133 research outputs found
Genetics of vestibular schwannoma : Genetic landscape of irradiated and radiation-naïve benign and malignant vestibular schwannoma
Background: Vestibular schwannoma (VS) is a benign intracranial neoplasm associated with reduced quality of life. Malignant peripheral nerve sheath tumor of the vestibular nerve (VN-MPNST) is the malignant counterpart, an exceedingly rare cancer associated with high mortality. The genetics underlying VS and its etiology is not well understood and the genome of irradiated VS and VN-MPNST has not been characterized. We addressed these shortcomings in this thesis.
Material and methods: Tumor specimens from the Bergen neurosurgical tissue bank were subjected to a combination of whole-exome sequencing (WES), whole-genome sequencing and microarray, MLPA, transcriptome sequencing, ViroChip and Sanger sequencing.
Results: A median of 14 (4-57) genes were mutated and a median of 0.17% of the autosome was affected by copy number aberrations (CNA) in VS. NF2 mutation was observed in 89%. Tumors with wildtype NF2 harbored mutations in genes linked to NF2. Novel genes and pathways identified in VS included CDC27 (11%), USP8 (7%) and axonal guidance pathway (54%). One clinically aggressive VS was identified and correlated with high mutational burden (231) and mutated RAD54L. Variant allele frequencies for both small mutations and CNAs indicated intratumoral heterogeneity. No plausible virus was associated with VS. We identified a premalignant VS characterized by large chromosomal aberrations and mutated NF2. Malignant transformation was accompanied by whole-genome doubling and mutations in GNAQ, FOXO4 and PDGFRB. VN-MPNST is characterized by gross chromosomal aberrations and homozygous loss of CDKN2A. Previous treatment with GKRS in VS and VN-MPNST did not correlate with neither specific mutations nor genome wide signatures. COSMIC mutational signature 3 contributes to VN-MPNST while signature 6 contributes to a subset of VS.
Conclusion: VS is characterized by intratumoral genetic heterogeneity and relatively few mutations. We found recurrent mutations in NF2 and the axonal guidance pathway in addition to novel genes in subsets. Mutated RAD54L might correlate with a hypermutator phenotype and worse clinical course. We identified CDKN2A as a likely tumor suppressor in both premalignant VS and VN-MPNST. Premalignant VS showed signs of chromosomal instability making it prone to malignant transformation. No biomarker of radioresistance or signature of exposure to ionizing radiation was identified in neither VS nor VN-MPNST. We found no evidence of a viral etiology in VS.Doktorgradsavhandlin
CIRA annual report FY 2015/2016
Reporting period April 1, 2015-March 31, 2016
Thoracic aortic aneurysms and dissections: genetic analysis of Mendelian and complex cases
The present doctoral thesis deals with the still partially unraveled genetic component of thoracic aortic aneurysms and dissections, a frequently asymptomatic but potentially lethal condition and major cause of sudden death. Our main objective was to contribute to further elucidate the genetics behind it, from both Mendelian and complex perspectives. We analyzed single and familial, forensic and clinical mendelian cases applying either a candidate-gene or whole exome massive parallel sequencing approach, respectively. We were able to solve approximately 23% of the forensic single cases and identified two strong candidate mutations in TGFB2 and PRKG1 genes in the two non-syndromic familial cases analyzed. For the analysis of complex cases we chose a population-based approach. We selected bicuspid aortic valve patients with and without concomitant thoracic aortic dilation and faced them against general population controls. We were not able to identify any consistently significant association, though a promising one arose involving HMCN2 and calcium metabolism that should be considered in future studies. The direct clinical consequences some of these results had supported molecular diagnosis, reliable genotype-phenotype correlations, and risk stratification as important tools for clinical management of these patients and family members at risk, as well as the need of research to continue
Improving Outcomes in Prostate Cancer
The results of the STAMPEDE trial demonstrate that intensified systemic therapy with docetaxel or abiraterone added to androgen deprivation therapy (ADT) improves overall survival (OS). Treatment benefit and tolerance vary, therefore prognostic and predictive biomarkers able to inform treatment selection are required to improve patient outcomes. Through performing a systematic review and meta-analysis I contextualised the celecoxib and zoledronic acid (celecoxib-ZA) results with the aim of understanding the intriguing synergistic therapeutic effect that was only seen in metastatic disease. Secondly, I explored whether prostate-specific antigen (PSA) response, assessed after commencing ADT, and PSA nadir assessed after completion of docetaxel, are prognostic of OS. Through collaboration with industry partners, I assessed the feasibility of performing targeted nextgeneration sequencing (tNGS) using formalin-fixed paraffin embedded (FFPE) prostate tumour samples. I explored the genomic profile of mCSPC and sought prevalence data to inform the evaluation of therapies such as poly ADP ribose polymerase inhibitors (PARPi) in homologous recombination deficient (HRD) cancers. No other trials evaluating a cox-2 inhibitor with a bisphosphonate were identified. However, supported by pre-clinical data, an immunological mechanism mediated by γδ T cells is proposed to explain the observed synergy. This strengthens the need for future trials and informs parallel translational research. PSA response can be used to risk-stratify patients shortly after commencing ADT and may be useful in informing the use of docetaxel. PSA nadir, assessed after completion of docetaxel, was also prognostic of OS and may be used to identify patients who remain at high risk where additional systemic therapies e.g. abiraterone, should be evaluated. The genomic study revealed that 94% (108/115) of sequenced samples had ≥1 pathogenic mutation although individual mutation frequencies remain low and pathway aberrations often co-exist, which would necessitate hierarchical allocation if used to guide treatment. The prevalence of HRD is clinically significant (15%) however the screening burden is considerable, compounded by variable sample quality, compromising the sequencing success rate (64%). These data support the further evaluation of the combination of cox-2 inhibitors and bisphosphonates and the use of PSA-based outcomes in risk-stratification, whilst the genomic feasibility and prevalence data will inform future trial designs incorporating molecular stratification
The identification of new familial pheochromocytoma/paraganglioma genes using whole exome sequencing
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 30-04-2015Los feocromocitomas (PCC) son tumores neuroendocrinos, desarrollados a partir del tejido cromafín de la
médula adrenal, que suelen causar hipertensión arterial por sobre-secreción de catecolaminas. Los
paragangliomas (PGL) son PCCs, en su mayoría secretores y con un gran riesgo de malignizar, que se
desarrollan a partir de paraganglios localizados fundamentalmente en la región intra-abdominal o
torácica. Algunos PGLs pueden desarrollarse en la región de la cabeza y el cuello, y en esa localización
suelen comportarse como masas benignas no secretoras. Los PCCs presentan una incidencia anual en
población española de 2 casos por millón de habitantes y son por tanto una enfermedad rara. Tanto la
secuenciación masiva del genoma completo, como la limitada a las regiones codificantes (secuenciación
exómica, SE), se han convertido a lo largo de los últimos años en herramientas de gran utilidad para el
descubrimiento de genes de susceptibilidad responsables de enfermedades mendelianas. De este modo,
el objetivo principal de esta tesis doctoral fue la identificación de nuevos genes de susceptibilidad
implicados en el desarrollo de PCC/PGL mediante el uso de la SE aplicada a 3 proyectos independientes.
En el primer proyecto, se llevó a cabo la SE de tres pacientes no relacionados, con antecedentes
familiares de la enfermedad y sin mutaciones en ninguno de los genes conocidos. Los pacientes fueron
seleccionados como candidatos para el estudio debido a que sus correspondientes tumores presentaban
perfiles de expresión muy homogéneos. El filtrado y posterior análisis de los datos de secuenciación
permitió identificar mutaciones germinales patogénicas en el gen MAX en los tres pacientes. Además, la
pérdida de heterocigosidad del alelo silvestre, la ausencia de proteína MAX en los tumores y el
descubrimiento de otras 5 mutaciones en pacientes aquejados de la enfermedad permitió demostrar que
MAX constituía un nuevo gen supresor de tumores asociado con el desarrollo de PCC hereditario. En un
segundo trabajo, el estudio de una serie compuesta por más de 1500 pacientes no relacionados permitió
establecer tanto la prevalencia de las mutaciones en MAX en pacientes con PCC/PGL (1,12%), como el
fenotipo asociado a dichas mutaciones. Por último, con el objeto de determinar la patogenicidad de las
variantes con significado desconocido halladas en el gen MAX, se llevó a cabo un estudio funcional de las
mismas en células de PCC de rata (PC12) y se implementó una herramienta de predicción in silico basada
en el consenso de 5 predictores. En el segundo proyecto, los candidatos a estudio mediante SE (3 tríos
paciente/madre/padre) fueron seleccionados en base a la presencia de una característica fenotípica poco
frecuente en pacientes con PCC/PGL: policitemia idiopática. Durante el análisis de los datos, se publicó el
descubrimiento de mutaciones somáticas post-zigóticas en el gen EPAS1 en pacientes con PCC/PGL
múltiple y policitemia idiopática. El análisis de EPAS1 en los correspondientes tumores de los pacientes
seleccionados para la SE, reveló la presencia de mutaciones somáticas en mosaico en el gen EPAS1 en
todos ellos. Además, el estudio de una serie adicional de tumores identificó mutaciones somáticas en
tumores de pacientes sin policitemia. Finalmente, se identificó la ganancia de la región 2p como exclusiva
de tumores con mutación en EPAS1. En el tercer proyecto, se llevó a cabo una selección de pacientes
basada en la presencia de tumores múltiples (más de 5) como indicador de la existencia de una
enfermedad hereditaria. Durante el filtrado de las variantes encontradas en uno de los pacientes
seleccionados, se identificó una mutación en el gen MDH2, implicado en el ciclo de Krebs. La ausencia de
RNAm, proteína y actividad enzimática malato deshidrogenasa, así como el diagnóstico de la enfermedad
en un pariente portador de la variante permitió concluir que el gen MDH2 es un nuevo gen supresor
tumoral responsable de susceptibilidad a desarrollar PCC/PGL. La identificación de este segundo gen de
susceptibilidad a desarrollar PCC/PGL demuestra la eficacia de la SE en la identificación de nuevos genes
responsables de enfermedades mendelianas.Pheochromocytomas (PCCs) are neuroendocrine tumors arising from the medulla of the adrenal gland
that usually cause hypertension due to oversecretion of cathecolamines. On the other hand,
paragangliomas (PGLs) are normally secretor tumors with high risk of malignancy that arise from the
paraganglia of the intra-abdominal or thoracic regions. Some PGLs arise in the head and neck region,
usually as benign, non-secretor tumors. PCC/PGL is a rare disease, with an incidence in the Spanish
population of approximately 2 cases per million habitants. High-throughput techniques such as wholegenome
sequencing (WGS) and whole-exome sequencing (WES) are nowadays widely applied to discover
susceptibility genes involved in mendelian diseases. Thus, the main objective of this thesis was to identify
new genes related to the susceptibility to develop PCC/PGL by applying WES in three independent
projects. In the first project, we applied WES to three unrelated patients with a family history of disease
testing negative for mutations in the major PCC/PGL susceptibility genes. The three patients were
selected because they shared a common homogeneous transcriptional profile, suggesting a common
underlying genetic alteration. The variant filtering process and the posterior analysis of the WES data
allowed us to identify pathogenic germline mutations in the MAX (MYC associated factor X) gene in all
three patients. Moreover, loss of heterozygosity of the wild type allele, loss of the protein in the tumors
and the identification of MAX mutations in additional patients with the disease, together indicated that
MAX constitutes a novel tumor suppressor gene. In a posterior international collaborative study, we
recruited more than 1500 unrelated patients and established the prevalence of MAX mutations to be
1.12%; we also characterized the associated phenotype. Finally, we developed a functional model to
determine the pathogenicity of variants of unknown significant (VUS) found in MAX. This model was
implemented based on a consensus in silico prediction obtained from five algorithms available online. In
the second project, the selection of three trios (patient/mother/father) was based on the presence of a
very infrequent phenotypic characteristic in patients suffering for PCC/PGL: idiopathic polycythemia.
While the analysis of the WES data was underway, a study was published reporting the presence of
somatic post-zygotic mutations in the gene EPAS1 (HIF2A) in patients presenting multiple PCC/PGL and
idiopathic polycythemia. Our posterior screening for EPAS1 mutations in the tumors from the index
patients revealed the presence of somatic mutations in all of them. In addition, the analysis of an
additional series of tumors identified somatic mutations in tumors from patients without polycythemia.
Finally, we indentified a gain of the chromosomic region 2p exclusive to tumors harboring EPAS1
mutations. In the last WES Project, we selected patients based on the presence of multiple (more than
five) tumors as phenotypic marker of hereditary disease. We identified a splice-site mutation affecting
the MDH2 gene involved in the Krebs cycle. The observed absence of the messenger RNA, protein and
malate dehydrogenase enzymatic activity, as well as the positive diagnosis for the disease of a family
member, led us to conclude that MDH2 is a novel tumor suppressor gene implicated in susceptibility to
develop PCC/PGL. The identification of this second susceptibility gene implicated in PCC/PGL
development demonstrates the efficacy of WES in discovering susceptibility genes involved in Mendelian
disease
Abstracts from the 50th European Society of Human Genetics Conference: Posters
International audienc
Investigation of external refrigeration systems for long term cryogenic storage Final report
Data on external refrigeration systems for space storage of cryogens for long period
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