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2.
Nat Protoc ; 16(2): 841-871, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33318691

RESUMO

Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time.

3.
Nature ; 2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33361815

RESUMO

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.

4.
Haematologica ; Online ahead of print2020 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-33054126

RESUMO

Nodal peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) remains a diagnosis encompassing a heterogenous group of PTCL cases not fitting criteria for more homogeneous subtypes. They are characterized by a poor clinical outcome when treated with anthracycline-containing regimens. A better understanding of their biology could improve prognostic stratification and foster the development of novel therapeutic approaches. Recent targeted and whole exome sequencing studies have shown recurrent copy number abnormalities (CNAs) with prognostic significance. Here, investigating 5 formalin-fixed, paraffin embedded cases of PTCL-NOS by whole genome sequencing (WGS), we found a high prevalence of structural variants and complex events, such as chromothripsis likely responsible for the observed CNAs. Among them, CDKN2A and PTEN deletions emerged as the most frequent aberration, as confirmed in a final cohort of 143 patients with nodal PTCL. The incidence of CDKN2A and PTEN deletions among PTCL-NOS was 46% and 26%, respectively. Furthermore, we found that co-occurrence of CDKN2A and PTEN deletions is an event associated with PTCL-NOS with absolute specificity. In contrast, these deletions were rare and never co-occurred in angioimmunoblastic and anaplastic lymphomas. CDKN2A deletion was associated with shorter overall survival in multivariate analysis corrected by age, IPI, transplant eligibility and GATA3 expression (adjusted HR =2.53; 95% CI 1.006-6.3; p=0.048). These data suggest that CDKN2A deletions may be relevant for refining the prognosis of PTCL-NOS and their significance should be evaluated in prospective trials.

5.
Nat Genet ; 52(11): 1178-1188, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33020667

RESUMO

Somatic mutations in driver genes may ultimately lead to the development of cancer. Understanding how somatic mutations accumulate in cancer genomes and the underlying factors that generate somatic mutations is therefore crucial for developing novel therapeutic strategies. To understand the interplay between spatial genome organization and specific mutational processes, we studied 3,000 tumor-normal-pair whole-genome datasets from 42 different human cancer types. Our analyses reveal that the change in somatic mutational load in cancer genomes is co-localized with topologically-associating-domain boundaries. Domain boundaries constitute a better proxy to track mutational load change than replication timing measurements. We show that different mutational processes lead to distinct somatic mutation distributions where certain processes generate mutations in active domains, and others generate mutations in inactive domains. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation-rate variations observed in human cancers.

6.
Nat Commun ; 11(1): 5040, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028839

RESUMO

Bringing together cancer genomes from different projects increases power and allows the investigation of pan-cancer, molecular mechanisms. However, working with whole genomes sequenced over several years in different sequencing centres requires a framework to compare the quality of these sequences. We used the Pan-Cancer Analysis of Whole Genomes cohort as a test case to construct such a framework. This cohort contains whole cancer genomes of 2832 donors from 18 sequencing centres. We developed a non-redundant set of five quality control (QC) measurements to establish a star rating system. These QC measures reflect known differences in sequencing protocol and provide a guide to downstream analyses and allow for exclusion of samples of poor quality. We have found that this is an effective framework of quality measures. The implementation of the framework is available at: https://dockstore.org/containers/quay.io/jwerner_dkfz/pancanqc:1.2.2 .


Assuntos
Genoma Humano/genética , Genômica/normas , Neoplasias/genética , Controle de Qualidade , Mapeamento Cromossômico/normas , Cromossomos Humanos/genética , Análise Mutacional de DNA/normas , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Masculino , Mutação , Software , Sequenciamento Completo do Genoma/normas
7.
Science ; 370(6512): 75-82, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33004514

RESUMO

The extent of somatic mutation and clonal selection in the human bladder remains unknown. We sequenced 2097 bladder microbiopsies from 20 individuals using targeted (n = 1914 microbiopsies), whole-exome (n = 655), and whole-genome (n = 88) sequencing. We found widespread positive selection in 17 genes. Chromatin remodeling genes were frequently mutated, whereas mutations were absent in several major bladder cancer genes. There was extensive interindividual variation in selection, with different driver genes dominating the clonal landscape across individuals. Mutational signatures were heterogeneous across clones and individuals, which suggests differential exposure to mutagens in the urine. Evidence of APOBEC mutagenesis was found in 22% of the microbiopsies. Sequencing multiple microbiopsies from five patients with bladder cancer enabled comparisons with cancer-free individuals and across histological features. This study reveals a rich landscape of mutational processes and selection in normal urothelium with large heterogeneity across clones and individuals.


Assuntos
Genes Neoplásicos , Mutagênese , Seleção Genética , Neoplasias da Bexiga Urinária/genética , Bexiga Urinária/patologia , Urotélio/patologia , Desaminases APOBEC/genética , Adulto , Idoso , Biópsia , Montagem e Desmontagem da Cromatina/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutagênicos/análise , Mutação
8.
J Pathol ; 252(4): 433-440, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32866294

RESUMO

The rare benign giant cell tumour of bone (GCTB) is defined by an almost unique mutation in the H3.3 family of histone genes H3-3A or H3-3B; however, the same mutation is occasionally found in primary malignant bone tumours which share many features with the benign variant. Moreover, lung metastases can occur despite the absence of malignant histological features in either the primary or metastatic lesions. Herein we investigated the genetic events of 17 GCTBs including benign and malignant variants and the methylation profiles of 122 bone tumour samples including GCTBs. Benign GCTBs possessed few somatic alterations and no other known drivers besides the H3.3 mutation, whereas all malignant tumours harboured at least one additional driver mutation and exhibited genomic features resembling osteosarcomas, including high mutational burden, additional driver event(s), and a high degree of aneuploidy. The H3.3 mutation was found to predate the development of aneuploidy. In contrast to osteosarcomas, malignant H3.3-mutated tumours were enriched for a variety of alterations involving TERT, other than amplification, suggesting telomere dysfunction in the transformation of benign to malignant GCTB. DNA sequencing of the benign metastasising GCTB revealed no additional driver alterations; polyclonal seeding in the lung was identified, implying that the metastatic lesions represent an embolic event. Unsupervised clustering of DNA methylation profiles revealed that malignant H3.3-mutated tumours are distinct from their benign counterpart, and other bone tumours. Differential methylation analysis identified CCND1, encoding cyclin D1, as a plausible cancer driver gene in these tumours because hypermethylation of the CCND1 promoter was specific for GCTBs. We report here the genomic and methylation patterns underlying the rare clinical phenomena of benign metastasising and malignant transformation of GCTB and show how the combination of genomic and epigenomic findings could potentially distinguish benign from malignant GCTBs, thereby predicting aggressive behaviour in challenging diagnostic cases. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

9.
Nature ; 587(7832): 126-132, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32879494

RESUMO

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

10.
Nat Genet ; 52(11): 1189-1197, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32989322

RESUMO

Epidemiological studies have identified many environmental agents that appear to significantly increase cancer risk in human populations. By analyzing tumor genomes from mice chronically exposed to 1 of 20 known or suspected human carcinogens, we reveal that most agents do not generate distinct mutational signatures or increase mutation burden, with most mutations, including driver mutations, resulting from tissue-specific endogenous processes. We identify signatures resulting from exposure to cobalt and vinylidene chloride and link distinct human signatures (SBS19 and SBS42) with 1,2,3-trichloropropane, a haloalkane and pollutant of drinking water, and find these and other signatures in human tumor genomes. We define the cross-species genomic landscape of tumors induced by an important compendium of agents with relevance to human health.

11.
Nat Commun ; 11(1): 4306, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855398

RESUMO

Metastatic melanoma carries a poor prognosis despite modern systemic therapies. Understanding the evolution of the disease could help inform patient management. Through whole-genome sequencing of 13 melanoma metastases sampled at autopsy from a treatment naïve patient and by leveraging the analytical power of multi-sample analyses, we reveal evidence of diversification among metastatic lineages. UV-induced mutations dominate the trunk, whereas APOBEC-associated mutations are found in the branches of the evolutionary tree. Multi-sample analyses from a further seven patients confirmed that lineage diversification was pervasive, representing an important mode of melanoma dissemination. Our analyses demonstrate that joint analysis of cancer cell fraction estimates across multiple metastases can uncover previously unrecognised levels of tumour heterogeneity and highlight the limitations of inferring heterogeneity from a single biopsy.


Assuntos
Evolução Clonal , Heterogeneidade Genética , Melanoma/genética , Neoplasias Cutâneas/genética , Idoso , Biópsia , Análise Mutacional de DNA , Humanos , Masculino , Melanoma/secundário , Estudos Prospectivos , Pele/patologia , Neoplasias Cutâneas/patologia , Sequenciamento Completo do Genoma
13.
Nat Genet ; 52(9): 884-890, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719516

RESUMO

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1-5. Here we examine the mechanism underlying chromothripsis and kataegis by using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In the absence of TREX1, the genome alterations induced by telomere crisis primarily involve breakage-fusion-bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.


Assuntos
Citidina Desaminase/genética , Exodesoxirribonucleases/genética , Fosfoproteínas/genética , Telômero/genética , Linhagem Celular Tumoral , Cromotripsia , Citosina Desaminase/genética , Instabilidade Genômica/genética , Humanos , Mutação/genética , Neoplasias/genética , Células U937
14.
Hemasphere ; 4(3): e371, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32647796

RESUMO

Myeloproliferative neoplasms (MPNs) are characterized by deregulation of mature blood cell production and increased risk of myelofibrosis (MF) and leukemic transformation. Numerous driver mutations have been identified but substantial disease heterogeneity remains unexplained, implying the involvement of additional as yet unidentified factors. The inflammatory microenvironment has recently attracted attention as a crucial factor in MPN biology, in particular whether inflammatory cytokines and chemokines contribute to disease establishment or progression. Here we present a large-scale study of serum cytokine profiles in more than 400 MPN patients and identify an essential thrombocythemia (ET)-specific inflammatory cytokine signature consisting of Eotaxin, GRO-α, and EGF. Levels of 2 of these markers (GRO-α and EGF) in ET patients were associated with disease transformation in initial sample collection (GRO-α) or longitudinal sampling (EGF). In ET patients with extensive genomic profiling data (n = 183) cytokine levels added significant prognostic value for predicting transformation from ET to MF. Furthermore, CD56+CD14+ pro-inflammatory monocytes were identified as a novel source of increased GRO-α levels. These data implicate the immune cell microenvironment as a significant player in ET disease evolution and illustrate the utility of cytokines as potential biomarkers for reaching beyond genomic classification for disease stratification and monitoring.

15.
Nat Commun ; 11(1): 3390, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636395

RESUMO

Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.


Assuntos
Genes de Imunoglobulinas , Hibridização in Situ Fluorescente/métodos , Oncogenes , Translocação Genética , Algoritmos , Estudos de Coortes , Genoma Humano , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Switching de Imunoglobulina , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Software , Sequenciamento Completo do Genoma
16.
Cell ; 182(3): 672-684.e11, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32697969

RESUMO

Inflammatory bowel disease (IBD) is a chronic inflammatory disease associated with increased risk of gastrointestinal cancers. We whole-genome sequenced 446 colonic crypts from 46 IBD patients and compared these to 412 crypts from 41 non-IBD controls from our previous publication on the mutation landscape of the normal colon. The average mutation rate of affected colonic epithelial cells is 2.4-fold that of healthy colon, and this increase is mostly driven by acceleration of mutational processes ubiquitously observed in normal colon. In contrast to the normal colon, where clonal expansions outside the confines of the crypt are rare, we observed widespread millimeter-scale clonal expansions. We discovered non-synonymous mutations in ARID1A, FBXW7, PIGR, ZC3H12A, and genes in the interleukin 17 and Toll-like receptor pathways, under positive selection in IBD. These results suggest distinct selection mechanisms in the colitis-affected colon and that somatic mutations potentially play a causal role in IBD pathogenesis.

17.
Cell Stem Cell ; 27(2): 326-335.e4, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32673568

RESUMO

DNA methyltransferase 3A (DNMT3A) is the most commonly mutated gene in clonal hematopoiesis (CH). Somatic DNMT3A mutations arise in hematopoietic stem cells (HSCs) many years before malignancies develop, but difficulties in comparing their impact before malignancy with wild-type cells have limited the understanding of their contributions to transformation. To circumvent this limitation, we derived normal and DNMT3A mutant lymphoblastoid cell lines from a germline mosaic individual in whom these cells co-existed for nearly 6 decades. Mutant cells dominated the blood system, but not other tissues. Deep sequencing revealed similar mutational burdens and signatures in normal and mutant clones, while epigenetic profiling uncovered the focal erosion of DNA methylation at oncogenic regulatory regions in mutant clones. These regions overlapped with those sensitive to DNMT3A loss after DNMT3A ablation in HSCs and in leukemia samples. These results suggest that DNMT3A maintains a conserved DNA methylation pattern, the erosion of which provides a distinct competitive advantage to hematopoietic cells.

18.
Cancer Discov ; 10(10): 1489-1499, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32690541

RESUMO

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

19.
Nat Commun ; 11(1): 2169, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358516

RESUMO

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Animais , Caenorhabditis elegans/genética , Dano ao DNA/genética , Reparo do DNA/genética , Genômica/métodos , Humanos , Mutação/genética , Mutação Puntual/genética
20.
Nature ; 580(7805): 640-646, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32350471

RESUMO

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.


Assuntos
Análise Mutacional de DNA , Endométrio/citologia , Endométrio/metabolismo , Epitélio/metabolismo , Saúde , Mutação , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Carcinogênese/genética , Células Clonais/citologia , Neoplasias do Endométrio/genética , Endométrio/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Paridade/genética , Fatores de Tempo , Adulto Jovem
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