Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: Molecular mechanisms and phenotypic correlations of rare forms of Neurofibromatosis type 1: mosaicisms, partial phenotypes and phenocopies

Introduction. Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder caused by monoallelic loss-of-function sequence and structural variants in the NF1 gene, whose product is a regulator of the RAS pathway. Clinical features of NF1 are café-au-lait macules (CaLMs), axillary/inguinal freckling, neurofibromas, Lisch nodules, plexiform neurofibromas, increased risk of neoplasms and developmental delay. Some of these features are shared with other RASopathies. The NF1 gene, due to its size and structure, presents high mitotic mutation rates, resulting in a relatively high frequency of mosaicism. Mosaicisms and phenocopies pose significant challenges in providing molecular diagnoses and appropriate clinical management in these individuals, due to the lack of scientific knowledge concerning the molecular bases, pathophysiology and subsequent implication of these conditions. Mosaic NF1 (mNF1) is due to a postzygotic variant occurred in NF1. Clinical findings are localized to the affected area of the body and the molecular investigations performed on genomic DNA from peripheral blood usually do not identify a causative variant in NF1. Few genotype-phenotype correlations have been described in individuals with germline pathogenic variant in NF1, including families without neurofibromas and increased susceptibility to neoplasms. CaLMs represent the earlier clinical findings in individuals with NF1, but they can be present in a broad spectrum of genetic conditions. Aims of the study. This project on rare forms of neurofibromatosis and phenocopies has been structured with different research lines on different populations of individuals with NF1 features. It aims to provide a molecular characterization for patients with clinical suspicion of NF1 not meeting the classical criteria and/or with negative testing for NF1 germline variants and to introduce genotype-phenotype correlations for the novel molecular findings. Materials and Methods. We clinically re-evaluated 770 individuals who underwent NF1/SPRED1 sequencing and MLPA. Next Generation Sequencing (NGS) and digital PCR were performed in bioptic samples of cases with clinical suspicion of mNF1. Cases without a suspicion of mNF1 underwent NGS RASopathy panel. To assess the impact of the changes in the RAS-mitogen-activated protein kinase (MAPK) pathway, we performed functional studies of the detected candidate variants. 5 Results. A monoallelic pathogenic variant in NF1 was detected in 419 cases. New genotype-phenotype correlation. Eight of the individuals bearing NF1 pathogenic variants did not present neurofibromas. Specifically, they carried a variant in codon 1436 in the NF1 gene. Functional studies revealed the inefficiency of the mutant neurofibromin to inactivate RAS, an anomalous interaction with SPRED1 and a more widespread cytoplasmic localization. mNF1 somatic mutational spectrum. Sequencing performed on the tissue specimen of individuals with mNF1 suspicion and negative results on peripheral blood revealed the first and second hit in a relevant portion of cases, thereby demonstrating the speculated underlying mechanism. Heterozygous LZTR1 variants are associated with CaLMs. Patients with NF1/SPRED1 negative results and without a mosaic phenotype underwent RASopathy panel; a heterozygous LZTR1 variant was detected in 18 cases. To evaluate the penetrance of the LZTR1 variants, additional individuals were collected, resulting in a total cohort of 125 participants. The molecular spectrum of LZTR1 mainly consisted of truncating variants, indicating loss-of-function. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate MAPK signaling. These findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive Noonan Syndrome, appear to occur with statistical significance in individuals with isolated multiple CaLMs. Conclusions. The findings obtained enable to characterize the phenotypic differences form classical NF1 resulting from specific variants, and disclosed novel molecular associations for patients with NF1-like clinical features due to different biological mechanisms, showing the broad variability of neurocutaneous phenotypes. The research on the molecular bases of these conditions, through clinical characterization and functional assays, can guide in defining the most appropriate diagnostic approach and in planning the surveillance of this group of individuals. Knowing the biological bases of a disease plays a critical role in defining follow-up strategies for tumour surveillance and reproductive risk management, offering the possibility of being taken care of at a reference centre and using target therapies, if available.