Evaluation of GFM1 mutations pathogenicity through in silico tools, RNA sequencing and mitophagy pahtway in GFM1 knockout cells

GFM1 is a nuclear gene that plays a role in mitochondrial function. In recent decades, various homozygous and compound heterozygous mutations have been identified, leading to significant health issues in patients and often resulting in early death. There is a few experimental research on this gene, particularly regarding its pathogenicity through in silico methods and RNA sequencing and experimental validation in GFM1 knockout cells. This study aims to explore how high-risk pathogenic variants affect protein stability and function using a comprehensive bioinformatics approach. Analyses with Align-GVGD, PolyPhen-2, MupRo, and SIFT indicated that most variants are likely to be highly pathogenic and destabilize the protein structure. The variants were consistently classified as high-risk by Align-GVGD and were deemed "probably damaging" or "possibly damaging" by PolyPhen-2. MupRo analysis suggested a reduction in protein stability, while SIFT indicated functional impacts for all variants. Further analysis with MetaRNN and structural assessments showed that these variants affect protein size, charge, and hydrophobicity, which may disrupt inter-domain interactions and hinder protein function. Differential gene expression analysis in GFM1 knockout HK2 and 293 T cells revealed significant changes in gene expression, particularly in areas related to translation, mitochondrial function, and cellular responses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that the affected genes are linked to neurodegenerative diseases, cancer, and various signaling pathways. GFM1 knockout cells displayed notable pathway changes, including those related to oxidative phosphorylation and neurodegenerative diseases (e.g., Parkinson's, Alzheimer's, Huntington's). Upregulation of mitochondrial electron transport chain components (COX17, NDUFB1, ATP5MC1) suggests a compensatory mechanism in response to impaired mitochondrial function. Disruptions in proteostasis and protein synthesis were highlighted by dysregulated proteasome and ribosomal pathways. Markers of mitophagy, such as increased HSP90 and decreased TOMM20 levels, along with changes in PINK1 protein, emphasize GFM1's involvement in mitophagy. Protein-protein interaction analysis connected GFM1 to key mitophagy proteins (e.g., OPTN, Park2/Parkin). Functional experiments confirmed increased mitophagy, indicating a protective response. These results highlight the negative impact of high-risk pathogenic variants on protein stability and cellular function, shedding light on their potential roles in disease progression. This study offers valuable insights into the pathogenic mechanisms linked to GFM1 mutations, underscoring its critical role in mitochondrial function and cellular balance. The findings highlight the gene's involvement in mitophagy, oxidative phosphorylation, and neurodegenerative pathways, laying the groundwork for future research into therapeutic approaches targeting GFM1-related dysfunctions.