RESUMO
Salmonella Typhimurium is a zoonotic pathogen that poses a major threat to public health. This generalist serotype can be found in many hosts and the environment where varying selection pressures may result in the accumulation of antimicrobial resistance determinants. However, the transmission of this serotype between food-producing hosts, specifically between poultry layer flocks and nearby dairy herds, was never demonstrated. We investigated an outbreak at a dairy in Israel to determine the role of nearby poultry houses to be sources of infection. The 2-month outbreak resulted in a 47% mortality rate among 15 calves born in that period. Routine treatment of fluid therapy, a nonsteroidal anti-inflammatory, and cefquinome was ineffective, and control was achieved by the introduction of vaccination of dry cows against Salmonella (Bovivac S, MSD Animal Health) and a strict colostrum regime. Whole genome sequencing and antimicrobial sensitivity tests were performed on S. Typhimurium strains isolated from the dairy (n = 4) and strains recovered from poultry layer farms (n = 10). We identified acquired antimicrobial-resistant genes, including the blaCTX-M-55 gene, conferring resistance to extended-spectrum cephalosporins, which was exclusive to dairy isolates. Genetic similarity with less than five single nucleotide polymorphism differences between dairy and poultry strains suggested a transmission link. This investigation highlights the severe impact of S. Typhimurium on dairy farms and the transmission risk from nearby poultry farms. The accumulation of potentially transferable genes conferring resistance to critically important antimicrobials underscores the increased public health risk associated with S. Typhimurium circulation between animal hosts.IMPORTANCESalmonella Typhimurium is one of the major causes of food-borne illness globally. Infections may result in severe invasive disease, in which antimicrobial treatment is warranted. Therefore, the emergence of multi-drug-resistant strains poses a significant challenge to successful treatment and is considered one of the major threats to global health. S. Typhimurium can be found in a variety of animal hosts and environments; however, its transmission between food-producing animals, specifically poultry layers flocks and dairy herds, was never studied. Here, we demonstrate the transmission of the pathogen from poultry to a nearby dairy farm. Alarmingly, the multi-drug-resistant strains collected during the outbreak in the dairy had acquired resistance to extended-spectrum cephalosporins, antibiotics critically important in treating Salmonellosis in humans. The findings of the study emphasize the increased risk to public health posed by zoonotic pathogens' circulation between animal hosts.
Assuntos
Antibacterianos , Fazendas , Saúde Pública , Salmonelose Animal , Salmonella typhimurium , Animais , Salmonella typhimurium/genética , Salmonella typhimurium/efeitos dos fármacos , Salmonelose Animal/microbiologia , Salmonelose Animal/epidemiologia , Salmonelose Animal/transmissão , Bovinos , Antibacterianos/farmacologia , Aves Domésticas/microbiologia , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/transmissão , Israel/epidemiologia , Indústria de Laticínios , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/transmissão , Doenças dos Bovinos/epidemiologia , Farmacorresistência Bacteriana/genética , Surtos de Doenças/veterinária , Galinhas/microbiologia , Humanos , Farmacorresistência Bacteriana Múltipla/genéticaRESUMO
Nonsense-mediated mRNA decay (NMD) is of universal biological significance. It has emerged as an important global RNA, DNA and translation regulatory pathway. By systematically sequencing 737 genes (annotated in the Vertebrate Genome Annotation database) on the human X chromosome in 250 families with X-linked mental retardation, we identified mutations in the UPF3 regulator of nonsense transcripts homolog B (yeast) (UPF3B) leading to protein truncations in three families: two with the Lujan-Fryns phenotype and one with the FG phenotype. We also identified a missense mutation in another family with nonsyndromic mental retardation. Three mutations lead to the introduction of a premature termination codon and subsequent NMD of mutant UPF3B mRNA. Protein blot analysis using lymphoblastoid cell lines from affected individuals showed an absence of the UPF3B protein in two families. The UPF3B protein is an important component of the NMD surveillance machinery. Our results directly implicate abnormalities of NMD in human disease and suggest at least partial redundancy of NMD pathways.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Linhagem Celular Transformada , Códon sem Sentido , Análise Mutacional de DNA , Saúde da Família , Feminino , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Dados de Sequência Molecular , Linhagem , Estabilidade de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , SíndromeRESUMO
We examined the coding sequence of 518 protein kinases, approximately 1.3 Mb of DNA per sample, in 25 breast cancers. In many tumors, we detected no somatic mutations. But a few had numerous somatic mutations with distinctive patterns indicative of either a mutator phenotype or a past exposure.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Mutação , Proteínas Quinases/genética , Idoso , Análise Mutacional de DNA , Feminino , Humanos , Família MultigênicaRESUMO
Cancers arise owing to mutations in a subset of genes that confer growth advantage. The availability of the human genome sequence led us to propose that systematic resequencing of cancer genomes for mutations would lead to the discovery of many additional cancer genes. Here we report more than 1,000 somatic mutations found in 274 megabases (Mb) of DNA corresponding to the coding exons of 518 protein kinase genes in 210 diverse human cancers. There was substantial variation in the number and pattern of mutations in individual cancers reflecting different exposures, DNA repair defects and cellular origins. Most somatic mutations are likely to be 'passengers' that do not contribute to oncogenesis. However, there was evidence for 'driver' mutations contributing to the development of the cancers studied in approximately 120 genes. Systematic sequencing of cancer genomes therefore reveals the evolutionary diversity of cancers and implicates a larger repertoire of cancer genes than previously anticipated.
Assuntos
Genes Neoplásicos/genética , Genoma Humano/genética , Genômica , Mutação/genética , Neoplasias/genética , Sequência de Aminoácidos , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Quinases/química , Proteínas Quinases/genéticaRESUMO
The protein-kinase family is the most frequently mutated gene family found in human cancer and faulty kinase enzymes are being investigated as promising targets for the design of antitumour therapies. We have sequenced the gene encoding the transmembrane protein tyrosine kinase ERBB2 (also known as HER2 or Neu) from 120 primary lung tumours and identified 4% that have mutations within the kinase domain; in the adenocarcinoma subtype of lung cancer, 10% of cases had mutations. ERBB2 inhibitors, which have so far proved to be ineffective in treating lung cancer, should now be clinically re-evaluated in the specific subset of patients with lung cancer whose tumours carry ERBB2 mutations.
Assuntos
Neoplasias Pulmonares/genética , Mutação/genética , Receptor ErbB-2/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA , Ativação Enzimática , Receptores ErbB/química , Receptores ErbB/genética , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/genética , Estrutura Terciária de Proteína , Quinazolinas/uso terapêutico , Receptor ErbB-2/química , Receptor ErbB-2/metabolismoRESUMO
We have previously demonstrated haploinsufficiency of the ribosomal gene RPS14, which is required for the maturation of 40S ribosomal subunits and maps to the commonly deleted region, in the 5q- syndrome. Patients with Diamond-Blackfan anaemia (DBA) show haploinsufficiency of the closely related ribosomal protein RPS19, and show a consequent downregulation of multiple ribosomal- and translation-related genes. By analogy with DBA, we have investigated the expression profiles of a large group of ribosomal- and translation-related genes in the CD34(+) cells of 15 myelodysplastic syndrome (MDS) patients with 5q- syndrome, 18 MDS patients with refractory anaemia (RA) and a normal karyotype, and 17 healthy controls. In this three-way comparison, 55 of 579 ribosomal- and translation-related probe sets were found to be significantly differentially expressed, with approximately 90% of these showing lower expression levels in the 5q- syndrome patient group. Using hierarchical clustering, patients with the 5q- syndrome could be separated both from other patients with RA and healthy controls solely on the basis of the deregulated expression of ribosomal- and translation-related genes. Patients with the 5q- syndrome have a defect in the expression of genes involved in ribosome biogenesis and in the control of translation, suggesting that the 5q- syndrome represents a disorder of aberrant ribosome biogenesis.
Assuntos
Cromossomos Humanos Par 5/genética , Síndromes Mielodisplásicas/genética , Biossíntese de Proteínas/genética , Proteínas Ribossômicas/genética , Deleção Cromossômica , Regulação para Baixo , Haploidia , Humanos , CariotipagemRESUMO
Malignant gliomas have a very poor prognosis. The current standard of care for these cancers consists of extended adjuvant treatment with the alkylating agent temozolomide after surgical resection and radiotherapy. Although a statistically significant increase in survival has been reported with this regimen, nearly all gliomas recur and become insensitive to further treatment with this class of agents. We sequenced 500 kb of genomic DNA corresponding to the kinase domains of 518 protein kinases in each of nine gliomas. Large numbers of somatic mutations were observed in two gliomas recurrent after alkylating agent treatment. The pattern of mutations in these cases showed strong similarity to that induced by alkylating agents in experimental systems. Further investigation revealed inactivating somatic mutations of the mismatch repair gene MSH6 in each case. We propose that inactivating somatic mutations of MSH6 confer resistance to alkylating agents in gliomas in vivo and concurrently unleash accelerated mutagenesis in resistant clones as a consequence of continued exposure to alkylating agents in the presence of defective mismatch repair. The evidence therefore suggests that when MSH6 is inactivated in gliomas, alkylating agents convert from induction of tumor cell death to promotion of neoplastic progression. These observations highlight the potential of large scale sequencing for revealing and elucidating mutagenic processes operative in individual human cancers.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/genética , Proteínas de Ligação a DNA/genética , Dacarbazina/análogos & derivados , Glioma/genética , Mutação , Recidiva Local de Neoplasia/genética , Idoso , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Dacarbazina/uso terapêutico , Feminino , Glioma/tratamento farmacológico , Glioma/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/enzimologia , Proteínas Quinases/genética , TemozolomidaRESUMO
Protein kinases are frequently mutated in human cancer and inhibitors of mutant protein kinases have proven to be effective anticancer drugs. We screened the coding sequences of 518 protein kinases (approximately 1.3 Mb of DNA per sample) for somatic mutations in 26 primary lung neoplasms and seven lung cancer cell lines. One hundred eighty-eight somatic mutations were detected in 141 genes. Of these, 35 were synonymous (silent) changes. This result indicates that most of the 188 mutations were "passenger" mutations that are not causally implicated in oncogenesis. However, an excess of approximately 40 nonsynonymous substitutions compared with that expected by chance (P = 0.07) suggests that some nonsynonymous mutations have been selected and are contributing to oncogenesis. There was considerable variation between individual lung cancers in the number of mutations observed and no mutations were found in lung carcinoids. The mutational spectra of most lung cancers were characterized by a high proportion of C:G > A:T transversions, compatible with the mutagenic effects of tobacco carcinogens. However, one neuroendocrine cancer cell line had a distinctive mutational spectrum reminiscent of UV-induced DNA damage. The results suggest that several mutated protein kinases may be contributing to lung cancer development, but that mutations in each one are infrequent.
Assuntos
Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Mutação , Proteínas Quinases/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Tumor Carcinoide/enzimologia , Tumor Carcinoide/genética , Carcinoma de Células Grandes/enzimologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Análise Mutacional de DNA , HumanosRESUMO
The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacologically and at the molecular level more extensively than any other set of cell lines. However, no systematic mutation analysis of genes causally implicated in oncogenesis has been reported. This study reports the sequence analysis of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examined, with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacologic and molecular profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.
Assuntos
Linhagem Celular Tumoral , Genes Neoplásicos , Mutação , Análise Mutacional de DNA , Éxons , Deleção de Genes , Perfilação da Expressão Gênica , Homozigoto , Humanos , Sítios de Splice de RNAAssuntos
Anemia Refratária/genética , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Regulação da Expressão Gênica , Proteínas Nucleares/biossíntese , Deleção de Sequência , Anemia Refratária com Excesso de Blastos/genética , Cromossomos Humanos Par 5/ultraestrutura , Progressão da Doença , Regulação para Baixo , Instabilidade Genômica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , NucleofosminaRESUMO
PURPOSE: The diagnosis of patients with myelodysplastic syndromes (MDS) is largely dependent on morphologic examination of bone marrow aspirates. Several criteria that form the basis of the classifications and scoring systems most commonly used in clinical practice are affected by operator-dependent variation. To identify standardized molecular markers that would allow prediction of prognosis, we have used gene expression profiling (GEP) data on CD34+ cells from patients with MDS to determine the relationship between gene expression levels and prognosis. PATIENTS AND METHODS: GEP data on CD34+ cells from 125 patients with MDS with a minimum 12-month follow-up since date of bone marrow sample collection were included in this study. Supervised principal components and lasso penalized Cox proportional hazards regression (Coxnet) were used for the analysis. RESULTS: We identified several genes, the expression of which was significantly associated with survival of patients with MDS, including LEF1, CDH1, WT1, and MN1. The Coxnet predictor, based on expression data on 20 genes, outperformed other predictors, including one that additionally used clinical information. Our Coxnet gene signature based on CD34+ cells significantly identified a separation of patients with good or bad prognosis in an independent GEP data set based on unsorted bone marrow mononuclear cells, demonstrating that our signature is robust and may be applicable to bone marrow cells without the need to isolate CD34+ cells. CONCLUSION: We present a new, valuable GEP-based signature for assessing prognosis in MDS. GEP-based signatures correlating with clinical outcome may significantly contribute to a refined risk classification of MDS.
Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Síndromes Mielodisplásicas/diagnóstico , Células-Tronco Neoplásicas/metabolismo , Idoso , Antígenos CD34/metabolismo , Feminino , Seguimentos , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Síndromes Mielodisplásicas/terapia , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Prognóstico , Taxa de SobrevidaRESUMO
For decades, cytogenetic studies have demonstrated that somatically acquired structural rearrangements of the genome are a common feature of most classes of human cancer. However, the characteristics of these rearrangements at sequence-level resolution have thus far been subject to very limited description. One process that is dependent upon somatic genome rearrangement is gene amplification, a mechanism often exploited by cancer cells to increase copy number and hence expression of dominantly acting cancer genes. The mechanisms underlying gene amplification are complex but must involve chromosome breakage and rejoining. We sequenced 133 different genomic rearrangements identified within four cancer amplicons involving the frequently amplified cancer genes MYC, MYCN, and ERBB2. The observed architectures of rearrangement were diverse and highly distinctive, with evidence for sister chromatid breakage-fusion-bridge cycles, formation and reinsertion of double minutes, and the presence of bizarre clusters of small genomic fragments. There were characteristic features of sequences at the breakage-fusion junctions, indicating roles for nonhomologous end joining and homologous recombination-mediated repair mechanisms together with nontemplated DNA synthesis. Evidence was also found for sequence-dependent variation in susceptibility of the genome to somatic rearrangement. The results therefore provide insights into the DNA breakage and repair processes operative in somatic genome rearrangement and illustrate how the evolutionary histories of individual cancers can be reconstructed from large-scale cancer genome sequencing.
Assuntos
Dano ao DNA , Reparo do DNA , DNA de Neoplasias , Rearranjo Gênico , Genoma Humano , Neoplasias/genética , Pareamento de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Aberrações Cromossômicas , Cromossomos Artificiais Bacterianos , Cromossomos Humanos , DNA de Neoplasias/biossíntese , Feminino , Dosagem de Genes , Variação Genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Técnicas de Amplificação de Ácido Nucleico , Recombinação Genética , Cariotipagem EspectralRESUMO
We have identified one frameshift mutation, one splice-site mutation, and two missense mutations in highly conserved residues in ZDHHC9 at Xq26.1 in 4 of 250 families with X-linked mental retardation (XLMR). In three of the families, the mental retardation phenotype is associated with a Marfanoid habitus, although none of the affected individuals meets the Ghent criteria for Marfan syndrome. ZDHHC9 is a palmitoyltransferase that catalyzes the posttranslational modification of NRAS and HRAS. The degree of palmitoylation determines the temporal and spatial location of these proteins in the plasma membrane and Golgi complex. The finding of mutations in ZDHHC9 suggests that alterations in the concentrations and cellular distribution of target proteins are sufficient to cause disease. This is the first XLMR gene to be reported that encodes a posttranslational modification enzyme, palmitoyltransferase. Furthermore, now that the first palmitoyltransferase that causes mental retardation has been identified, defects in other palmitoylation transferases become good candidates for causing other mental retardation syndromes.
Assuntos
Aciltransferases/genética , Síndrome de Marfan/complicações , Síndrome de Marfan/genética , Deficiência Intelectual Ligada ao Cromossomo X/complicações , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação , Aciltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , Feminino , Humanos , Masculino , Síndrome de Marfan/enzimologia , Deficiência Intelectual Ligada ao Cromossomo X/enzimologia , Dados de Sequência Molecular , Linhagem , Fenótipo , Homologia de Sequência de Aminoácidos , Proteínas ras/metabolismoRESUMO
We have identified three truncating, two splice-site, and three missense variants at conserved amino acids in the CUL4B gene on Xq24 in 8 of 250 families with X-linked mental retardation (XLMR). During affected subjects' adolescence, a syndrome emerged with delayed puberty, hypogonadism, relative macrocephaly, moderate short stature, central obesity, unprovoked aggressive outbursts, fine intention tremor, pes cavus, and abnormalities of the toes. This syndrome was first described by Cazebas et al., in a family that was included in our study and that carried a CUL4B missense variant. CUL4B is a ubiquitin E3 ligase subunit implicated in the regulation of several biological processes, and CUL4B is the first XLMR gene that encodes an E3 ubiquitin ligase. The relatively high frequency of CUL4B mutations in this series indicates that it is one of the most commonly mutated genes underlying XLMR and suggests that its introduction into clinical diagnostics should be a high priority.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Culina/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação , Ubiquitina-Proteína Ligases/genética , Agressão , Sequência de Aminoácidos , Criança , Pré-Escolar , Deformidades do Pé/genética , Cabeça/anormalidades , Humanos , Hipogonadismo/genética , Masculino , Dados de Sequência Molecular , Obesidade/genética , Subunidades Proteicas/genética , Convulsões/genética , Tremor/genéticaRESUMO
In the course of systematic screening of the X-chromosome coding sequences in 250 families with nonsyndromic X-linked mental retardation (XLMR), two families were identified with truncating mutations in BRWD3, a gene encoding a bromodomain and WD-repeat domain-containing protein. In both families, the mutation segregates with the phenotype in affected males. Affected males have macrocephaly with a prominent forehead, large cupped ears, and mild-to-moderate intellectual disability. No truncating variants were found in 520 control X chromosomes. BRWD3 is therefore a new gene implicated in the etiology of XLMR associated with macrocephaly and may cause disease by altering intracellular signaling pathways affecting cellular proliferation.
Assuntos
Anormalidades Múltiplas/genética , Cabeça/anormalidades , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação , Fatores de Transcrição/genética , Humanos , Masculino , Linhagem , Alinhamento de SequênciaRESUMO
In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.
Assuntos
Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação , Subunidades sigma do Complexo de Proteínas Adaptadoras/metabolismo , Adulto , Criança , Endossomos/metabolismo , Feminino , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Deficiência Intelectual Ligada ao Cromossomo X/psicologia , LinhagemRESUMO
The protein kinase gene family is the most frequently mutated in human cancer. Previous work has documented activating mutations in the KIT receptor tyrosine kinase in testicular germ-cell tumors (TGCT). To investigate further the potential role of mutated protein kinases in the development of TGCT and to characterize the prevalence and patterns of point mutations in these tumors, we have sequenced the coding exons and splice junctions of the annotated protein kinase family of 518 genes in a series of seven seminomas and six nonseminomas. Our results show a remarkably low mutation frequency, with only a single somatic point mutation, a K277E mutation in the STK10 gene, being identified in a total of more than 15 megabases of sequence analyzed. Sequencing of STK10 in an additional 40 TGCTs revealed no further mutations. Comparative genomic hybridization and LOH analysis using SNP arrays demonstrated that the 13 TGCTs mutationally screened through the 518 protein kinase genes were uniformly aneuploid with consistent chromosomal gains on 12p, 8q, 7, and X and losses on 13q, 18q, 11q, and 4q. Our results do not provide evidence for a mutated protein kinase implicated in the development of TGCT other than KIT. Moreover, they demonstrate that the general prevalence of point mutations in TGCT is low, in contrast to the high frequency of copy number changes.