A novel diagnostic method to detect truncated neurofibromin in neurofibromatosis 1

Abstract

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition caused by dominant loss-of-function mutations of the tumor suppressor gene NF1 that encodes neurofibromin, a negative regulator of RAS activity. Mutation analysis of NF1 located at 17q11.2 has been hampered by the large size of the gene, the high rate of new mutations, the lack of mutational clustering, and the presence of several homologous loci. To date, about 80% of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the protein level. We evaluated a novel diagnostic method to detect truncated forms of neurofibromin in a large cohort of unrelated subjects suspected of having NF1, according to the NIH consensus criteria. Western blot analysis was carried out on protein extracts from patients' leukocytes to highlight the possible presence of altered neurofibromin as a result of mutations in NF1. Truncated neurofibromin was identified in 274/336 patients (81%), confirming the usefulness and reproducibility of the proposed diagnostic approach. Our methodology can be routinely applied in the diagnostic setting, thanks to its simplicity and reliability. Combined with molecular approaches, it may increase the accuracy and efficiency of NF1 genetic testing.

Understanding the Biological Activities of Vitamin D in Type 1 Neurofibromatosis: New Insights into Disease Pathogenesis and Therapeutic Design

“…NF1 exhibits one of the highest spontaneous mutation rates in the human genome; indeed, almost half of the patients present de novo pathogenic variants [ 254 , 255 , 256 , 257 ]. The majority (>80%) of constitutional NF1 mutations result in truncated forms of neurofibromin, which consequently shows loss of function or decreased activity [ 256 , 257 , 258 , 259 ]. The extreme clinical variability of NF1 disease suggests that random events intervene in determining the phenotype.…”

“…Although genetic tests are available [ 259 , 369 ], NF1 remains a clinical diagnosis and the majority of affected subjects are identified in infancy or childhood [ 370 ].…”

CRMP2 and voltage-gated ion channels: potential roles in neuropathic pain

“…Strikingly, neurofibromatosis type-1 (NF1) patients, in whom neurofibromin is often mutated or truncated, experience idiopathic chronic pain for which opioids often fail to provide relief [106–109]. Observations of DRGs in Nf1 +/− haploinsufficient mice revealed augmented sodium (Na + ) current densities (both tetrodotoxin (TTX)-sensitive and TTX-resistant), alongside increased N-type Ca 2+ currents and enhanced stimulus-evoked release of glutamate, substance P, and CGRP [110–113].…”

Neurofibromin (NF1 ) genetic variant structure-function analyses using a full-length mouse cDNA

“…Finally, the heterologous cDNA expression system has allowed us to validate commercially available antibodies that recognize mature and truncated versions of neurofibromin proteins with different specificity to either C‐terminus (SC‐67, rabbit polyclonal) or N‐terminal amino acids 241–540 (sc‐376886, mouse monoclonal). While the C‐terminal antibody had been validated previously (Esposito et al., ), we now describe using the N‐terminal sc‐376886 to detect truncated proteins. Ultimately with further validation, this assay system might be used to transition likely pathogenic to pathogenic variants in clinical lab assessments.…”