Intermediate Molecular Phenotypes to Identify Genetic Markers of Anthracycline-Induced Cardiotoxicity Risk.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice (n = 165) generated by backcrossing were treated with doxorubicin and docetaxel. We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients (n = 71) and women with breast cancer (n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.

Intermediate molecular phenotypes to identify genetic markers of anthracycline-induced cardiotoxicity risk.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex disease whose polygenic component is mainly unidentified. We propose that levels of intermediate molecular phenotypes in the myocardium associated with histopathological damage could explain CDA susceptibility; so that variants of genes encoding these intermediate molecular phenotypes could identify patients susceptible to this complication. A genetically heterogeneous cohort of mice generated by backcrossing (N = 165) was treated with doxorubicin and docetaxel. Cardiac histopathological damage was measured by fibrosis and cardiomyocyte size by an Ariol slide scanner. We determine intramyocardial levels of intermediate molecular phenotypes of CDA associated with histopathological damage and quantitative trait loci (ipQTLs) linked to them. These ipQTLs seem to contribute to the missing heritability of CDA because they improve the heritability explained by QTL directly linked to CDA (cda-QTLs) through genetic models. Genes encoding these molecular subphenotypes were evaluated as genetic markers of CDA in three cancer patient cohorts (N = 517) whose cardiac damage was quantified by echocardiography or Cardiac Magnetic Resonance. Many SNPs associated with CDA were found using genetic models. LASSO multivariate regression identified two risk score models, one for pediatric cancer patients and the other for women with breast cancer. Molecular intermediate phenotypes associated with heart damage can identify genetic markers of CDA risk, thereby allowing a more personalized patient management. A similar strategy could be applied to identify genetic markers of other complex trait diseases.

A hotspot mutation targeting the R-RAS2 GTPase acts as a potent oncogenic driver in a wide spectrum of tumors.

A missense change in RRAS2 (Gln72 to Leu), analogous to the Gln61-to-Leu mutation of RAS oncoproteins, has been identified as a long-tail hotspot mutation in cancer and Noonan syndrome. However, the relevance of this mutation for in vivo tumorigenesis remains understudied. Here we show, using an inducible knockin mouse model, that R-Ras2Q72L triggers rapid development of a wide spectrum of tumors when somatically expressed in adult tissues. These tumors show limited overlap with those originated by classical Ras oncogenes. R-Ras2Q72L-driven tumors can be classified into different subtypes according to therapeutic susceptibility. Importantly, the most relevant R-Ras2Q72L-driven tumors are dependent on mTORC1 but independent of phosphatidylinositol 3-kinase-, MEK-, and Ral guanosine diphosphate (GDP) dissociation stimulator. This pharmacological vulnerability is due to the extensive rewiring by R-Ras2Q72L of pathways that orthogonally stimulate mTORC1 signaling. These findings demonstrate that RRAS2Q72L is a bona fide oncogenic driver and unveil therapeutic strategies for patients with cancer and Noonan syndrome bearing RRAS2 mutations.

Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL. METHODS: Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3'UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL. RESULTS: RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3'UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL. CONCLUSIONS: The results indicate that overexpression of wild type RRAS2 is behind the development of CLL.

Immunosuppression-Independent Role of Regulatory T Cells against Hypertension-Driven Renal Dysfunctions.

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39(+) regulatory T (TREG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in TREG/TH cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that TREG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.

[Study of AZF regions of Y chromosome in males with idiopathic infertility. Analysis of two methods of molecular diagnosis]. / Estudio de las regiones AZF del cromosoma Y en varones con infertilidad idiopática. Comparación de dos métodos de diagnóstico molecular.

BACKGROUND AND OBJECTIVE: It is well known that both azoospermia and oligozoospermia are associated to microdeletions of single tagged sites (STS) in the long arm of the Y chromosome. Characterization of deletions is carried out by polymerase chain reaction, although the number and regions included in the analysis varies between laboratories. The aim of this study was to analyze the presence of chromosome Y microdeletions using 2 different sets of STSs. PATIENTS AND METHOD: We analysed the presence of microdeletions in the Yq chromosome in 30 patients with idiopathic male infertility, using 2 sets of STSs, those proposed by the European Molecular Genetics Quality Network (EMQN) as first choice and those of the Y Chromosome Deletion Detection System (Promega). RESULTS: AZF microdeletions were detected in 4 patients (13%). Only one case was detected simultaneously with both sets. CONCLUSION: In patients with idiopathic male infertility detection of AZF microdeletion in Y chromosome has important methodological problems. Further studies are needed to achieve a more reliable method to be used by clinical laboratories.

Estudio de las regiones AZF del cromosoma Y en varones con infertilidad idiopática. Comparación de dos métodos de diagnóstico molecular / Study of AZF regions of Y chromosome in males with idiopathic infertility. Analysis of two methods of molecular diagnosis

Fundamento y objetivo: La azoospermia y la oligospermia causantes de infertilidad masculina se asocian con deleciones en regiones que contienen secuencias no repetitivas en el brazo largo del cromosoma Y. La caracterización de estas deleciones se realiza mediante reacción en cadena de la polimerasa, aunque varían el número y la localización de las regiones estudiadas dependiendo de los laboratorios. En este estudio nos planteamos analizar la presencia de deleciones en el cromosoma Y de pacientes infértiles empleando dos métodos diferentes. Pacientes y método: Se ha estudiado a 30 pacientes con infertilidad idiopática masculina, siguiendo las recomendaciones de la EMQN (European Molecular Genetics Quality Network) y, en paralelo, con el test Y Chromosome Deletion Detection System® (Promega). Resultados: Hemos detectado deleciones en la región AZF en 4 pacientes (13%). En sólo un caso ambos sistemas la detectaron simultáneamente. Conclusiones: La detección de microdeleciones de las regiones AZF del cromosoma Y presenta problemas metodológicos. Se necesitan más estudios para conseguir un consenso sobre el método más fiable para aplicar este tipo de estudios a los laboratorios clínicos

Background and objective: It is well known that both azoospermia and oligozoospermia are associated to microdeletions of single tagged sites (STS) in the long arm of the Y chromosome. Characterization of deletions is carried out by polimerase chain reaction, although the number and regions included in the analysis varies between laboratories. The aim of this study was to analyze the presence of chromosome Y microdeletions using 2 different sets of STSs. Patients and Method: We analysed the presence of microdeletions in the Yq chromosome in 30 patients with idiopathic male infertility, using 2 sets of STSs, those proposed by the European Molecular Genetics Quality Network (EMQN) as first choice and those of the Y Chromosome Deletion Detection System® (Promega). Results: AZF microdeletions were detected in 4 patients (13%). Only one case was detected simultaneously with both sets. Conclusion: In patients with idiopathic male infertility detection of AZF microdeletion in Y chromosome has important methodological problems. Further studies are needed to achieve a more reliable method to be used by clinical laboratories