NEUROFIBROMATOSI DI TIPO 1 E GENI MODIFICATORI PREDISPONENTI L’INSORGENZA DI TUMORI (GLIOMA DELLE VIE OTTICHE)

Introduction. Neurofibromatosis type I, the most common form of Neurofibromatosis, presents with a wide phenotypic variability, both inter- and intra-familiar; a comparative analysis between type/site of mutation and phenotype cannot identify a correlation between genotype and clinical features in affected patients. Pilocytic astrocytomas (PAs) are the tumors that occur most often in NF1 patients; they affect children and young adults and occur preferentially along the optic pathway. These tumors are defined as Grade I gliomas (low-grade) and their growth is slow, but a small number of these tumors continue to grow, behave in an aggressive fashion and cause loss of vision or hypothalamic dysfunction. It is not known which changes determine whether an NF1-associated pilocytic astrocytoma will remain stable or exhibit a clinical progression: genetic alterations associated with NF1-associated optic glioma pathogenesis have not been well characterized. Aim of the study. The aim of the study was to analyze a group of NF1 patients affected with optic glioma and a group of patients affected with optic glioma in which the diagnosis of NF1 was excluded, in order to evaluate the presence of specific mutations in the NF1 gene and in the tumor-suppressor gene CDKN2A; to analyze polymorphisms in the tumor-suppressor genes CDKN2A and TP53, that are reported in the literature to be associated with risk and tumor progression, in the study patients and in a healthy control group; to evaluate results with statistical methods; to establish if the glioma formation is associated with some specific mutations or polymorphisms in the studied genes. Materials and methods. A mutation/SNP screening in NF1, CDKN2A and TP53 genes was performed in 25 NF1 patients affected with optic glioma diagnosis and 21 non-NF1 patients affected with optic glioma. Blood samples were obtained after informed consent from all of the patients and a control group of 50 unrelated adult healthy individuals from the Padova Clinical Genetic Service database. Mutation analysis was done by DHPLC after amplification by PCR of all the exons of NF1 and CDKN2A genes, in all of the patients, and direct sequencing of the samples showing some alteration at the DHPLC analysis. The SNPs analysis was done in CDKN2A and TP53 genes in all the patients and in the control group by RFLP and HRMA, respectively, after amplification by PCR of the target sequences. Results. Mutation scanning in the NF1 gene by DHPLC analysis has identified mutations in 60% of the NF1 patients with optic glioma diagnosis. A preferential type of nucleotidic alteration was not observed but the majority (67%) of them causes the formation of a truncated protein. No mutations were found in the non-NF1 patients with optic glioma diagnosis. These results confirm the lack of a genotype–phenotype (optic glioma) correlation in patients affected with NF1. Mutation scanning in the CDKN2A gene by DHPLC analysis has identified two nucleotidic alterations in three NF1 patients: the G442A polymorphism in exon 2, known in the literature, and a new substitution in 3’ UTR of the gene (C520G) that could represent a new polymorphism. SNPs analysis (C500G and C540T in 3’ UTR of CDKN2A gene and IVS 6+62 G/A and 12256 G/C, in the promoter region of the TP53 gene) was done by two different techniques: RFLP and HRMA, respectively. We have compared the two methods and define that HRMA is a better instrument for SNPs analysis because of its sensitivity and specificity and because it is a cost-effective and simple post-PCR technique. A statistical analysis of allelic and genotypic frequences of the CDKN2A and TP53 genes polymorphisms in patients and control groups was done using the ?2 test. The results seem to suggest a non-association between the presence of these SNPs and an increased risk of optic glioma growth in the studied population (P>0,05). However the number of individuals analized is not sufficient for exhaustive conclusions. Future studies should be done with more polymorphisms and a larger patients group.