RESUMEN
The All of Us Research Program (AoURP) is a historic effort to accelerate research and improve healthcare by generating and collating data from one million people in the United States. Participants will have the option to receive results from their genome analysis, including actionable findings in 59 gene-disorder pairs for which disorder-associated variants are recommended for return by the American College of Medical Genetics and Genomics. To ensure consistent reporting across the AoURP, in a prelaunch study the four participating clinical laboratories shared all variant classifications in the 59 genes of interest from their internal databases. Of the 11,813 unique variants classified by at least two of the four laboratories, classifications were concordant with regard to reportability for 99.1% (11,711), with only 0.9% (102) having reportability differences. Through variant reassessment, data sharing, and discussion of rationale, participating laboratories resolved all 102 reportable differences. These approaches will be maintained during routine AoU reporting to ensure continuous classification harmonization and consistent reporting within AoURP.
Asunto(s)
Genoma Humano , Salud Poblacional , Pruebas Genéticas/métodos , Variación Genética , Genoma Humano/genética , Genómica/métodos , Humanos , Estados UnidosRESUMEN
PURPOSE: In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) published consensus standardized guidelines for sequence-level variant classification in Mendelian disorders. To increase accuracy and consistency, the Clinical Genome Resource Familial Hypercholesterolemia (FH) Variant Curation Expert Panel was tasked with optimizing the existing ACMG/AMP framework for disease-specific classification in FH. In this study, we provide consensus recommendations for the most common FH-associated gene, LDLR, where >2300 unique FH-associated variants have been identified. METHODS: The multidisciplinary FH Variant Curation Expert Panel met in person and through frequent emails and conference calls to develop LDLR-specific modifications of ACMG/AMP guidelines. Through iteration, pilot testing, debate, and commentary, consensus among experts was reached. RESULTS: The consensus LDLR variant modifications to existing ACMG/AMP guidelines include (1) alteration of population frequency thresholds, (2) delineation of loss-of-function variant types, (3) functional study criteria specifications, (4) cosegregation criteria specifications, and (5) specific use and thresholds for in silico prediction tools, among others. CONCLUSION: Establishment of these guidelines as the new standard in the clinical laboratory setting will result in a more evidence-based, harmonized method for LDLR variant classification worldwide, thereby improving the care of patients with FH.
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Genoma Humano , Hiperlipoproteinemia Tipo II , Pruebas Genéticas/métodos , Variación Genética/genética , Genoma Humano/genética , Genómica/métodos , Humanos , Hiperlipoproteinemia Tipo II/genéticaRESUMEN
In silico approaches for next-generation sequencing (NGS) data modeling have utility in the clinical laboratory as a tool for clinical assay validation. In silico NGS data can take a variety of forms, including pure simulated data or manipulated data files in which variants are inserted into existing data files. In silico data enable simulation of a range of variants that may be difficult to obtain from a single physical sample. Such data allow laboratories to more accurately test the performance of clinical bioinformatics pipelines without sequencing additional cases. For example, clinical laboratories may use in silico data to simulate low variant allele fraction variants to test the analytical sensitivity of variant calling software or simulate a range of insertion/deletion sizes to determine the performance of insertion/deletion calling software. In this article, the Working Group reviews the different types of in silico data with their strengths and limitations, methods to generate in silico data, and how data can be used in the clinical molecular diagnostic laboratory. Survey data indicate how in silico NGS data are currently being used. Finally, potential applications for which in silico data may become useful in the future are presented.
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Patólogos , Patología Molecular , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biología Computacional/métodos , Programas InformáticosRESUMEN
The relevance of large copy number variants (CNVs) to hereditary disorders has been long recognized, and population sequencing efforts have chronicled many common structural variants (SVs). However, limited data are available on the clinical contribution of rare germline SVs. Here, a detailed characterization of SVs identified using targeted next-generation sequencing was performed. Across 50 genes associated with hereditary cancer and cardiovascular disorders, a minimum of 828 unique SVs were reported, including 584 fully characterized SVs. Almost 40% of CNVs were <5 kb, with one in three deletions impacting a single exon. Additionally, 36 mid-range deletions/duplications (50 to 250 bp), 21 mobile element insertions, 6 inversions, and 27 complex rearrangements were detected. This data set was used to model SV detection in a bioinformatics pipeline solely relying on read depth, which revealed that genome sequencing (30×) allows detection of 71%, a 500× panel only targeting coding regions 53%, and exome sequencing (100×) <20% of characterized SVs. SVs accounted for 14.1% of all unique pathogenic variants, supporting the importance of SVs in hereditary disorders. Robust SV detection requires an ensemble of variant-calling algorithms that utilize sequencing of intronic regions. These algorithms should use distinct data features representative of each class of mutational mechanism, including recombination between two sequences sharing high similarity, covariants inserted between CNV breakpoints, and complex rearrangements containing inverted sequences.
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Rotura Cromosómica , Cromosomas Humanos/genética , Enfermedad/genética , Genoma Humano , Mutación de Línea Germinal , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Intrones , Algoritmos , HumanosRESUMEN
PURPOSE: Genetic tests for the most commonly mutated genes in dilated cardiomyopathy (DCM) can confirm a clinical diagnosis in the proband and inform family management. Presymptomatic family members can be identified, allowing for targeted clinical monitoring to minimize adverse outcomes. However, the marked locus and allelic heterogeneity associated with DCM have made clinical genetic testing challenging. Novel sequencing platforms have now opened up avenues for more comprehensive diagnostic testing while simultaneously decreasing test cost and turn around time. METHODS: By using a custom design based on triplicate resequencing and separate genotyping of known disease-causing variants, we developed the DCM CardioChip for efficient analysis of 19 genes previously implicated in causing DCM. RESULTS: The chip's analytical sensitivity for known and novel substitution variants is 100% and 98%, respectively. In screening 73 previously tested DCM patients who did not carry clinically significant variants in 10 genes, 7 variants of likely clinical significance were identified in the remaining 9 genes included on the chip. Compared with traditional Sanger-based sequencing, test cost and turn around time were reduced by approximately 50%. CONCLUSIONS: The DCM CardioChip is a highly efficient screening test with a projected clinical sensitivity of 26-29%.
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Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/genética , Familia , Femenino , Pruebas Genéticas , Humanos , Mutación , EmbarazoRESUMEN
Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.
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Difosfonatos/química , Difosfonatos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Lípidos/química , Ratones , Ratones Desnudos , Invasividad Neoplásica , Resonancia Magnética Nuclear Biomolecular , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/enzimologíaRESUMEN
Hereditary mixed polyposis syndrome (HMPS) is a hereditary syndrome that is characterized by multiple colon polyps of mixed pathologic subtypes and an increased risk for colorectal cancer. A 40 kb duplication in the 5' regulatory region of the GREM1 gene was recently found to be the causal mutation in a subset of Ashkenazi Jewish families with HMPS. Given this discovery, the GREM1 5' regulatory region is now analyzed on many different multi-gene cancer panels, however the data on duplications distinct from the 40 kb duplication remains minimal. Herein we report a novel 24 kb tandem duplication of the 5' regulatory region of GREM1 in a patient without Ashkenazi Jewish heritage, who had a family history that was concerning for Lynch syndrome and satisfied Amsterdam II criteria. This is only the third reported GREM1 duplication separate from the 40 kb Ashkenazi Jewish duplication, and is the only reported duplication to selectively involve exon 1 of GREM1. This finding supports comprehensive testing of the GREM1 regulatory region in families of all ethnicities with multiple colon polyps or colon cancer, and when Lynch syndrome is suspected.
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Poliposis Adenomatosa del Colon/genética , Duplicación de Gen , Péptidos y Proteínas de Señalización Intercelular/genética , Poliposis Adenomatosa del Colon/diagnóstico , Poliposis Adenomatosa del Colon/patología , Poliposis Adenomatosa del Colon/cirugía , Colon/diagnóstico por imagen , Colon/patología , Colon/cirugía , Colonoscopía , Neoplasias Colorrectales Hereditarias sin Poliposis/diagnóstico , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Femenino , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Humanos , Persona de Mediana Edad , LinajeRESUMEN
Recent advancements in next-generation sequencing have greatly expanded the use of multi-gene panel testing for hereditary cancer risk. Although genetic testing helps guide clinical diagnosis and management, testing recommendations are based on personal and family history of cancer and ethnicity, and many carriers are being missed. Herein, we report the results from 23,179 individuals who were referred for 30-gene next-generation sequencing panel testing for hereditary cancer risk, independent of current testing guidelines-38.7% of individuals would not have met National Comprehensive Cancer Network criteria for genetic testing. We identified a total of 2811 pathogenic variants in 2698 individuals for an overall pathogenic frequency of 11.6% (9.1%, excluding common low-penetrance alleles). Among individuals of Ashkenazi Jewish descent, three-quarters of pathogenic variants were outside of the three common BRCA1 and BRCA2 founder alleles. Across all ethnic groups, pathogenic variants in BRCA1 and BRCA2 occurred most frequently, but the contribution of pathogenic variants in other genes on the panel varied. Finally, we found that 21.7% of individuals with pathogenic variants in genes with well-established genetic testing recommendations did not meet corresponding National Comprehensive Cancer Network criteria. Taken together, the results indicate that more individuals are at genetic risk for hereditary cancer than are identified by current testing guidelines and/or use of single-gene or single-site testing.
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Biomarcadores de Tumor , Pruebas Genéticas , Heterocigoto , Síndromes Neoplásicos Hereditarios/diagnóstico , Síndromes Neoplásicos Hereditarios/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Mutación , Síndromes Neoplásicos Hereditarios/mortalidad , Guías de Práctica Clínica como Asunto , Pronóstico , Adulto JovenRESUMEN
Novel bis-heterocyclic bisphosphonates/phosphonamidates were synthesized utilizing the Pudovick reaction. The employment of Nb(2)O(5) as catalyst was found to increase the yields and purity of the bisbenzoxazaphosphine derivatives (13a-h). Their anticancer activity studies in vitro, on three human tumor cell lines NCI-H460 (lung large cell), MCF-7 (breast adenocarcinoma), and SF-268 (central nervous system glioblastoma), showed that bis-[3-(3-chloro-4-fluorophenyl)-2-oxo-3,4-dihydro-2H-2lambda(5)-benzo[e][1,3,2]oxazaphosphinin-2-yl]arylmethanes (13a-h) and [(4-chlorophenyl)-(hydroxyamidophosphinoyl)-methyl]phosphonic acid (14) exhibited significant anticancer activity.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Hidrocarburos Clorados/síntesis química , Hidrocarburos Clorados/farmacología , Neoplasias/tratamiento farmacológico , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Clorobencenos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Neoplasias/patología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Bioinformatics pipelines are an integral component of next-generation sequencing (NGS). Processing raw sequence data to detect genomic alterations has significant impact on disease management and patient care. Because of the lack of published guidance, there is currently a high degree of variability in how members of the global molecular genetics and pathology community establish and validate bioinformatics pipelines. Improperly developed, validated, and/or monitored pipelines may generate inaccurate results that may have negative consequences for patient care. To address this unmet need, the Association of Molecular Pathology, with organizational representation from the College of American Pathologists and the American Medical Informatics Association, has developed a set of 17 best practice consensus recommendations for the validation of clinical NGS bioinformatics pipelines. Recommendations include practical guidance for laboratories regarding NGS bioinformatics pipeline design, development, and operation, with additional emphasis on the role of a properly trained and qualified molecular professional to achieve optimal NGS testing quality.
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Biología Computacional/normas , Guías como Asunto , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Patología Molecular/normas , Humanos , Laboratorios , Reproducibilidad de los Resultados , Estados UnidosRESUMEN
We investigated three series of sulfonium bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines. The first series consisted of 6 cyclic sulfonium bisphosphonates, the most active species having an (average) IC50 of 89 microM. The second consisted of 10 phenylalkyl and phenylalkoxy bisphosphonates, the most active species having an IC50 of 18 microM. The third series consisted of 17 n-alkyl sulfonium bisphosphonates, the most active species having an IC50 of approximately 240 nM. Three QSAR models showed that the experimental cell growth inhibition results could be well predicted. We also determined the structures of one sulfonium bisphosphonate bound to farnesyl diphosphate synthase, finding that it binds exclusively to the dimethylallyl diphosphate binding site. These results are of interest since they show that sulfonium bisphosphonates can have potent activity against a variety of tumor cell lines, the most active species having IC50 values much lower than conventional nitrogen-containing bisphosphonates.
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Difosfonatos/síntesis química , Sulfonas/síntesis química , Animales , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Difosfonatos/química , Difosfonatos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Geraniltranstransferasa/química , Humanos , Modelos Moleculares , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Sulfonas/química , Sulfonas/farmacología , Trypanosoma brucei brucei/enzimologíaRESUMEN
We synthesized and tested three series of bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines: MCF-7 (breast), NCI-H460 (lung), and SF-268 (CNS). The first series of compounds consisted of 49 nitrogen-containing bisphosphonates, the most active species being a tetrakispivaloyloxymethyl (POM) ester, having an (average) IC(50) of 6.8 microM. The second series of compounds consisted of nine terphenylbisphosphonates, the most active species also being a POM ester, having an IC(50) of 2.2 microM. The third series of compounds consisted of seven halogen or cyanophenylbisphosphonates, the most active species again being a POM ester, having an IC(50) of 500 nM. Taken together, these results are of interest because they show that bisphosphonate esters can have potent activity against a variety of tumor cell lines, with the most active terphenyl- and halophenyl-containing species having IC(50) values approximately 10-40x lower than the most potent commercially available bisphosphonates.
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Antineoplásicos/síntesis química , Derivados del Benceno/síntesis química , Difosfonatos/síntesis química , Isoquinolinas/síntesis química , Piridinas/síntesis química , Quinolinas/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Derivados del Benceno/química , Derivados del Benceno/farmacología , Línea Celular Tumoral , Difosfonatos/química , Difosfonatos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoquinolinas/química , Isoquinolinas/farmacología , Piridinas/química , Piridinas/farmacología , Quinolinas/química , Quinolinas/farmacología , Relación Estructura-Actividad , Compuestos de Terfenilo/síntesis química , Compuestos de Terfenilo/química , Compuestos de Terfenilo/farmacologíaRESUMEN
We screened a library of 117 bisphosphonates for antibacterial activity against Escherichia coli. The most potent growth inhibitors where N-[methyl(4-phenylalkyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonates, known potent bone resorption inhibitors, and there was a generally good correlation between cell growth inhibition and E. coli farnesyl diphosphate synthase (FPPS) inhibition. However, some potent FPPS inhibitors had no activity in cell growth inhibition, and based on the result of Catalyst pharmacophore modeling, this could be attributed to the requirement of a large hydrophobic feature for cellular activity (due most likely to transport). The activity of the most potent compound, N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (13), was strongly potentiated by the drug fosmidomycin. The transcription profiles for 13 or fosmidomycin alone were different from those found with carbenicillin or ciprofloxacin alone, but there were many similarities between the combination (13-fosmidomycin) and carbenicillin or ciprofloxacin, reflecting the more potent bactericidal activity of the drug combination on bacterial growth.
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Antibacterianos/farmacología , Difosfonatos/farmacología , Escherichia coli K12/efectos de los fármacos , Fosfomicina/análogos & derivados , Terpenos/antagonistas & inhibidores , Antibacterianos/química , Análisis por Conglomerados , Difosfonatos/química , Sinergismo Farmacológico , Escherichia coli K12/crecimiento & desarrollo , Escherichia coli K12/metabolismo , Fosfomicina/farmacología , Expresión Génica , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/química , Modelos Moleculares , Análisis de Secuencia por Matrices de Oligonucleótidos , Relación Estructura-Actividad CuantitativaRESUMEN
The effect of the administration of a commercial preparation of human gamma globulins has been evaluated in a mouse model of intranasal infection with BCG. First, we demonstrated the passage of specific antibodies to saliva and lung lavage following the intranasal or intraperitoneal administration to mice of human gamma globulins. This treatment of mice inhibited BCG colonization of the lungs (p < 0.01). A similar inhibitory effect was observed after infection of mice with gamma globulin opsonized BCG organisms (p < 0.01). These results are relevant for the development of new strategies for the control and treatment of tuberculosis.
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Mycobacterium bovis , Tuberculosis/prevención & control , gammaglobulinas/uso terapéutico , Administración Intranasal , Animales , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Infusiones Parenterales , Pulmón/inmunología , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Mycobacterium bovis/aislamiento & purificación , Fagocitosis , Saliva/inmunología , Tuberculosis/inmunología , gammaglobulinas/administración & dosificación , gammaglobulinas/farmacocinéticaRESUMEN
Cytogenomic microarray analysis (CMA) offers high resolution, genome-wide copy number information and is widely used in clinical laboratories for diagnosis of constitutional abnormalities. The Cancer Genomics Consortium (CGC) conducted a multiplatform, multicenter clinical validation project to compare the reliability and inter- and intralaboratory reproducibility of this technology for clinical oncology applications. Four specimen types were processed on three different microarray platforms-from Affymetrix, Agilent, and Illumina. Each microarray platform was employed at two independent test sites. The results were compared in a blinded manner with current standard methods, including karyotype, FISH, or morphology. Twenty-nine chronic lymphocytic leukemia blood, 34 myelodysplastic syndrome bone marrow, and 30 fresh frozen renal epithelial tumor samples were assessed by all six laboratories. Thirty formalin fixed paraffin embedded renal tumor samples were analyzed at the Affymetrix and Agilent test sites only. All study samples were initial diagnostic samples. Array data were analyzed at each participating site and were submitted to caArray for central analysis. Laboratory interpretive results were submitted to the central analysis team for comparison with the standard-of-care assays and for calculation of intraplatform reproducibility and cross-platform concordance. The results demonstrated that the three microarray platforms 1) detect clinically actionable genomic changes in cancer compatible to standard-of-care methods; 2) further define cytogenetic aberrations; 3) identify submicroscopic alterations and loss of heterozygosity (LOH); and 4) yield consistent results within and between laboratories. Based on this study, the CGC concludes that CMA is a sensitive and reliable technique for copy number and LOH assessment that may be used for clinical oncology genomic analysis.
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Hibridación Genómica Comparativa/métodos , Análisis Citogenético/métodos , Neoplasias/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Aberraciones Cromosómicas , Dosificación de Gen , Humanos , Hibridación Fluorescente in Situ , Cariotipo , Neoplasias Renales/diagnóstico , Neoplasias Renales/genética , Leucemia Linfocítica Crónica de Células B/diagnóstico , Leucemia Linfocítica Crónica de Células B/genética , Pérdida de Heterocigocidad , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Neoplasias/genética , Neoplasias Glandulares y Epiteliales/diagnóstico , Neoplasias Glandulares y Epiteliales/genética , Reproducibilidad de los Resultados , Nivel de AtenciónRESUMEN
A cohort of 338 patients diagnosed with myeloproliferative neoplasms was investigated by conventional cytogenetics and evaluated for the presence of the JAK2 V617F mutation. A t(1;9)(p10;q10) in addition to two extra der(1;9)(q10;p10) chromosomes was observed in two patients of essential thrombocythemia that transformed to acute myelogenous leukemia or to myelofibrosis. These findings suggest that the presence of extra derivative chromosomes der(1q;9p) in combination with the JAK2 V617F mutation may play a role in the progression of myeloproliferative neoplasms and supports the use of cytogenetics in the follow-up of the disease.
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Cromosomas Humanos Par 1 , Cromosomas Humanos Par 9 , Janus Quinasa 2/genética , Trombocitemia Esencial/genética , Translocación Genética , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Aberraciones Cromosómicas , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 9/genética , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense/genética , Mutación Missense/fisiología , Fenilalanina/genética , Recurrencia , Trombocitemia Esencial/patología , Valina/genéticaRESUMEN
Poco se ha estudiado acerca del papel de los anticuerpos específicos, presentes en las secreciones del aparato respiratorio, en la defensa contra patógenos intracelulares, como es el caso de las micobacterias causantes de la tuberculosis en el hombre: Micobacterium tuberculosis, bovis y africanum. Con el objetivo de desarrollar modelos adecuados para evaluar el posible papel de la inmunidad secretoria en la defensa contra la tuberculosis, se desarrollaron dos modelos animales con la utilización de un anticuerpo monoclonal IgA dirigido contra la proteína de 16 kD de M. tuberculosis y M. bovis. En el primer modelo se inocularon ratones Balb/c, por vía subcutánea al nivel de la nuca, con diferentes cantidades de células del hibridoma TBA61, productor de la IgA específica. En un segundo modelo, se inoculó por vía intraperitoneal líquido ascítico correspondiente a este hibridoma obtenido en ratón. En ambos casos se determinó, a diferentes tiempos, la concentración del monoclonal en saliva y sólo en suero para el segundo. En los dos modelos se demostró el paso del monoclonal a la saliva, donde alcanzó la máxima concentración: a los 21 días en los animales inoculados con el hibridoma, y a las 2 horas en saliva y suero en los animales inoculados con el líquido ascítico. Se sugiere, por su sencillez y mayor inocuidad, el uso del segundo modelo para la realización de estudios de reto por vía mucosal(AU)