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Tumor genomic profiling (TGP) has the potential to identify germline variants in addition to its primary use of informing cancer treatment based on genetic alterations within the tumor. However, there are no formal consensus guidelines to identify patients who would be eligible for genetic counseling (GC) and germline testing (GT) testing in patients undergoing TGP. The purpose of this study is to describe an institutionally developed Germline Review Protocol (GRP) to evaluate adult cancer patient cases already undergoing TGP to determine GC referral eligibility. We report on our retrospective experience implementing this protocol into practice wherein 172 patients out of 638 patients reviewed (27%) were recommended for a GC referral over a 17-month time period. Of those 172 patients recommended for a GC referral, only 34 patients (20%) completed GC and GT. Among patients who received GT, 15 (44%) were positive for at least one pathogenic or likely pathogenic (P/LP) variant, seven patients (21%) were negative and 12 patients (35%) had at least 1 variant of uncertain significance (VUS). The primary reason GC and GT was not completed was because the patient moved to hospice care or was deceased. This is one of the first studies outlining the process and results of a formalized institutional protocol to facilitate patient referrals for GC and GT based on TGP results.
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Testes Genéticos , Neoplasias , Adulto , Predisposição Genética para Doença , Genômica , Células Germinativas , Humanos , Neoplasias/genética , Estudos RetrospectivosRESUMO
Screening guidelines in patients with inherited endocrine syndromes and others at high risk for developing endocrine cancers have significant implications as early tumor detection is associated with improved outcomes. Given the unique challenges in diagnosis and treatment, patients at high risk for developing endocrine cancers require multidisciplinary care. The complexity of decision making and rarity of these syndromes support referral to high-volume centers with the experience and knowledge to treat these at-risk patients.
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Neoplasias das Glândulas Endócrinas/diagnóstico , Neoplasia Endócrina Múltipla/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Detecção Precoce de Câncer/métodos , Humanos , Neoplasias Pancreáticas/diagnóstico , Guias de Prática Clínica como Assunto , Neoplasias da Glândula Tireoide/diagnósticoRESUMO
BACKGROUND: Current guidelines recommend genetic testing for all patients with pancreatic cancer (PC). METHODS: Patients with localized PC who received neoadjuvant therapy between 2009 and 2018 were identified. Genetic consultation (including personal and family history of cancer), genetic testing, and variant data were abstracted. RESULTS: Of 510 patients identified, 163 (32%) underwent genetic counseling and genetic testing was performed in 127 (25%). Patients who underwent genetic testing were younger (median age: 63 vs. 67, p = 0.01). Multi-gene testing was performed in 114 (90%) of 127 patients, targeted gene testing was performed in 8 (6%), and not specified in 5 (4%). Of 127 patients who underwent genetic testing, 20 (16%) had pathogenic (P)/likely pathogenic (LP) variants, observed in ATM (n = 7/105,7%), CHEK2 (n = 3/98, 3%), BRCA1 (n = 2/117, 2%), BRCA2 (n = 2/122, 2%), PALB2 (n = 1/115, 1%), MUTYH (n = 1/98, 1%), CDKN2A (n = 1/94, 1%), STK11 (n = 1/97, 1%), NBN (n = 1/98, 1%), and MSH6 (n = 1/97, 1%). Of 20 patients with either a P/LP variant, nine (45%) had a prior cancer, three (15%) had a first-degree relative with PC, and six (30%) had an any-degree relative with PC. CONCLUSION: Pathogenic/likely pathogenic variants were identified in 16% of patients who underwent genetic testing, 60% of which occurred in the homologous recombination pathway.
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Mutação em Linhagem Germinativa , Neoplasias Pancreáticas , Predisposição Genética para Doença , Testes Genéticos , Células Germinativas , Humanos , Pessoa de Meia-Idade , Neoplasias Pancreáticas/genéticaRESUMO
OBJECTIVE: The National Comprehensive Cancer Network recommends all women with ovarian cancer be offered genetic testing. Despite a decade of endorsement, many oncology practitioners have yet to make this a part of routine practice. Referral to genetic counseling and completion of genetic testing among patients at substantial risk of germline mutations are significantly lacking, adversely affecting patient care and squandering an opportunity to maximize cancer prevention efforts. This project determined the impact and feasibility of implementing a basic model for universal referral to genetic counseling and completion of genetic testing in women with a diagnosis of ovarian cancer in an academic gynecology oncology practice with access to electronic health records (EHRs). METHODS: Patients diagnosed with ovarian cancer from January 2008 to November 2013 were retrospectively reviewed to determine the baseline referral rate for genetic counseling and testing completion in our practice. Implementation of a process change model combining provider training, patient education, enhanced electronic health record documentation and improved patient appointment scheduling strategies were implemented. We then prospectively collected data on all newly diagnosed ovarian cancer patients that had not already undergone genetic testing presenting from December 1, 2013 to November 30, 2016. RESULTS: Genetic referral rates, genetic counseling and testing completion rates were markedly improved. Pre-implementation our genetic testing rate was 27% and post implementation our testing rate was 82% (p-value≤0.001). CONCLUSIONS: Low cost interventions that target education of both providers and patients regarding the importance of genetic testing along with utilization of the EHR and streamlined patient appointment services can significantly increase rates of genetic testing completion.
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Testes Genéticos/métodos , Testes Genéticos/normas , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário , Feminino , Aconselhamento Genético/métodos , Aconselhamento Genético/normas , Humanos , Pessoa de Meia-Idade , Melhoria de Qualidade , Adulto JovemRESUMO
Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.
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Janus Quinase 2/metabolismo , Leucemia Mielomonocítica Juvenil/etiologia , Transtornos Mieloproliferativos/etiologia , Neurofibromina 1/deficiência , Fator de Transcrição STAT5/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Leucemia Mieloide/etiologia , Leucemia Mieloide/genética , Leucemia Mielomonocítica Juvenil/genética , Camundongos , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
Juvenile Myelomonocytic Leukemia (JMML) is a relentlessly progressive myeloproliferative/myelodysplastic (MPD/MDS) hematopoietic disorder more common in patients with any one of at least three distinct genetic lesions, specifically NF1 gene loss and PTPN11 and NRAS mutations. NF1 and PTPN11 are molecular lesions associated with Neurofibromatosis Syndrome Type I (NF1 Syndrome) and Noonan's Syndrome, respectively. The occurrence of JMML is rare; even among those predisposed with these syndromes to development of disease, and secondary genetic events likely contribute to the development and progression of disease. In NF1 syndrome, loss of p53 function is a common event in solid tumors, but uncommon in JMML, suggesting that the p53 pathway may be modified by other events in this hematopoietic disorder. The work presented here investigates the possible role of the p19(Arf) (p19) tumor suppressor in development of MPD associated with Nf1 gene loss in mice. We find that Nf1 mutant hematopoietic cells with loss of p19 develop accelerated hematopoietic disease similar to acute leukemia with a variable phenotype. This suggests that p19 may play a role in development of JMML and evaluation of the human p19 homolog (p14(ARF)) in JMML may be informative.
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Inibidor p16 de Quinase Dependente de Ciclina/genética , Leucemia Mielomonocítica Juvenil/genética , Neurofibromina 1/genética , Doença Aguda , Animais , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Leucemia Mielomonocítica Juvenil/metabolismo , Leucemia Mielomonocítica Juvenil/patologia , Camundongos , Camundongos Mutantes , Neurofibromatose 1/genética , Neurofibromatose 1/metabolismo , Neurofibromatose 1/patologia , Neurofibromina 1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Historically, germline testing of patients with pancreatic cancer was performed selectively in patients with a strong family history of cancer. Current guidelines recommend universal testing because some patients may have actionable germline pathogenic variants without family history. METHODS: We conducted a cost-effectiveness analysis using a decision-tree model to compare universal versus selective testing strategies for patients with pancreatic cancer. Costs, probabilities, and overall survival were estimated from the published literature and institutional data. One-way and probabilistic sensitivity analyses explored model uncertainty. RESULTS: Universal germline genetic testing had an incremental cost of $310 with an increase of 0.003 life-years. The incremental cost-effectiveness ratio was $121,924/life-years. Parameters which were most impactful (sensitivity analysis) included the median overall survival of patients with advanced disease treated with personalized therapy, cost of personalized therapy for advanced disease, and the probability of receiving personalized therapy in advanced disease. A strategy of selective testing was more cost-effective in 59% of iterations when the willingness-to-pay threshold was set to $100,000/life-years. CONCLUSION: Our model suggested that selective germline testing of patients with newly diagnosed pancreatic cancer is more cost-effective than universal testing. Additional research is needed to explore the impact of cascade testing of relatives on cost-effectiveness.
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Análise Custo-Benefício , Testes Genéticos/economia , Testes Genéticos/normas , Células Germinativas/metabolismo , Neoplasias Pancreáticas/epidemiologia , Análise Custo-Benefício/métodos , Técnicas de Apoio para a Decisão , Árvores de Decisões , Gerenciamento Clínico , Testes Genéticos/métodos , Humanos , Oncologia/economia , Oncologia/métodos , Modelos Teóricos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Medicina de Precisão , Vigilância em Saúde PúblicaRESUMO
Inherited syndromes are important to recognize in the setting of a pancreatic neuroendocrine tumor (PNET) as there are significant implications for the patient's medical management and opportunity for early detection of subsequent manifestations. Although most PNETs are sporadic, approximately 10% are due to an inherited syndrome, which include multiple endocrine neoplasia type 1 (MEN1), multiple endocrine neoplasia type 4 (MEN4), von Hippel-Lindau disease (VHL), neurofibromatosis type 1 (NF1), and tuberous sclerosis complex (TSC). The general hallmarks of a hereditary endocrine neoplasia predisposition syndrome include any one of the following: multiple primary tumors (in the same or different organs), rare tumors (prevalence of less than 1 in 1,000 people in the general population), earlier age of diagnosis (usually under the age of 40), characteristic pattern of disease in the individual or family (phenotype and inheritance pattern). These syndromes are monogenic (due to a single gene disorder), highly penetrant (with all carriers of the disease exhibiting at least part of the phenotype) and can display variable expressivity (where affected individuals may have different presentations and features of the disease). A thoughtful approach to management is required, even if the presenting symptom is resolved, as these syndromes often involve multi-organ disease with a lifelong risk for tumor development. Additionally, the natural history of tumors in the setting of a hereditary condition may be different than would be expected in a sporadic form of the disease. For example, in some circumstances the risk of metastatic disease is lower, and therefor longer observation is the preferred approach over early surgical intervention. The unique aspects to management, challenges in hereditary disease recognition and accurate diagnosis, and rarity of these syndromes are all reasons to support referral to high-volume centers with the experience and knowledge to treat patients with hereditary endocrine neoplasia syndromes.
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The Genetic Counseling Working Group from the 16th International Workshop on Multiple Endocrine Neoplasia (MEN 2019) convened to discuss contemporary challenges and opportunities in the area of genetic counseling for individuals and families affected by hereditary endocrine neoplasia syndromes. As healthcare professionals with multidisciplinary training in human genetics, risk assessment, patient education, psychosocial counseling, and research methodology, genetic counselors bring a unique perspective to working toward addressing these challenges and identifying their subsequent opportunities. This Working Group focused on the following broad areas: (1) genetic counseling resources for endocrine neoplasias, (2) candidate gene discovery, (3) implications of increasingly sensitive and expansive genetic testing technologies for both the germline and the tumors, and (4) situating clinical diagnoses for hereditary endocrine neoplasia syndromes in the context of present-day knowledge.
Assuntos
Neoplasias das Glândulas Endócrinas/genética , Aconselhamento Genético/métodos , Predisposição Genética para Doença/genética , Neoplasia Endócrina Múltipla/genética , HumanosRESUMO
BACKGROUND: Recent studies documented a shortage of direct patient care (DPC) genetic counselors in the United States. We aimed to survey genetic counselor members of the Wisconsin Genetic Counselors Association (WIGCA) to determine if the supply and demand was met within the state and where access to services can improve. METHODS: An email invitation was sent to all genetic counselor members of the WIGCA with a link to a confidential online survey. Survey questions addressed the workforce composition, elements that impact services, and professional satisfaction of practicing genetic counselors. RESULTS: The Wisconsin workforce currently has half of the projected need for full-time DPC genetic counselors. One-third of genetic counselors reported changing from direct to non-direct patient care positions. In-person services are concentrated within Milwaukee and Madison. Appointment wait times are decreased when patients meet with a genetic counselor only, and half of the genetic counselors reported moderate to high stress levels. DISCUSSION/CONCLUSION: A shortage of DPC genetic counselors in Wisconsin is confirmed due to the total full-time effort in direct patient care. Data provided here can be used to identify targets for increasing the number of DPC genetic counselors, maximizing time spent on patient care, and improving access.
Assuntos
Conselheiros , Aconselhamento Genético , Humanos , Assistência ao Paciente , Estados Unidos , Wisconsin , Recursos HumanosRESUMO
Germline variants in tumor suppressor genes (TSGs) can result in RNA mis-splicing and predisposition to cancer. However, identification of variants that impact splicing remains a challenge, contributing to a substantial proportion of patients with suspected hereditary cancer syndromes remaining without a molecular diagnosis. To address this, we used capture RNA-sequencing (RNA-seq) to generate a splicing profile of 18 TSGs (APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53) in 345 whole-blood samples from healthy donors. We subsequently demonstrated that this approach can detect mis-splicing by comparing splicing profiles from the control dataset to profiles generated from whole blood of individuals previously identified with pathogenic germline splicing variants in these genes. To assess the utility of our TSG splicing profile to prospectively identify pathogenic splicing variants, we performed concurrent capture DNA and RNA-seq in a cohort of 1000 patients with suspected hereditary cancer syndromes. This approach improved the diagnostic yield in this cohort, resulting in a 9.1% relative increase in the detection of pathogenic variants, demonstrating the utility of performing simultaneous DNA and RNA genetic testing in a clinical context.
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Previous studies of the Sleeping Beauty (SB) transposon system, as an insertional mutagen in the germline of mice, have used reverse genetic approaches. These studies have led to its proposed use for regional saturation mutagenesis by taking a forward-genetic approach. Thus, we used the SB system to mutate a region of mouse Chromosome 11 in a forward-genetic screen for recessive lethal and viable phenotypes. This work represents the first reported use of an insertional mutagen in a phenotype-driven approach. The phenotype-driven approach was successful in both recovering visible and behavioral mutants, including dominant limb and recessive behavioral phenotypes, and allowing for the rapid identification of candidate gene disruptions. In addition, a high frequency of recessive lethal mutations arose as a result of genomic rearrangements near the site of transposition, resulting from transposon mobilization. The results suggest that the SB system could be used in a forward-genetic approach to recover interesting phenotypes, but that local chromosomal rearrangements should be anticipated in conjunction with single-copy, local transposon insertions in chromosomes. Additionally, these mice may serve as a model for chromosome rearrangements caused by transposable elements during the evolution of vertebrate genomes.
Assuntos
Elementos de DNA Transponíveis/fisiologia , Mutagênese Insercional , Mutação , Animais , Animais Recém-Nascidos , Aberrações Cromossômicas , Cromossomos , Cromossomos de Mamíferos , Cruzamentos Genéticos , DNA Concatenado/química , Genes Dominantes , Genes Recessivos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Linhagem , Fenótipo , Sindactilia/genética , Transposases/genéticaRESUMO
OBJECTIVE: To report a patient with the hyperparathyroidism-jaw tumor syndrome (HPT-JT) who was found to have a rare ovarian tumor (granulosa cell tumor [GCT]). HPT-JT is caused by pathogenic variants in the CDC73 gene and results in primary hyperparathyroidism (PHPT), benign fibro-osseous jaw tumors, benign or malignant renal tumors and cysts, and benign or malignant uterine tumors. We believe this is the first reported case of HPT-JT and GCT. METHODS: The patient was a 31-year-old woman with abdominal pain who was found to have adult GCT. Her history was significant for a single gland parathyroidectomy at age 23 for PHPT. Her mother also had PHPT with 1-gland removal, as well as a history of renal cysts. Because of the personal and familial history of PHPT, she underwent germline sequencing of genes associated with PHPT including CASR, CDC73, CDKN1B, MEN1 and RET. RESULTS: Genetic testing revealed a CDC73 gene pathogenic variant (c.687_688dupAG) which creates a premature translational stop signal causing loss-of-function. CONCLUSION: We report a case of ovarian GCT in a young patient with primary hyperparathyroidism and a CDC73 gene mutation. Ovarian granulosa cell tumor may be another CDC73-related tumor.
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BACKGROUND: Mutations in BRCA1 or BRCA2 genes results in an elevated risk for developing both breast and ovarian cancers over the lifetime of affected carriers. General surgeons may be faced with questions about surgical risk reduction and survival benefit of prophylactic surgery. METHODS: A systematic literature review was performed using the electronic databases PubMed, OVID MEDLINE, and Scopus comparing prophylactic surgery vs observation with respect to breast and ovarian cancer risk reduction and mortality in BRCA mutation carriers. RESULTS: Bilateral risk-reducing mastectomy provides a 90% to 95% risk reduction in BRCA mutation carriers, although the data do not demonstrate improved mortality. The reduction in ovarian and breast cancer risks using risk-reducing bilateral salpingo-oophorectomy has translated to improvement in survival. CONCLUSIONS: Clinical management of patients at increased risk for breast cancer requires consideration of risk, patient preference, and quality of life.
Assuntos
Neoplasias da Mama , Genes BRCA1 , Genes BRCA2 , Heterozigoto , Neoplasias Ovarianas , Mastectomia Profilática , Procedimentos Cirúrgicos Profiláticos , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/prevenção & controle , Feminino , Humanos , Mutação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/prevenção & controle , Ovariectomia , SalpingectomiaRESUMO
Next generation sequencing (NGS) technology is rapidly being implemented into clinical practice. Qualitative research was performed to gain an improved understanding of the landscape surrounding the use of NGS in cancer genetics. A focus group was conducted at the Wisconsin Cancer Risk Programs Network biannual meeting. Free flowing discussion with occasional open-ended questions provided insights into the use of NGS. 19 genetic counselors and medical professionals participated. Three major themes were identified with respect to NGS and its use in cancer genetics: knowledge gaps, the evolving clinician role, and uncertain utility. Several corresponding subthemes were identified. With respect to knowledge gaps, participants expressed concern regarding unexpected results and variants of unknown significance, lack of data about NGS findings, absence of standardization regarding use of NGS and guidelines for interpretation, and discomfort with new technology. Regarding the evolving clinician role, necessary changes to the roles of genetic counselors and physicians were noted, as was the resultant impact on care received by patients and their families. Finally, the clinical and economic utility of NGS was questioned. While a shift from traditional Sanger sequencing to NGS is occurring in molecular genetic testing for disease susceptibility, there are several obstacles that need to be overcome before widespread adoption of this technology can occur. Furthermore, key aspects of NGS and it utility remain unexplored. Continued investigation into these subjects is necessary before this technology will consistently be of benefit to patients and their families.
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Atitude do Pessoal de Saúde , Biomarcadores Tumorais/genética , Testes Genéticos/estatística & dados numéricos , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Aconselhamento Genético , Predisposição Genética para Doença , Testes Genéticos/métodos , Genoma Humano , Humanos , Síndromes Neoplásicas Hereditárias/psicologia , Medição de RiscoRESUMO
Neurofibromatosis type 1 (NF1) syndrome is caused by germline mutations in the NF1 tumor suppressor, which encodes neurofibromin, a GTPase activating protein for Ras. Children with NF1 are predisposed to juvenile myelomonocytic leukemia (JMML) and lethally irradiated mice given transplants with homozygous Nf1 mutant (Nf1-/-) hematopoietic stem cells develop a fatal myeloproliferative disorder (MPD) that models JMML. We investigated the requirement for signaling through the GM-CSF receptor to initiate and sustain this MPD by generating Nf1 mutant hematopoietic cells lacking the common beta chain (Beta c) of the GM-CSF receptor. Mice reconstituted with Nf1-/-, beta c-/- stem cells did not develop evidence of MPD despite the presence of increased number of immature hematopoietic progenitors in the bone marrow. Interestingly, when the Mx1-Cre transgene was used to inactivate a conditional Nf1 mutant allele in hematopoietic cells, concomitant loss of beta c-/- reduced the severity of the MPD, but did not abrogate it. Whereas inhibiting GM-CSF signaling may be of therapeutic benefit in JMML, our data also demonstrate aberrant proliferation of Nf1-/-myeloid progenitors that is independent of signaling through the GM-CSF receptor.
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Leucemia Mielomonocítica Crônica/etiologia , Transtornos Mieloproliferativos/etiologia , Neurofibromatose 1/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Animais , Modelos Animais de Doenças , Transplante de Células-Tronco Hematopoéticas , Leucemia Mielomonocítica Crônica/prevenção & controle , Camundongos , Camundongos Mutantes , Transtornos Mieloproliferativos/prevenção & controle , Transdução de SinaisRESUMO
Sleeping Beauty (SB) is a DNA transposon capable of mediating gene insertion and long-term expression in vertebrate cells when co-delivered with a source of transposase. In all previous reports of SB-mediated gene insertion in somatic cells, the transposase component has been provided by expression of a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to transposon re-mobilization and reintegration. We addressed this potential problem by supplying the transposase-encoding molecule in the form of mRNA. We show that transposase-encoding mRNA can effectively mediate transposition in vitro in HT1080 cells and in vivo in mouse liver following co-delivery with a recoverable transposon or with a luciferase transposon. We conclude that in vitro-transcribed mRNA can be used as an effective source of transposase for SB-mediated transposition in mammalian cells and tissues.
Assuntos
Elementos de DNA Transponíveis , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , RNA Mensageiro/genética , Transposases/genética , Animais , Linhagem Celular Tumoral , Humanos , Fígado/enzimologia , Dados de Sequência Molecular , Mutagênese Insercional , Especificidade de Órgãos/genética , RNA Mensageiro/administração & dosagem , Recombinação GenéticaRESUMO
The Sleeping Beauty transposon system (SB) has been shown to mediate nonviral integration of expression constructs resulting in long-term gene expression in several mammalian targets. Often, however, it is difficult to discern long-term expression resulting from transposition vs nonhomologous chromosomal recombination or maintenance of plasmid DNA in an extrachromosomal form. We have designed a system to silence expression from nontransposed sequences, making it possible to determine more specifically the amount of expression resulting from transposition. A transposon plasmid, pT2F/Cage (carrying a murine erythropoietin (Epo) gene transcriptionally regulated by the ubiquitously expressed CAGS promoter), was engineered to contain LoxP sites positioned so as to interrupt expression upon Cre-mediated recombination. Upon transposition these sites become segregated, thus protecting the expression construct from Cre-mediated recombination and subsequent silencing. Interferon-inducible Mx1Cre mice were administered pT2F/Cage with or without transposase-encoding plasmid. At 2 to 4 weeks postinjection, in the absence of SB transposase, Cre induction reduced Epo expression to about 1% of that seen in the group that was administered transposase-encoding plasmid, which maintained Epo levels near those of the uninduced groups. Southern hybridization analysis and plasmid rescue of transfected tissue supported the efficient Cre-mediated silencing of nontransposed sequences. These results indicate a substantial level of DNA-mediated expression not associated with transposition, but which can be quantitatively distinguished from transposition by its sensitivity to Cre recombinase. The results also provide additional evidence for the effectiveness of the Sleeping Beauty transposon system as an in vivo DNA-mediated gene transfer strategy for achieving long-term expression.