Bularreko minbiziaren prebentziorako BRCA1 gene mutatuaren edizioa CRISPR/Cas9 teknikaren bidez

[EUS] Gaur egungo gizartean gero eta minbizi kasu gehiago diagnostikatzen dira, eta horien artean % 23a bular-minbiziari dagokio. Gainera, nagusienetarikoa da emakumeen heriotza kausen artean, % 14a alegia. Hori dela eta, azterketa asko egin izan dira bular minbiziaren inguruan eta bi gene izan dira, nagusiki, bular minbiziarekin lotu direnak, BRCA1 eta BRCA2, hain zuzen. Lan honetan bi hauetako bat aztertuko da, BRCA1 genea, gizakiotan 17. kromosoman kokatzen dena (17q21 ), gene honen zenbait mutazio komunak direlako bular-minbizia duten gaixoen artean eta baita familia berekoen artean. Gene honek tumore-supresorea den p220 proteina kondetzen du. Proteina horren ziklo zelularraren kontrolean funtsezko papera betetzen du, izan ere, zelulen zikloko zenbait bidezidor konplexutan parte hartzen du, beste proteina batzuekin elkar eraginez, p53 proteinarekin adibidez. BRCA1 genean mutazioak gertatzean, baina, ziklo zelularraren kontrola inhibitu egiten da eta zelulen gehiegizko proliferazioa gertatzen da, bular zein obario minbizia dakarrena. Gene honetan deskribatutako mutazioen artean bi dira nagusitzen direnak: 2. exonean ematen den 2 nukleotidotako delezioa, 187delAG, eta 19. exonean ematen den nukleotido baten insertzioa, 5382insC. Lan honetan azken mutazio hau landu da, izan ere, handia da mutazio honengatik bular-minbizia garatzeko arriskua (% 67). Lan honetan beraz, mutazioa honen edizioak bular minbizia garatzeko arrisku murrizten inpaktua izango duela proposatzen dugu. Hortaz, kontzeptuen proba moduan hurrengo proiektu pilotua proposatzen da. Mutatutako HCC 1937 lerro zelularra erabiliko dugu, BRCA1 5382insC mutaziorako homozigotoa dena, in vitro modelo esperimental moduan. Edizioa aurrera eramateko CRISPR/Cas9 edizio sistema erabiltzea proposatzen da mutatutako zelulak basati bihurtzeko helburuarekin. Edizioa zuzentzeko, HCC 1937 zelulak 2 plasmido erabilita transfektatuko dira: bat RNA gudaren eta Cas9 nukleasaren ko-espreziorako, eta bestea DNA emailea eramango duena, basatia BRCA1 generako, DNAren konponketa faboratzeko. Edizioaren ondorioak aztertzeko, proliferazio indizea eta kimioterapiarekiko sentikortasuna analizatuko dira HCC 1937 zelula editatuetan.[EN] Nowadays, a large amount of cancer cases are diagnosed every year, of which 23% correspond to breast cancer. In addition, breast cancer is the main cause (14%) of death among women and therefore, many studies have been accomplished in it. Those studies have proven that two genes are mainly related to breast cancer, namely BRCA1 and BRCA2. In this project we are going to focus on one of these two genes will be analysed, the BRCA1 gene, which in humans is found in chromosome 17 (17q21), because some of the mutations of this gene are common among breast cancer patients and among the people from the same family. This gene encodes for a tumor-suppressor protein, known as p220. This protein has a key role in the control of cell cycle, as it interacts with other proteins, like p53. Moreover, when mutations in the BRCA1 gene appears cell cycle control is inhibited, and an over-proliferation of cells occurs. This can lead to the developing of a breast or ovarian cancer. Among the mutations described in this gene, two are those that prevail: the 2-nucleotide deletion in exon 2, 187delAG, and the insertion of a nucleotide in exon 19, 5382insC. Because of the high risk of developing cancer in carriers of this latest mutation (67%), BRCA1 5382insC has been addressed in the present work. Here we propose that editing this mutation would have an impact in reducing the risk of developing cancer. Thus, as a proof of principle the following pilot project is proposed. We will use HCC 1937 mutated cell line is proposed, which is a homozygous for BRCA1 5382insC mutation, as an experimental in vitro model. It is proposed to perform the edition using the CISPR/Cas9 edition-system in order to make the mutated cell line become wild-type. To conduct the edition, we will transfect HCC 1937 cells with 2 plasmids: one coexpressing a guide-RNA to the mutant sequence and Cas9 nuclease, and another one providing the donor DNA wild-type for BRCA1 to facilitate the repair of the cleaved DNA by the cells. In order to test the outcome of the edition, we will analyzed the proliferative index and the sensitivity to chemotherapy in edited HCC 1937 cells

Bularreko minbiziaren prebentziorako BRCA1 gene mutatuaren edizioa CRISPR/Cas9 teknikaren bidez

[EUS] Gaur egungo gizartean gero eta minbizi kasu gehiago diagnostikatzen dira, eta horien artean % 23a bular-minbiziari dagokio. Gainera, nagusienetarikoa da emakumeen heriotza kausen artean, % 14a alegia. Hori dela eta, azterketa asko egin izan dira bular minbiziaren inguruan eta bi gene izan dira, nagusiki, bular minbiziarekin lotu direnak, BRCA1 eta BRCA2, hain zuzen. Lan honetan bi hauetako bat aztertuko da, BRCA1 genea, gizakiotan 17. kromosoman kokatzen dena (17q21 ), gene honen zenbait mutazio komunak direlako bular-minbizia duten gaixoen artean eta baita familia berekoen artean. Gene honek tumore-supresorea den p220 proteina kondetzen du. Proteina horren ziklo zelularraren kontrolean funtsezko papera betetzen du, izan ere, zelulen zikloko zenbait bidezidor konplexutan parte hartzen du, beste proteina batzuekin elkar eraginez, p53 proteinarekin adibidez. BRCA1 genean mutazioak gertatzean, baina, ziklo zelularraren kontrola inhibitu egiten da eta zelulen gehiegizko proliferazioa gertatzen da, bular zein obario minbizia dakarrena. Gene honetan deskribatutako mutazioen artean bi dira nagusitzen direnak: 2. exonean ematen den 2 nukleotidotako delezioa, 187delAG, eta 19. exonean ematen den nukleotido baten insertzioa, 5382insC. Lan honetan azken mutazio hau landu da, izan ere, handia da mutazio honengatik bular-minbizia garatzeko arriskua (% 67). Lan honetan beraz, mutazioa honen edizioak bular minbizia garatzeko arrisku murrizten inpaktua izango duela proposatzen dugu. Hortaz, kontzeptuen proba moduan hurrengo proiektu pilotua proposatzen da. Mutatutako HCC 1937 lerro zelularra erabiliko dugu, BRCA1 5382insC mutaziorako homozigotoa dena, in vitro modelo esperimental moduan. Edizioa aurrera eramateko CRISPR/Cas9 edizio sistema erabiltzea proposatzen da mutatutako zelulak basati bihurtzeko helburuarekin. Edizioa zuzentzeko, HCC 1937 zelulak 2 plasmido erabilita transfektatuko dira: bat RNA gudaren eta Cas9 nukleasaren ko-espreziorako, eta bestea DNA emailea eramango duena, basatia BRCA1 generako, DNAren konponketa faboratzeko. Edizioaren ondorioak aztertzeko, proliferazio indizea eta kimioterapiarekiko sentikortasuna analizatuko dira HCC 1937 zelula editatuetan.[EN] Nowadays, a large amount of cancer cases are diagnosed every year, of which 23% correspond to breast cancer. In addition, breast cancer is the main cause (14%) of death among women and therefore, many studies have been accomplished in it. Those studies have proven that two genes are mainly related to breast cancer, namely BRCA1 and BRCA2. In this project we are going to focus on one of these two genes will be analysed, the BRCA1 gene, which in humans is found in chromosome 17 (17q21), because some of the mutations of this gene are common among breast cancer patients and among the people from the same family. This gene encodes for a tumor-suppressor protein, known as p220. This protein has a key role in the control of cell cycle, as it interacts with other proteins, like p53. Moreover, when mutations in the BRCA1 gene appears cell cycle control is inhibited, and an over-proliferation of cells occurs. This can lead to the developing of a breast or ovarian cancer. Among the mutations described in this gene, two are those that prevail: the 2-nucleotide deletion in exon 2, 187delAG, and the insertion of a nucleotide in exon 19, 5382insC. Because of the high risk of developing cancer in carriers of this latest mutation (67%), BRCA1 5382insC has been addressed in the present work. Here we propose that editing this mutation would have an impact in reducing the risk of developing cancer. Thus, as a proof of principle the following pilot project is proposed. We will use HCC 1937 mutated cell line is proposed, which is a homozygous for BRCA1 5382insC mutation, as an experimental in vitro model. It is proposed to perform the edition using the CISPR/Cas9 edition-system in order to make the mutated cell line become wild-type. To conduct the edition, we will transfect HCC 1937 cells with 2 plasmids: one coexpressing a guide-RNA to the mutant sequence and Cas9 nuclease, and another one providing the donor DNA wild-type for BRCA1 to facilitate the repair of the cleaved DNA by the cells. In order to test the outcome of the edition, we will analyzed the proliferative index and the sensitivity to chemotherapy in edited HCC 1937 cells

Survival in male COVID-19 patients linked to testosterone recovery

Infection with SARS-CoV-2 portends a broad range of outcomes, from a majority of asymptomatic cases or mild clinical courses to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty and co-morbidities such as obesity, diabetes or cardiovascular disease. A precise knowledge is still lacking of the molecular and biological mechanisms that may explain the association of severe disease with male sex. Here, we show that testosterone trajectories are highly accurate individual predictors (AUC of ROC = 0.928, p < 0.0001) of survival in male COVID-19 patients. Longitudinal determinations of blood levels of luteinizing hormone (LH) and androstenedione suggest an early modest inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by either full recovery in survivors or a peripheral failure in lethal cases. Moreover, failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and decrease of non-classical monocytes. The strong association of recovery or failure to reinstate testosterone levels with survival or death from COVID-19 in male patients is suggestive of a significant role of testosterone status in the immune responses to COVID-19.This study was funded by grants from the Ministerio de Ciencia e Innovacion (RTI2018-096055-B-I00), Consejo Superior de Investigaciones Cientificas COVID-19 Research Fund (CSIC-COV19-006, CSIC-COV19-201), Agencia de Gestio Ajuts Universitaris i de Recerca (2020PANDE00048 and 2017SGR 1411 GRC), Plan Nacional de I+D (PID-107139RB-C21) and Instituto Nacional de la Salud Carlos III (PI18/00346 and COVID-19_00416).N

Additional file 1 of Recovery of serum testosterone levels is an accurate predictor of survival from COVID-19 in male patients [Dataset]

Additional file 1: Table ST1. Treatments comparison by outcome in male patients. Table ST2. Treatments comparison by outcome in female patients. Table ST3. WHO classification of disease outcome. Table ST4. Panels and antibodies used for immunophenotyping. Figure SF1. Patients distribution by outcome, age, and comorbidities. Figure SF2. Distribution of male patients with comorbidities according to age and testosterone levels. Figure SF3. Longitudinal analysis of serum levels of IL-6, C-reactive protein (CRP), ferritin and lactate dehydrogenase (LDH) in male patients. Figure SF4. Bioavailable testosterone serum levels and correlation between age and sex-hormone binding globulin (SHBG). Figure SF5. Flow cytometry analysis of circulating immune subpopulations in three illustrative cases with moderate, severe survivor and severe deceased outcomes.Ministerio de Ciencia, Innovación y Universidades Consejo Superior de Investigaciones Científicas Agència de Gestió d’Ajuts Universitaris i de Recerca Conselleria d'Educació, Investigació, Cultura i Esport Instituto de Salud Carlos IIIPeer reviewe

Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters; A Retrospective Cohort Analysis and Pilot Study

BackgroundTwo years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. ObjectivesTo measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. ConclusionsLaboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible

Bularreko minbiziaren prebentziorako BRCA1 gene mutatuaren edizioa CRISPR/Cas9 teknikaren bidez

[EUS] Gaur egungo gizartean gero eta minbizi kasu gehiago diagnostikatzen dira, eta horien artean % 23a bular-minbiziari dagokio. Gainera, nagusienetarikoa da emakumeen heriotza kausen artean, % 14a alegia. Hori dela eta, azterketa asko egin izan dira bular minbiziaren inguruan eta bi gene izan dira, nagusiki, bular minbiziarekin lotu direnak, BRCA1 eta BRCA2, hain zuzen. Lan honetan bi hauetako bat aztertuko da, BRCA1 genea, gizakiotan 17. kromosoman kokatzen dena (17q21 ), gene honen zenbait mutazio komunak direlako bular-minbizia duten gaixoen artean eta baita familia berekoen artean. Gene honek tumore-supresorea den p220 proteina kondetzen du. Proteina horren ziklo zelularraren kontrolean funtsezko papera betetzen du, izan ere, zelulen zikloko zenbait bidezidor konplexutan parte hartzen du, beste proteina batzuekin elkar eraginez, p53 proteinarekin adibidez. BRCA1 genean mutazioak gertatzean, baina, ziklo zelularraren kontrola inhibitu egiten da eta zelulen gehiegizko proliferazioa gertatzen da, bular zein obario minbizia dakarrena. Gene honetan deskribatutako mutazioen artean bi dira nagusitzen direnak: 2. exonean ematen den 2 nukleotidotako delezioa, 187delAG, eta 19. exonean ematen den nukleotido baten insertzioa, 5382insC. Lan honetan azken mutazio hau landu da, izan ere, handia da mutazio honengatik bular-minbizia garatzeko arriskua (% 67). Lan honetan beraz, mutazioa honen edizioak bular minbizia garatzeko arrisku murrizten inpaktua izango duela proposatzen dugu. Hortaz, kontzeptuen proba moduan hurrengo proiektu pilotua proposatzen da. Mutatutako HCC 1937 lerro zelularra erabiliko dugu, BRCA1 5382insC mutaziorako homozigotoa dena, in vitro modelo esperimental moduan. Edizioa aurrera eramateko CRISPR/Cas9 edizio sistema erabiltzea proposatzen da mutatutako zelulak basati bihurtzeko helburuarekin. Edizioa zuzentzeko, HCC 1937 zelulak 2 plasmido erabilita transfektatuko dira: bat RNA gudaren eta Cas9 nukleasaren ko-espreziorako, eta bestea DNA emailea eramango duena, basatia BRCA1 generako, DNAren konponketa faboratzeko. Edizioaren ondorioak aztertzeko, proliferazio indizea eta kimioterapiarekiko sentikortasuna analizatuko dira HCC 1937 zelula editatuetan.[EN] Nowadays, a large amount of cancer cases are diagnosed every year, of which 23% correspond to breast cancer. In addition, breast cancer is the main cause (14%) of death among women and therefore, many studies have been accomplished in it. Those studies have proven that two genes are mainly related to breast cancer, namely BRCA1 and BRCA2. In this project we are going to focus on one of these two genes will be analysed, the BRCA1 gene, which in humans is found in chromosome 17 (17q21), because some of the mutations of this gene are common among breast cancer patients and among the people from the same family. This gene encodes for a tumor-suppressor protein, known as p220. This protein has a key role in the control of cell cycle, as it interacts with other proteins, like p53. Moreover, when mutations in the BRCA1 gene appears cell cycle control is inhibited, and an over-proliferation of cells occurs. This can lead to the developing of a breast or ovarian cancer. Among the mutations described in this gene, two are those that prevail: the 2-nucleotide deletion in exon 2, 187delAG, and the insertion of a nucleotide in exon 19, 5382insC. Because of the high risk of developing cancer in carriers of this latest mutation (67%), BRCA1 5382insC has been addressed in the present work. Here we propose that editing this mutation would have an impact in reducing the risk of developing cancer. Thus, as a proof of principle the following pilot project is proposed. We will use HCC 1937 mutated cell line is proposed, which is a homozygous for BRCA1 5382insC mutation, as an experimental in vitro model. It is proposed to perform the edition using the CISPR/Cas9 edition-system in order to make the mutated cell line become wild-type. To conduct the edition, we will transfect HCC 1937 cells with 2 plasmids: one coexpressing a guide-RNA to the mutant sequence and Cas9 nuclease, and another one providing the donor DNA wild-type for BRCA1 to facilitate the repair of the cleaved DNA by the cells. In order to test the outcome of the edition, we will analyzed the proliferative index and the sensitivity to chemotherapy in edited HCC 1937 cells

Recovery of serum testosterone levels is an accurate predictor of survival from COVID-19 in male patients

SARS-CoV-2 infection portends a broad range of outcomes, from a majority of asymptomatic cases to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty, and co-morbidities such as obesity, diabetes, and cardiovascular disease. A precise knowledge of the molecular and biological mechanisms that may explain the association of severe disease with male sex is still lacking. Here, we analyzed the relationship of serum testosterone levels and the immune cell skewing with disease severity in male COVID-19 patients. Biochemical and hematological parameters of admission samples in 497 hospitalized male and female COVID-19 patients, analyzed for associations with outcome and sex. Longitudinal (in-hospital course) analyses of a subcohort of 114 male patients were analyzed for associations with outcome. Longitudinal analyses of immune populations by flow cytometry in 24 male patients were studied for associations with outcome. We have found quantitative differences in biochemical predictors of disease outcome in male vs. female patients. Longitudinal analyses in a subcohort of male COVID-19 patients identified serum testosterone trajectories as the strongest predictor of survival (AUC of ROC = 92.8%, p < 0.0001) in these patients among all biochemical parameters studied, including single-point admission serum testosterone values. In lethal cases, longitudinal determinations of serum luteinizing hormone (LH) and androstenedione levels did not follow physiological feedback patterns. Failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and augmented circulating classical monocytes. Recovery or failure to reinstate testosterone levels is strongly associated with survival or death, respectively, from COVID-19 in male patients. Our data suggest an early inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by full recovery in survivors or a peripheral failure in lethal cases. These observations are suggestive of a significant role of testosterone status in the immune responses to COVID-19 and warrant future experimental explorations of mechanistic relationships between testosterone status and SARS-CoV-2 infection outcomes, with potential prophylactic or therapeutic implications. The online version contains supplementary material available at 10.1186/s12916-022-02345-

Exposing and Overcoming Limitations of clinical laboratory tests in COVID-19 by adding immunological parameters; A Retrospective cohort analysis and pilot study

Background: Two years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. Objectives: To measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings: 1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. Conclusions: Laboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible