Neurofibromatosis type 1 patients present a high variability in their clinical expressivity. The most common manifestation is the appearance of dermal neurofibromas, benign tumors that arise in the peripheral nervous system. They appear at puberty and increase their number throughout life, with patients showing a great variation in their number, ranging from tens to thousands. The main objective of this thesis consisted in the identification of genes and variants influencing the number of dermal neurofibromas developed by NF1 patients. We centered our studies only to Schwann cells (neurofibromas develop due to a double inactivation of the NF1 gene, but only Schwann cells bear it), and the HR mechanism (HR has been found to be responsible for a high percentage of somatic NF1 inactivations in neurofibromas). In the first part of this project we characterized our cohort of 117 NF1 patients at clinical (age, sex, the number of dermal neurofibromas developed) and tumor molecular (estimating the LOH frequencies together with the identification of the mechanisms generating these LOHs) level. 23.7% of tumors showed LOH. 37% of tumors exhibited LOH due to deletion, and 63% due to HR. LOH frequencies were very variable, ranging from less than 10% to more than 50% of LOH. In addition, our studies suggested that patients with the highest rates of HR frequency showed the highest rates of nº of dNFs (with a p value close to significance). We developed the Microsatellite Multiplex PCR Analysis (MMPA) that improved and facilitated neurofibroma analysis. With this technique it was possible to obtain: data regarding the tumor sample allelic imbalance (AI) status and extension, the percentage of normal cells present in the tumor sample, the copy-number status of specific alleles of heterozygous loci showing AI and the mechanisms generating these AIs, in only one PCR reaction. The re-analysis of 29 neurofibromas showed a good agreement between the information generated by MMPA and the data generated for the same tumors by other techniques. In the second part of this project we selected candidate genes, involved in the HR mechanism, as possible modifiers of the number of dermal neurofibromas. We developed the HoReYe assay to model HR in yeast. With this technique we were able to determine the HR rate for the yeast strain BMA64. Once more yeast strains were characterized for the HR rate, the X-QTL assay would be performed to determine genetic variation responsible for high or low HR rates. In addition, due to the complexity of the HoReYe setting up, a surrogate of this technique was proposed to determine, in an easier way, the HR rate of yeast strains. In the third part of this project genetic variation of candidate genes would be analyzed by direct sequencing to identify both common and rare variants. Sanger sequencing was first used to analyze the BLM gene in 12 NF1 patients, but not variant found was affecting the protein structure. We would employ Next-generation sequencing to analyze genetic variation the 18 NF1 patients characterized. However, until now, only data from patient P027 was recovered showing 845 variants, which will be further analyzed in the near future. This thesis has established the basis to identify candidate genes related to HR rate, which will be studied in the NF1 patients previously characterized in order to identify allelic variants responsible for the number of dermal neurofibromas developed. / Els pacients amb Neurofibromatosi tipus 1 presenten una gran variabilitat en les seves manifestacions clíniques. El tret més característic és l’aparició de neurofibromes dèrmics, els quals poden aparèixer a decenes o milers en un pacient. L’objectiu principal de la present tesi ha estat identificar aquells gens i variants al•lèliques responsables del nombre de neurofibromes desenvolupats pels pacients NF1. Per a realitzar aquest treball ens hem centrat en estudiar les cèl•lules de Schwann, les portadores de la doble inactivació del gen NF1, i el mecanisme de recombinació homòloga, responsable d’un alt percentatge de les inactivacions somàtiques del gen NF1. En la primera part del treball vam caracteritzar els pacients NF1 de forma clínica, analitzant el sexe, edat i el nombre de neurofibromes desenvolupats, i molecular a nivell tumoral, determinant la presència de LOH i els mecanismes mutacionals generadors d’aquesta. Així, vam establir una prevalença de LOH als pacients d’un 23.7%, éssent el mecanisme de recombinació homòloga el més frequent, i vam obtenir una possible correlació entre tenir un elevat percentatge de recombinació homòloga generant LOHs i un elevat nombre de neurofibromes desenvolupats. A més, vam desenvolupar la tècnica de MMPA per a facilitar l’anàlisi de neurofibromes dèrmics, la qual pot ser aplicada a l’anàlisi d’altres tumors. La segona part del treball consistia en identificar gens candidats responsables del nombre de neurofibromes desenvolupats pels pacients NF1. Vam decidir utilitzar el llevat com a organisme model per a estudiar el mecanisme de recombinació homòloga, i obtenir gens candidats relacionats amb aquest mecanisme. Vam desenvolupar la tècnica HoReYe per a obtenir la taxa de recombinació homòloga en diferents soques de llevat. A més, vam idear les tècniques que s’haurien d’utilitzar posteriorment per a determinar les variants al•lèliques responsables d’aquestes taxes. En la tercera part del treball els gens candidats es van analitzar, tant per sequenciació per Sanger, com per seqüenciació de próxima generació. La intenció era trobar variants tant rares com comunes, per a no perdre cap tipus de variabilitat en l’anàlisi. En aquest treball s’han introduit les bases per a identificar, en pacients prèviament caracteritzats, els gens responsables del nombre de neurofibromes desenvolupats en pacients NF1.

The aetiology of common diseases is shaped by the effects of genetic and environmental factors. Big efforts have been devoted to unravel the genetic basis of disease with the hope that it will help to develop new therapeutic treatments and to achieve personalized medicine. With the development of high-throughput genotyping technologies, hundreds of association studies have described many loci associated to disease. However, the depiction of disease architecture remains incomplete. The aim of this work is to perform exhaustive comparisons across human populations to evaluate pressing questions. Our results provide new insights in the allele frequency of risk variants, their sharing across populations and the likely architecture of disease / La etiología de las enfermedades comunes está formada por factores genéticos y ambientales. Se ha puesto mucho empeño en describir sus bases genéticas. Este conocimiento será útil para desarrollar nuevas terapias y la medicina personalizada. Gracias a las técnicas de genotipado masivo, centenares de estudios de asociación han descrito una infinidad de genes asociados a enfermedad. Pese a ello, la arquitectura genética de las enfermedades no ha sido totalmente descrita. Esta tesis pretende llevar a cabo exhaustivas comparaciones entre poblaciones para responder diversas preguntas candentes. Nuestros resultados dan pistas sobre la frecuencia de los alelos de riesgo, su presencia entre poblaciones y la probable arquitectura de las enfermedades.