Finding genes related to homologous recombination as modifiers of the number of dermal neurofibromas in neurofibromatosis type 1 patients / Estudi sobre la implicació dels gens de recombinació homòloga com a modificadors del nombre de neurofibromes dèrmics en pacients amb Neurofibromatosi tipus 1

摘要

[eng] 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.