Identification of diagnostic, prognostic and new major and minor susceptibility genes to pheochromacytoma amd paragangliomas (PCC/PGL)

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 18-05-2017Esta tesis tiene embargado el acceso al texto completo hasta el 18-11-2018ANTECEDENTES: El diagnóstico genético se recomienda en todos los pacientes con feocromocitoma (FEO) y paraganglioma (PGL), (FPGL), ya que el 65-80% se explican por una mutación en uno de los 34 genes descritos. Se han propuesto distintos algoritmos de diagnóstico genético, pero suelen excluir los FPGL esporádicos (FPGL-E) y ninguno incluye el estudio de mutaciones somáticas (MS). Además, como la lista de genes relacionados con FPGL no para de crecer cada año, el diagnóstico genético implica cada vez más tiempo, y los paneles de genes mediante secuenciación masiva (PG-NGS) emergen como una herramienta rentable y efectiva. OBJETIVOS: Nuestro objetivo fue aclarar la heterogeneidad genética en el desarrollo de los FPGL mediante el estudio genético sistemático. El estudio se realizó en dos partes sucesivas. MATERIAL Y MÉTODOS: La parte I incluyó 329 propósitus y se centró en la caracterización genética de pacientes con FPGL-E mediante la secuenciación por Sanger (SS) y las grandes deleciones de los principales genes relacionados con FPGL. Noventa y nueve tumores de los pacientes sin mutación germinal (MG) se incluyeron en el estudio de MS en RET, VHL, HRAS, EPAS1, MAX y SDHB. En la parte II el estudio genético se realizó de forma “ciega” utilizando 2 PG-NGS. Uno permitía el estudio en ADN germinal y de tumor congelado y el segundo fue específicamente diseñado para DNA extraído de tumor parafinado. En el segundo estudio se incluyeron 453 pacientes con FPGL (30 de ellos controles con mutaciones patogénicas conocidas y 275 habían sido parcialmente estudiados mediante SS (WTPS)). RESULTADOS: Parte I: se encontraron MGs en 46 pacientes (14%), siendo más frecuentes en PGLs (28.7%) que en FEOs (4,5%) (p=6.62×10-10). Los PGLs de cabeza y cuello (CC-PGLs) y los torácicos (T-PGLs), más comúnmente presentaban MGs (p=2.0×10-4 y p=0.027, respectivamente), pero no los abdominales (A-PGLs). Se encontraron MSs en el 43% de los tumores estudiados, y fueron más frecuentes en FEOs (48,5%) que en PGLs (32.3%) (p=0.13). Cinco casos metastásicos y un cuarto de los FPGL-E presentaban una MS, independientemente de la edad. Parte II: el abordaje con NGS mostró una sensibilidad ≥99.4%, independientemente del tipo de ADN. Se identificaron 45 variantes de significado desconocido y 89 mutaciones, siendo MGs 29 (7,2%) y MSs 58 (31,7%) en los 183 tumores estudiados (37 se encontraron en los casos WTPS). CONCLUSIONES: Recomendamos priorizar el estudio de MG en los pacientes con un único CC-PGL y T PGL, y de MS en FEO. El fenotipo catecolaminérgico y la IHC-SDHB deberían guiar el estudio genético, principalmente en A-PGLs únicos. Los casos pediátricos y metastásicos no deberían excluirse del estudio somático. Ambos PG-NGS son una alternativa eficiente y precisa a la SS, que facilita el estudio de genes “minoritarios” de FPGL y el diagnóstico genético en pacientes con datos clínicos incongruentes o ausentes, que de otra manera no serían diagnosticados

A Large Case-Control Study Performed in Spanish Population Suggests That RECQL5 Is the Only RECQ Helicase Involved in Breast Cancer Susceptibility.

Around 50% of the familial breast cancer (BC) cases are estimated to be caused by germline variants in known low-, moderate-, and high-risk susceptibility genes, while the other half is of unknown genetic origin. In the present study, we wanted to evaluate the role of the RECQ helicases, some of which have been studied in the past as candidates, with unclear results about their role in the disease. Using next-generation sequencing (NGS) technology, we analyzed the whole coding sequence of BLM, RECQL1, RECQL4, RECQL5, and WRN in almost 2000 index cases from BC Spanish families that had previously tested negative for the known BC susceptibility genes (BRCAX) and compared the results with the controls extracted from gnomAD. Our results suggest that BLM, RECQL1, RECQL4, and WRN do not play a major role in BC susceptibility. However, in the combined analysis, joining the present results with those previously reported in a series of 1334 BC Spanish patients and controls, we found a statistically significant association between Loss of Function (LoF) variants in RECQL5 and BC risk, with an OR of 2.56 (p = 0.009; 95% CI, 1.18-4.98). Our findings support our previous work and places the RECQL5 gene as a new moderate-risk BC gene.A.O. is partially funded by FIS PI19/00640 supported by FEDER funds and the Spanish Network on Rare Diseases (CIBERER). M.d.l.H. is partially funded by FIS PI20/00110 supported by FEDER funds.S

SpadaHC: a database to improve the classification of variants in hereditary cancer genes in the Spanish population

Accurate classification of genetic variants is crucial for clinical decision-making in hereditary cancer. In Spain, genetic diagnostic laboratories have traditionally approached this task independently due to the lack of a dedicated resource. Here we present SpadaHC, a web-based database for sharing variants in hereditary cancer genes in the Spanish population. SpadaHC is implemented using a three-tier architecture consisting of a relational database, a web tool and a bioinformatics pipeline. Contributing laboratories can share variant classifications and variants from individuals in Variant Calling Format (VCF) format. The platform supports open and restricted access, flexible dataset submissions, automatic pseudo-anonymization, VCF quality control, variant normalization and liftover between genome builds. Users can flexibly explore and search data, receive automatic discrepancy notifications and access SpadaHC population frequencies based on many criteria. In February 2024, SpadaHC included 18 laboratory members, storing 1.17 million variants from 4306 patients and 16 343 laboratory classifications. In the first analysis of the shared data, we identified 84 genetic variants with clinically relevant discrepancies in their classifications and addressed them through a three-phase resolution strategy. This work highlights the importance of data sharing to promote consistency in variant classifications among laboratories, so patients and family members can benefit from more accurate clinical management.Database URL: https://spadahc.ciberisciii.es/ Overview of SpadaHC and its main views. (A) List of existing variants in SpadaHC (in the image, search for the ATM gene). The 'Expert Cl.' column shows the classification made by a group of experts; the 'Lab Cl.' column shows a summary of the classifications made by the laboratories. (B) Allele frequency of a variant in the SpadaHC population according to clinical suspicion and sex. (C) Classifications provided by the laboratories for a variant. (D) List of patients carrying a variant. (E) Histogram showing the coverage and frequency (allele balance) with which the variant was detected in carrier patients. Alt text: SpadaHC overview; laboratories can share datasets of variant classifications (Excel) and variants from individuals (VCFs + Excel). The datasets undergo quality control, bioinformatics pipeline annotation and database integration before being displayed in SpadaHC. The graphical abstract also shows five views of SpadaHC

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through models, and define specific therapeutic options according to tumor genomic features. : We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized . : A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, =4.67·10), and was found associated with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated a repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. : Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/ mTOR axis in metastatic pheochromocytoma/ paraganglioma

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients’ liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients’ management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors

Alterations in SLC4A2, SLC26A7 and SLC26A9 DriveAcid–Base Imbalance in Gastric Neuroendocrine Tumors and Uncover a Novel Mechanism for a Co-Occurring Polyautoimmune Scenario

bstract:Autoimmune polyendocrine syndrome (APS) is assumed to involve an immune systemmalfunction and entails several autoimmune diseases co-occurring in different tissues of the samepatient; however, they are orphans of its accurate diagnosis, as its genetic basis and pathogenicmechanism are not understood. Our previous studies uncovered alterations in the ATPase H+/K+Transporting Subunit Alpha (ATP4A) proton pump that triggered an internal cell acid–base imbal-ance, offering an autoimmune scenario for atrophic gastritis and gastric neuroendocrine tumors withsecondary autoimmune pathologies. Here, we propose the genetic exploration of APS involvinggastric disease to understand the underlying pathogenic mechanism of the polyautoimmune scenario.The whole exome sequencing (WES) study of five autoimmune thyrogastric families uncovered differ-ent pathogenic variants in SLC4A2, SLC26A7 and SLC26A9, which cotransport together with ATP4A.Exploratoryin vitrostudies suggested that the uncovered genes were involved in a pathogenicmechanism based on the alteration of the acid–base balance. Thus, we built a custom gene panelwith 12 genes based on the suggested mechanism to evaluate a new series of 69 APS patients. In total,64 filtered putatively damaging variants in the 12 genes of the panel were found in 54.17% of thestudied patients and none of the healthy controls. Our studies reveal a constellation of solute carriersthat co-express in the tissues affected with different autoimmune diseases, proposing a unique geneticorigin for co-occurring pathologies. These results settle a new-fangled genetics-based mechanismfor polyautoimmunity that explains not only gastric disease, but also thyrogastric pathology and disease co-occurrence in APS that are different from clinical incidental findings. This opens a newwindow leading to the prediction and diagnosis of co-occurring autoimmune diseases and clinicalmanagement of patients

PheoSeq: a targeted next-generation sequencing assay for pheochromocytoma and paraganglioma diagnostics

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed

Targeted sequencing reveals low-frequency variants in EPHA genes as markers of paclitaxel-induced peripheral neuropathy.

PURPOSE: Neuropathy is the dose limiting toxicity of paclitaxel and a major cause for decreased quality of life. Genetic factors have been shown to contribute to paclitaxel neuropathy susceptibility; however, the major causes for inter-individual differences remain unexplained. In this study we identified genetic markers associated with paclitaxel-induced neuropathy through massive sequencing of candidate genes. EXPERIMENTAL DESIGN: We sequenced the coding region of 4 EPHA genes, 5 genes involved in paclitaxel pharmacokinetics and 30 Charcot-Marie-Tooth genes, in 228 cancer patients with no/low neuropathy or high grade neuropathy during paclitaxel treatment. An independent validation series included 202 paclitaxel-treated patients. Variation-/ gene-based analyses were used to compare variant frequencies among neuropathy groups and Cox regression models were used to analyze neuropathy evolution along treatment. RESULTS: Gene-based analysis identified EPHA6 as the gene most significantly associated with paclitaxel-induced neuropathy. Low frequency non-synonymous variants in EPHA6 were present exclusively in patients with high neuropathy and all affected the ligand binding domain. Accumulated dose analysis in the discovery series showed a significantly higher neuropathy risk for EPHA5/6/8 low-frequency non-synonymous variant carriers (HR=14.60, 95%CI=2.33-91.62, P=0.0042) and an independent cohort confirmed an increased neuropathy risk (HR=2.07, 95%CI=1.14-3.77, P=0.017). Combining the series gave an estimated 2.50-fold higher risk of neuropathy (95%CI=1.46-4.31; P=9.1x10(-4)). CONCLUSION: This first study sequencing EPHA genes revealed that low frequency variants in EPHA6, EPHA5 and EPHA8 contribute to the susceptibility to paclitaxel-induced neuropathy. Furthermore, EPHAs neuronal injury repair function suggests that these genes might constitute important neuropathy markers for many neurotoxic drugs.Funding agencies: Spanish Ministry of Economy and Competiveness [SAF2015-64850-R]; Severo Ochoa Excellence Programme [SEV-2011-0191]; Fundacion AECC; Swedish Cancer Society; Swedish Research Council; LiU Cancer</p

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development

Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients