Confronto tra gli approcci metodologici utilizzati per individuare l'espansione esonucleotidica GGGGCC nel gene C9orf72 nei casi SLA/FTD per cui non sono state identificate mutazioni genetiche

The presence of GGGGCC hexanucleotide repeat expansion in a non-coding region of C9orf72 gene is one the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat expansion occurs in 37% of familial ALS cases, 25% of familial FTD cases and up to 88% of familial ALS/FTD cases, but there is a percentage of cases of both ALS and ALS/FTD for whom a genetic cause is not detected. The most used genetic testing for G4C2-repeat expansion detection are the Amplicon Length Analysis and the Repeat-Primed Polymerase Chain Reaction (RP-PCR) that are able to evaluate the allelic size and the presence of the expansion respectively and allow to establish the frequency in the population worldwide. Southern Blot Analysis can determine the real expansion size, but it is not routinely applied because it is expensive and time consuming. Actually, the estimation of the expansion size is challenging and, for this reason, limitation of standard methodological approaches could represent a hindrance for establishing a definite cut-off between healthy and pathogenic cases. Furthermore, the recent discovery of a small deletion in C9orf72, that hides the presence of the expansion in a portion of UK population, highlighted the necessity to make an accurate analysis of patients undergoing genetic testing, in order to avoid the possibility of false negative results. In this study we selected 48 Italian cases of fALS or probable fALS and ALS with cognitive involvement, previously tested by a RP-PCR protocol usually performed in our laboratory and negative for C9orf72 G4C2-hexanucleotide repeat expansion. Moreover, NGS analysis on DNA of these patients was performed and it revealed a negativity for mutations in 33 ALS-related genes. In order to confirm the findings of our previous C9orf72 analysis and evaluate the failure in identifying expansions, we performed Amplicon Length Analysis, an alternative validated protocol of RP-PCR able to bypass the small deletion, and a new commercial kit with a higher resolution genotyping. Results obtained by each technique confirmed the negativity for G4C2-repeat expansion in all samples and excluded the presence of the deletion which could make the expansion undetectable. These results have demonstrated the reliability of the methodological approach previously used in our laboratory and have also suggested the requirement to perform both amplicon length analysis and RP-PCR for each sample, in order to ensure a precise detection of G4C2- repeat expansions. For this reason, the new commercial kit meets this requirement, because it combines the two methods together. Our cohort of fALS still remains without a genetic evidence, indicating that other causative genes remain to be identified in order to provide insights about cellular mechanism underlying neuron degeneration in ALS/FTD pathogenesis. Finally, future studies will also help to develop effective therapeutic strategies.