A new mutation detected by NGS in MT-ATP6 gene associated with MELAS and in silico 3D protein analysis

Introduction The possibility to utilize today tools for the 3D structural analysis of proteins based on artificial intelligence such as AlphaFold, allows to obtain with relative ease, very useful functional indications related to the modifications caused by gene mutations, comparable with those that can be obtained experimentally. Objective The aim was to understand the impact of mitochondrial gene mutations identified by NGS on protein stability by means of an in silico structural approach. Materials and Methods Thirdy DNA samples, previously analyzed for canonical diagnostic mitochondrial DNA mutations were deep sequenced in order to identify the possible mtDNA genetic determinants. The Precision ID mtDNA Whole Genome Panel was utilized and an Ion 318 Chip Kit v2 was subjected to the ION Torrent PGM sequencing machine. The sequences were reviewed by IGV, v2.1.13 software. The variants were annotated and filtered considering the type of pathogenic variants, the population frequencies and presence in database and an heteroplasmy >5%. Protein structures of interest were loaded as PDB files from UniProt on-line database ((www.expasy.org/). The PDB files by AlphaFold, were open and analysed using SPDBV viewer software version 4.1. Results After the alignment, the coverage analysis reported 100% of the on target reads, with a uniformity of the 99.9% and a mean coverage of 574x. We identified 27 non-synonymous variants with a MAF <1%. Among these variants, we focused our attention on the MT-ATP6 gene carrying the mutation m.9124A> G; p.T200A and associated with MELAS. The related protein belongs to the ATP sinthase intramembrane unit F0. The 3D structure of this protein with a polar non-polar variation (T200A) shows a loss of the intrachain H-bond at the H6 domain between residue 200 and 197. Discussions and Conclusions The new gene variant let us to suggest that at protein level the resulting alteration could distorce the H6 helical structure inducing an anomalous protein functionality related to Melas. Of interest is the observation that an analogous variant already identified, the A177T, on the H5 domain, associated with MELAS, determines also an H-bond alteration. Because the H5 and H6 alpha-helical are in a very near region, we therefore suggest that the protein functionality linked to this region (H5-H6) may be related to the MELAS phenotype.