Spectrum of NF1 variants in Portugal and implementation of NF1 RNA-based approach

• Autor(a) principal: Pinho, Diana Isabel de Jesus Serrador Moreira de
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/36571
• topic: Neurofibromatosis type 1; NF1 gene; Mutational spectrum; RNA-based approach; ACMG variant classification

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic disorders and is caused by inactivating variants in the NF1 gene. The hallmarks of this chronic progressive disease are the development of benign neurofibromas as well as café-au-lait macules, axillary and inguinal freckling, Lisch nodules, optic nerve glioma and malignant peripheral nerve sheath tumours. Genetic analysis of NF1 may facilitate the identification of patients in early stages of the disease. This project reports a retrospective re-examination of the genetic variants identified in NF1 gene over a period of 10 years in the laboratory of Genetics Diagnostics of Ipatimup Diagnostics. The classification of 147 NF1 germline variants identified in 165 patients belonging to a cohort of 220 unrelated individuals with suspected neurofibromatosis has been reviewed. All genetic findings were reassessed according to the Guidelines for the Interpretation of Sequence Variants of the ACMG-AMP consensus and stratified into one of the five proposed classes (benign, likely benign, uncertain significance, likely pathogenic and pathogenic). The 147 variants included 53 frameshift alterations and 4 small inframe deletions, 32 nonsense and 25 missenses variants, 18 splicing alterations and 10 copy number variations including the entire NF1 gene, 4 complex variants and 1 synonymous. Most of these genetic changes are unique and distributed along the gene, although data suggest that five functional domains (CSRD, TBD, GRD and SEC-PH domains) are enriched in variants, which account for 46.8% of the NF1 alterations in this cohort. The second aim of this study was to implement a full-length RNA sequencing method, in order to detected splicing changes, which cannot be identified by the study of genomic DNA, commonly used. As such, in last 2 years, the search for variants in the NF1 gene in 28 unrelated patients has been investigated by studying the entire NF1 cDNA. According to the literature, the sensitivity, cost-effectiveness and reliability of a comprehensive RNA-based approach represents enormous advantages in comparison to specific DNA amplification. The detection rate is expected to be higher, which can be explained by the frequent occurrence of splicing defects in the NF1 gene that occur outside the canonical splice site. A total of 18 variants have been identified, of which 3 would not be detectable by the conventional technique previously used. Indeed, in 4 of the cases, the study of the complete RNA complemented with the gDNA allowed to identify 2 deep intronic variants (c.288+1139T>A and c.1642-449A>G), an Alu element insertion (c.587-4_587-3insAluYa5,587-15_587-4dupTTGTGTTTTTTC) and a complex indel variant (c.3975-1011_4110+1674delinsGGGCGGATCACGAGGTCAGGAGAT). Thus, this study demonstrates the advantage of an approach that integrates cDNA/gDNA analysis: the dual characterization of genetic alterations at both levels allows the detection of a broader spectrum of variants than any single-level approach, and provides a comprehensive understanding of its molecular pathogenesis.