small-molecular drugs. Here, a heterozygous diploid S. cerevisiae strain QSS4 was generated to determine whether drugs could induce chromosomal instability by determining the frequency of mitotic recombination. Using the combination of a custom SNP microarray and yeast screening system, the patterns of chromosomal instability induced by drugs were explored at the whole genome level in QSS4. We found that Zeocin (a member of the bleomycin family) treatment increased the rate of genomic alterations, including aneuploidy, loss of heterozygosity (LOH), and chromosomal rearrangement over a hundred-fold. Most recombination events are likely to be initiated by DNA double-stand breaks directly generated by Zeocin. Another remarkable finding is that G4-motifs and low GC regions were significantly underrepresented within the gene conversion tracts of Zeocin-induced LOH events, indicating that certain DNA regions are less preferred Zeocin-binding sites in vivo. This study provides a novel paradigm for evaluating genetic toxicity of small-molecular drugs using yeast models.


Assuntos
Instabilidade Cromossômica/efeitos dos fármacos , Cromossomos Fúngicos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Aneuploidia , Bleomicina/farmacologia , Divisão Celular , Rearranjo Gênico , Instabilidade Genômica , Perda de Heterozigosidade , Recombinação Genética
5.
Pol J Vet Sci ; 22(1): 133-141, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30997777

RESUMO

Characterisation of copy number variation (CNV) and loss of heterozygosity (LOH) has pro- vided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. The role of CNVs and germinal or somatic LOHs in canine mammary tumours is still unknown. Therefore, the aim of this study was to identify CNVs and LOHs in canine mammary tumours. Forty-eight samples obtained from normal (n=24) and tumour (n=24) tissues of dogs were analysed. In the study, we used CanineHD BeadChip assay (Illumina) and OncoSNP software to identify copy number alternations in genomes of dif- ferent dog breeds and in different mammary cancer types occurring in this species. The analyses revealed that, in the case of CNV, the amplification-type variants were longer and more frequent than deletions. Based on the analysis of the frequency of different types of aberrations in the in- dividual parts of the genome, regions that are particularly susceptible to structural aberrations were indicated. The fraction of genes identified within these regions was associated with major processes of neoplastic transformation. Association analysis of such traits as tumour grading as well as the size and age of dogs demonstrated that structural aberrations were more frequent in dogs diagnosed with tumour malignancy grade II and III, in dogs with a larger body size, and in large dogs aged 7-8. The promising results of these pioneering investigations prompt continuation thereof to analyse other types of cancer.


Assuntos
Doenças do Cão/genética , Variação Estrutural do Genoma , Neoplasias Mamárias Animais/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Cães , Feminino , Regulação Neoplásica da Expressão Gênica , Perda de Heterozigosidade , Neoplasias Mamárias Animais/genética
6.
Exp Oncol ; 41(1): 53-56, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30932413

RESUMO

AIM: To examine frequencies and spectrum of genomic alterations in Ukrainian patients diagnosed with primary myelofibrosis (PMF). MATERIALS AND METHODS: We enrolled 30 Ukrainian patients diagnosed with PMF who were previously tested for usual mutations in mye-loproliferative neoplasms driver genes (JAK2, MPL and CALR). Genomic DNA samples were obtained from peripheral blood leukocytes of these patients. Copy number alterations and copy-neutral loss of heterozygosity (cnLOH) were assessed using a high-density CytoScan HD microarray platform. Statistical significance was evaluated by the Fisher exact test. RESULTS: We identified frequent genomic alterations, but no significant difference in the rates of copy-number loss, copy-number gain, cnLOH, or multiple genomic alterations were found in the groups of PMF patients that were positive for one of the usual mutations in driver genes or negative for such mutations (33.3% and 55.6%, p = 0.4181, 19.0% and 11.1%, p = 1.0000, 61.9% and 44.4%, p = 0.4434, 33.3% and 55.6%, p = 0.4181, respectively). The most frequent alterations were cnLOH at 1p36-1p22, 9p24.3-9p13.3 and 11q12.3-11q25; copy number loss at 7q21-7q36.3 and 13q12.3-13q14.3. Copy number alterations and cnLOH commonly affected the EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53 genes, in addition to JAK2, MPL and CALR. CONCLUSION: We demonstrated the spectrum of genomic alterations in the groups of the Ukrainian PMF patients with or without the usual mutations in the specific driver genes. We identified several potential genes, which may be involved in the myeloproliferative neoplasms development and their phenotype modification (EZH2, LAMB4, CBL, CUX1, ATM, RB1 and TP53).


Assuntos
Variações do Número de Cópias de DNA , Estudos de Associação Genética , Predisposição Genética para Doença , Perda de Heterozigosidade , Mielofibrose Primária/genética , Biomarcadores , Mapeamento Cromossômico , Feminino , Humanos , Masculino , Ucrânia
7.
Leukemia ; 33(8): 1881-1894, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30816328

RESUMO

In more than 30% of B-cell precursor acute lymphoblastic leukaemia (B-ALL), chromosome 21 sequence is overrepresented through aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). Although frequent, the mechanisms by which these abnormalities promote B-ALL remain obscure. Intriguingly, we found copy number neutral loss of heterozygosity (CN-LOH) of 12q was recurrent in iAMP21-ALL, but never observed in B-ALL without some form of chromosome 21 gain. As a consequence of CN-LOH 12q, mutations or deletions of the adaptor protein, SH2B3, were converted to homozygosity. In patients without CN-LOH 12q, bi-allelic abnormalities of SH2B3 occurred, but only in iAMP21-ALL, giving an overall incidence of 18% in this sub-type. Review of published data confirmed a tight association between overrepresentation of chromosome 21 and both CN-LOH 12q and SH2B3 abnormalities in B-ALL. Despite relatively small patient numbers, preliminary analysis linked 12q abnormalities to poor outcome in iAMP21-ALL (p = 0.03). Homology modelling of a leukaemia-associated SH2 domain mutation and in vitro analysis of patient-derived xenograft cells implicated the JAK/STAT pathway as one likely target for SH2B3 tumour suppressor activity in iAMP21-ALL.


Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Perda de Heterozigosidade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas/genética , Humanos , Interleucina-7/farmacologia , Mutação , Fator de Transcrição STAT5/fisiologia
8.
Leukemia ; 33(8): 1895-1909, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30842609

RESUMO

Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup ('PAX5-plus', n = 19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis.


Assuntos
Mutação , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Cromossomos Humanos Par 9 , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Metabolismo Energético , Humanos , Perda de Heterozigosidade
9.
Int J Hematol ; 109(5): 572-577, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30887274

RESUMO

Light-chain plasma cell myeloma (LC-PCM) is a PCM subtype in which only immunoglobulin light-chain is secreted. However, the absence of immunoglobulin heavy-chain (IGH) production in this condition has not been fully elucidated. To address this issue, we retrospectively analyzed patients at our center with LC-PCM and found a group who had only split signals of IGH gene derived from 14q32/IGH translocations by fluorescence in situ hybridization (FISH). Six patients were identified with only split signals of the IGH gene derived from 14q32/IGH translocations. Five of these patients were newly diagnosed, while one had IgG-λ PCM at presentation, which transformed to λ LC-PCM after treatment. The translocation partners were identified in four patients: two cases of (11;14)(q13;q32) and two cases of (4;14)(p16;q32). The development of LC-PCM appears to be explained by the application of allelic exclusion in these patients, such that 14q32/IGH translocation in one allele contributes to the pathogenesis of PCM and the subsequent loss of the other allele is responsible for the loss of IGH production. These findings suggest that a FISH pattern of IGH with "split and loss" may constitute a unique subgroup of LC-PCM.


Assuntos
Cromossomos Humanos Par 14/genética , Cadeias lambda de Imunoglobulina , Leucemia Plasmocitária/genética , Perda de Heterozigosidade , Translocação Genética , Idoso , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 4/genética , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética
10.
Nat Commun ; 10(1): 1415, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926791

RESUMO

B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.


Assuntos
Elementos de DNA Transponíveis/genética , Testes Genéticos/métodos , Linfoma de Células B/genética , Animais , Sistemas CRISPR-Cas/genética , Células Clonais , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Genes Supressores de Tumor , Estudos de Associação Genética , Humanos , Perda de Heterozigosidade , Linfoma de Células B/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/metabolismo , Reprodutibilidade dos Testes
11.
Hum Immunol ; 80(4): 257-262, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30790598

RESUMO

INTRODUCTION: Chromosomal abnormalities are frequent events in hematological malignancies. The degree of HLA compatibility between donor and recipient in hematopoietic stem cell transplantation is critical. PURPOSE OF THE STUDY: In this report, we describe an acute myeloid leukemia case with loss of heterozygosity (LOH) encompassing the entire HLA. MATERIALS AND METHODS: HLA molecular typing was performed on peripheral blood (PB) and buccal swabs (BS). Chromosomal microarray analysis (CMA) was performed using a whole genome platform. RESULTS: Typing results on PB sample collected during blast crisis demonstrated homozygosity at the -A, -B, -C, -DR, and -DQ loci. A BS sample demonstrated heterozygosity at all loci. A subsequent PB sample drawn after count recovery confirmed heterozygosity. The CMA performed on PB samples collected during and after blast crisis revealed a large terminal region of copy-neutral LOH involving chromosome region 6p25.3p21.31, spanning approximately 35.9 Mb. The results of the CMA assay on sample collected after count recovery did not demonstrate LOH. CONCLUSIONS: LOH at the HLA gene locus may significantly influence the donor search resulting in mistakenly choosing homozygous donors. We recommend confirming the HLA typing of recipients with hematological malignancies when homozygosity is detected at any locus by using BS samples, or alternatively from PB when remission is achieved.


Assuntos
Medula Óssea/fisiologia , Genoma/genética , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/genética , Leucócitos Mononucleares/fisiologia , Perda de Heterozigosidade , Complexo Principal de Histocompatibilidade/genética , Idoso , Circulação Sanguínea , Feminino , Teste de Histocompatibilidade , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/terapia , Análise em Microsséries , Técnicas de Diagnóstico Molecular , Indução de Remissão
12.
Med Sci (Paris) ; 35(2): 138-151, 2019 Feb.
Artigo em Francês | MEDLINE | ID: mdl-30774081

RESUMO

The main objective of oncogenetics is to characterize a subpopulation of patients at high risk of cancer development at an early age in order to provide specific recommendations for an optimized follow-up and care path. Oncogenetic counselling helps to assess individual risk from a family history. By a family approach of formal genetics, the key issue is to identify families with a strong aggregation of cancers, and, in particular, suggesting a specific syndrome of inherited predisposition to cancer. This approach can lead to the proposal of germline genetic testing in search of causal mutations. As up to know, the search for a constitutional mutation in the BRCA genes has led to the identification of a causal deleterious mutation in less than 10% of index-cases analyzed. It is therefore important to evaluate the impact of new genes in the current panorama of inherited predisposition to breast and ovarian cancer.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Neoplasias da Mama/diagnóstico , Feminino , Genes BRCA1 , Genes BRCA2 , Testes Genéticos/métodos , Mutação em Linhagem Germinativa , Humanos , Perda de Heterozigosidade , Neoplasias Ovarianas/genética , Síndrome
13.
J Mol Neurosci ; 68(1): 11-18, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30778836

RESUMO

Scoliosis is a common manifestation of neurofibromatosis type 1, causing significant morbidity. The etiology of dystrophic scoliosis in neurofibromatosis type 1 is not fully understood and therapies are lacking. Somatic mutations in NF1 have been shown in tibial pseudarthrosis providing rationale for similar processes in neurofibromatosis type 1-associated dystrophic scoliosis. Spinal samples from surgical procedures with matched peripheral blood of two individuals with neurofibromatosis type 1 and dystrophic scoliosis were obtained and DNA extracted. Next generation sequencing of various spinal sections as well as the germline/blood sample were performed using a RASopathy gene panel (includes the NF1 gene). Variants were compared between the spinal tissue samples and the germline data. In addition, the next generation sequencing allele frequency data were used to detect somatic loss of heterozygosity. All samples had a detected potentially inactivating NF1 germline mutation. Both individuals demonstrated an allelic imbalance inclusive of NF1 in the next generation sequencing data. In addition, for the same two individuals, there was an increase in the % variant reads for the germline mutation in some of the surgical spinal samples corresponding to the allelic imbalance. Contra analysis did not show any deletion in Chromosome 17 next generation sequencing data. Microarray analysis verified somatic copy neutral loss of heterozygosity for these two individuals for the majority of the chromosome 17 q-arm, inclusive of the NF1 gene. These results suggest that the cause of dystrophic scoliosis is multifactorial and that a somatic NF1 mutation contributes to the etiology.


Assuntos
Neurofibromatose 1/genética , Neurofibromina 1/genética , Escoliose/genética , Criança , Feminino , Frequência do Gene , Humanos , Perda de Heterozigosidade , Masculino , Mutação , Neurofibromatose 1/complicações , Neurofibromatose 1/patologia , Escoliose/etiologia , Escoliose/patologia , Coluna Vertebral/metabolismo , Coluna Vertebral/patologia
14.
Br J Cancer ; 120(4): 444-452, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30739914

RESUMO

BACKGROUND: Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis. METHODS: In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed. RESULTS: Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes. CONCLUSIONS: Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.


Assuntos
Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Evolução Molecular , Genes BRCA1 , Genes BRCA2 , Humanos , Perda de Heterozigosidade , Masculino , Mutação , Metástase Neoplásica , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/patologia , Sequenciamento Completo do Genoma
15.
Ann Diagn Pathol ; 39: 63-68, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30785029

RESUMO

Hereditary leiomyomatosis and renal cell carcinoma-associated renal cell carcinoma (HLRCC)/fumarate hydratase deficient renal cell carcinoma (FHRCC) is defined by molecular genetic changes (mutation/LOH in fumarate hydratase (FH) gene). We investigated chromosomal numerical aberration pattern (CNV) in FHRCC/HLRCC using array comparative genomic hybridization analysis and low pass whole genome sequencing. Genetic analysis was successfully completed in 12 tumors. Most common chromosomal aberrations detected were a complete or partial loss of chromosome 4 (5/12 cases), chromosome 15 (4/12 cases), and chromosomes 9, 13, and 14 (each in 3/12 cases), as well as a complete or partial gain of chromosome 17 (in 4/12 cases). No chromosomal losses or gains were detected in 4 cases. Copy number variation pattern in FHRCC/HLRCC appears to be highly variable and does not provide a useful diagnostic tool in identifying these cases. Immunohistochemical staining and especially molecular genetic evaluation of FH gene mutations/LOH remain the gold standard in identifying FHRCC/HLRCC.


Assuntos
Carcinoma de Células Renais/genética , Aberrações Cromossômicas , Fumarato Hidratase/deficiência , Neoplasias Renais/genética , Adulto , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Sequenciamento Completo do Genoma
16.
mSphere ; 4(1)2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760617

RESUMO

The heterozygous diploid genome of Candida albicans is highly plastic, with frequent loss of heterozygosity (LOH) events. In the SC5314 laboratory strain, while LOH events are ubiquitous, a chromosome homozygosis bias is observed for certain chromosomes, whereby only one of the two homologs can occur in the homozygous state. This suggests the occurrence of recessive lethal allele(s) (RLA) preventing large-scale LOH events on these chromosomes from being stably maintained. To verify the presence of an RLA on chromosome 7 (Chr7), we utilized a system that allows (i) DNA double-strand break (DSB) induction on Chr7 by the I-SceI endonuclease and (ii) detection of the resulting long-range homozygosis. I-SceI successfully induced a DNA DSB on both Chr7 homologs, generally repaired by gene conversion. Notably, cells homozygous for the right arm of Chr7B were not recovered, confirming the presence of RLA(s) in this region. Genome data mining for RLA candidates identified a premature nonsense-generating single nucleotide polymorphism (SNP) within the HapB allele of C7_03400c whose Saccharomyces cerevisiae ortholog encodes the essential Mtr4 RNA helicase. Complementation with a wild-type copy of MTR4 rescued cells homozygous for the right arm of Chr7B, demonstrating that the mtr4K880* RLA is responsible for the Chr7 homozygosis bias in strain SC5314. Furthermore, we observed that the major repeat sequences (MRS) on Chr7 acted as hot spots for interhomolog recombination. Such recombination events provide C. albicans with increased opportunities to survive DNA DSBs whose repair can lead to homozygosis of recessive lethal or deleterious alleles. This might explain the maintenance of MRS in this species.IMPORTANCE Candida albicans is a major fungal pathogen, whose mode of reproduction is mainly clonal. Its genome is highly tolerant to rearrangements, in particular loss of heterozygosity events, known to unmask recessive lethal and deleterious alleles in heterozygous diploid organisms such as C. albicans By combining a site-specific DSB-inducing system and mining genome sequencing data of 182 C. albicans isolates, we were able to ascribe the chromosome 7 homozygosis bias of the C. albicans laboratory strain SC5314 to an heterozygous SNP introducing a premature STOP codon in the MTR4 gene. We have also proposed genome-wide candidates for new recessive lethal alleles. We additionally observed that the major repeat sequences (MRS) on chromosome 7 acted as hot spots for interhomolog recombination. Maintaining MRS in C. albicans could favor haplotype exchange, of vital importance to LOH events, leading to homozygosis of recessive lethal or deleterious alleles that inevitably accumulate upon clonality.


Assuntos
Candida albicans/genética , Genes Letais , Genes Recessivos , Genoma Fúngico , Sequências Repetitivas de Ácido Nucleico , Alelos , Cromossomos Fúngicos , Diploide , Perda de Heterozigosidade , Polimorfismo de Nucleotídeo Único , Recombinação Genética
17.
Oncogene ; 38(17): 3119-3133, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30622339

RESUMO

Esophageal squamous cells carcinoma (ESCC) is a major common thoracic tumor characterized by distinctly high incidences and mortality rates. Despite advances in multimodality therapy, the mortality rate of ESCC remains high and understanding of molecular alterations leading to the development and progression of ESCC is still very limited. In this study, a new tumor suppressor candidate, cell adhesion molecule with homology to L1CAM (CHL1), located at 3p26 which was frequently deleted in ESCC was identified. Reduced expression of CHL1 correlated with poor differentiation, increased invasion, and lymph-node metastasis, advanced tumor stage, and decreased overall survival. Methylation-specific PCR and FISH assays revealed that down-regulation of CHL1 in both ESCC cell lines and clinical samples were associated with promoter hypermethylation and loss of heterozygosity. Functional studies using lentiviral-based overexpression and knockdown systems provided direct support of CHL1 to function as an important tumor suppressor with both anti-proliferation and anti-metastasis abilities, through Merlin and SEMA3B-Np1-mediated inhibition of AKT signaling pathway. Further characterization of CHL1 may provide a novel therapeutic target in ESCC treatment.


Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Metilação de DNA , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Perda de Heterozigosidade , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Epigênese Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Transdução de Sinais , Análise de Sobrevida
18.
Methods Mol Biol ; 1908: 89-111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649723

RESUMO

The basis of cancer biology is built upon two fundamental processes that result in uncontrolled cell proliferation and tumor formation: loss of tumor suppressor gene function and gain of oncogene function. Somatic DNA copy number variants (CNVs), which generally range in size from kilobases to entire chromosomes, facilitate gains and losses of chromosomal material incorporating oncogenes and tumor suppressor genes, respectively. In fact, many cancer types are characterized by DNA copy number changes and relatively few single nucleotide mutations (Ciriello et al. Nat Genet 45:1127-1133, 2013). Currently, the optimal method to detect such somatic copy number changes across the cancer genome is whole-genome single nucleotide polymorphism (SNP) microarray analysis.


Assuntos
Variações do Número de Cópias de DNA , Perda de Heterozigosidade , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , Genômica/métodos , Humanos , Mutação , Inclusão em Parafina , Polimorfismo de Nucleotídeo Único , Fixação de Tecidos
19.
Methods Mol Biol ; 1908: 113-124, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649724

RESUMO

Copy number variants (CNVs) and copy neutral loss of heterozygosity (CN-LOH) represent important types of genomic abnormalities in cancer. Genomic DNA microarray serves as the current gold standard method for detecting genome-wide CNVs and CN-LOH. However, as next-generation sequencing (NGS) is widely used to detect gene variants in clinical testing, the ability of NGS to detect CNVs and CN-LOH has also been demonstrated. This chapter describes a protocol for detecting genome-wide large somatic CNVs and CN-LOH using a single nucleotide polymorphism (SNP) sequencing backbone. When combined with a targeted gene mutation panel, this strategy allows for simultaneous detection of somatic gene mutations and genome-wide CNVs and CN-LOH.


Assuntos
Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Perda de Heterozigosidade , Neoplasias/genética , Análise de Sequência de DNA/métodos , Software , Análise de Dados , Genômica/métodos , Humanos , Mutação , Polimorfismo de Nucleotídeo Único
20.
BMC Cancer ; 19(1): 69, 2019 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-30642281

RESUMO

BACKGROUND: Diffuse gastric cancer (DGC) is associated with the reduction or absence of the expression of the cell adhesion protein E-cadherin (encoded by the CDH1 gene). Molecular characteristics are less well described for mixed gastric cancer (MGC). The main somatic alterations that have been described in the CDH1 gene are mutations, loss of heterozygosity (LOH) and promoter methylation. The aim was to analyze CDH1 somatic alterations in Mexican patients with diffuse and mixed gastric cancer. METHODS: We searched for mutations in the CDH1 gene in tumor DNA from DGC (n = 13) and MGC (n = 7) patients by next generation sequencing (NGS). Validation of findings was performed using Sanger sequencing. LOH was analyzed using dinucleotide repeat markers surrounding the CDH1 gene, and methylation was investigated by DNA bisulfite conversion and sequencing. E-cadherin protein deficiency was analyzed by immunohistochemistry. RESULTS: Seventeen point variants were identified by NGS, 13 of them were validated by Sanger sequencing. Only 1/13 had not been previously reported (c.-137C > A), and 12/13 were already reported as polymorphisms. Two DGC cases presented LOH at the locus 16q22.1 (13.3%). CDH1 promoter methylation was positive in (7/11) 63.6% and (4/6) 66.6% of the cases with DGC and MGC, respectively. E-cadherin protein deficiency was observed in 58.3% of DGC cases while 100% in MGC cases. CONCLUSIONS: While no pathogenic somatic mutations were found that could explain the diffuse histology of gastric cancer in DGC and MGC, methylation was the most common somatic inactivation event of the CDH1 gene, and LOH was rare. The previously unreported c.-137C > A variant modify the CDH1 gene expression since it alters the binding sites for transcription factors.


Assuntos
Antígenos CD/genética , Caderinas/genética , Mutação , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Alelos , Metilação de DNA , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Perda de Heterozigosidade , Masculino , México , Polimorfismo Genético , Regiões Promotoras Genéticas
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