Missense Mutation in AIFM1 Gene Leads to Mitochondrial Dysfunction and Riboflavin Deficiency Intolerance

Research Background

Mitochondria are double-membrane organelles found in eukaryotic cells with nuclei, primarily producing adenosine triphosphate (ATP) through oxidative phosphorylation to provide cellular energy. Mitochondria have their own genome, encoding 13 proteins involved in mitochondrial respiratory chain complexes, while other mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria. Among them, Apoptosis-Inducing Factor (AIFM1) is a mitochondrial flavoprotein encoded by the X-linked AIFM1 gene, involved in caspase-independent cell death and regulating the biogenesis of respiratory chain complexes.

Studies have shown that mutations in the AIFM1 gene are associated with various clinical phenotypes, but the effectiveness of riboflavin (vitamin B2) treatment remains controversial. This study aims to explore the mechanism of mitochondrial dysfunction caused by the c.1019T > C variant of the AIFM1 gene and evaluate the effect of riboflavin supplementation on this variant.

Research Source

This study was jointly conducted by researchers from the Institute of Neuromuscular and Neurodegenerative Diseases at Qilu Hospital of Shandong University, Mitochondrial Medicine Laboratory at Qilu Hospital (Qingdao) of Shandong University, Institute of Brain Science at Shandong University, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, and other institutions. The main authors include Zhao Ying, Lin Yan, Wang Bin, Liu Fuchen, et al. The research findings were received on May 23, 2023, accepted on August 9, 2023, and published online in “Neuromolecular Medicine” on August 21, 2023.

Research Methods and Procedures

Subjects

The subject was a 7-year-old boy hospitalized for gait abnormalities, presenting with cerebellar ataxia, axonal sensorimotor neuropathy, and muscle weakness, with no significant family history. After approval by the ethics committee, whole-genome DNA sequencing and histological analysis were performed on the boy and his parents.

Genomic and Amino Acid Analysis

Total genomic DNA was extracted from peripheral blood and subjected to next-generation sequencing (NGS) for neurological disease genomic testing. Sanger sequencing was used to confirm variants and perform family verification. Amino acid conservation analysis was completed using MutationTaster.

Muscle Histology and Immunohistochemistry Studies

A biceps biopsy was performed on the patient, and 8μm thick frozen sections underwent a series of histochemical and immunohistochemical staining, including hematoxylin-eosin (HE) staining, modified Gomori trichrome (MGT) staining, cytochrome C oxidase (COX) staining, etc. Additionally, immunohistochemical staining with AIFM1 antibody (17984-1-AP, Proteintech) was performed on muscle sections.

Fibroblast Culture

Primary skin fibroblasts were obtained from skin biopsies and cultured according to standard methods. Fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. For riboflavin deficiency studies, custom DMEM medium without riboflavin was used. For riboflavin supplementation studies, equal amounts of riboflavin solutions at different concentrations were added.

Western Blotting

Muscle samples and skin fibroblasts were lysed and protein quantified. 30μg of protein was loaded for each sample, electrophoresed in 12% denatured SDS-PAGE, then transferred to PVDF membranes, and blotted with primary antibodies against AIFM1, OXPHOS Antibody Cocktail, and other mitochondrial respiratory chain complex subunits.

Oxygen Consumption Rate Measurement

Oxygen consumption rates of fibroblasts were measured using a Seahorse Bioscience XFe24 analyzer, and ATP production, maximum respiration, and non-mitochondrial respiration were measured after adding various inhibitors and uncouplers.

Mitochondrial Membrane Potential Measurement

Mitochondrial membrane potential (Δψm) was detected using the mitochondria-specific dye JC-1, observed under a fluorescence microscope and quantitatively analyzed.

Apoptosis Assay

Apoptosis levels in fibroblasts were measured using the Tunel staining method, observing Tunel-labeled cells at 488nm excitation and 530nm emission, with apoptosis induced after Staurosporine treatment.

Statistical Analysis

Statistical analysis was performed using SPSS 24.0 software and GraphPad Prism 8. Quantitative data are presented as mean ± standard deviation, analyzed using one-tailed Student’s t-test, with p-values less than 0.05 considered statistically significant.

Research Results

Identification of AIFM1 Gene Variant

Genomic DNA sequencing revealed a c.1019T > C missense variant in the AIFM1 gene in the patient. Amino acid conservation analysis showed that the sequence at this site is highly conserved across multiple species, and the variant leads to a change from methionine to threonine (p. Met340Thr). According to the American College of Medical Genetics and Genomics (ACMG) guidelines, this variant was classified as a “likely pathogenic” variant.

Mitochondrial Abnormalities

The patient’s biceps biopsy showed inconsistent muscle fiber sizes, with MGT staining revealing small red subsarcolemmal patches, indicating abnormal mitochondrial aggregation. COX staining showed a few fibers with reduced COX, and SDH/COX double staining confirmed mitochondrial abnormalities with abnormal blue fibers. ATPase staining showed mild reinnervation phenomena.

Decreased Expression of AIFM1 and Mitochondrial Complex Subunits

Immunohistochemical staining and Western Blot results showed significantly reduced expression of AIFM1 in the patient’s muscle tissue and skin fibroblasts. Simultaneously, the expression of the NDUFB8 subunit of complex I and the CYTB subunit of complex III were significantly decreased in muscle tissue.

Mitochondrial Respiratory Dysfunction

Seahorse analyzer measurements showed significantly reduced basal respiration, ATP production, proton leak, maximum respiration, and spare respiratory capacity in the patient’s fibroblasts. Complex I, II, and IV-mediated respiration was significantly reduced in mutant cells.

Riboflavin Deficiency Induces AIFM1 Downregulation

Under riboflavin-deficient conditions, AIFM1 levels gradually decreased in both mutant and wild-type fibroblasts, with a more pronounced decrease in mutant cells. High concentrations of riboflavin (2 and 20mg/L) could partially increase AIFM1 levels.

Improvement in Clinical Symptoms

After 10 months of treatment with 100mg of riboflavin daily, the patient showed significant improvement in ataxia and muscle weakness, with a 19% improvement in the International Cooperative Ataxia Rating Scale (ICARS).

Conclusion

This study validates the pathogenicity of the c.1019T > C variant in the AIFM1 gene, leading to mitochondrial dysfunction and intolerance to riboflavin deficiency in cells with this variant. Riboflavin supplementation helps maintain AIFM1 protein levels and mitochondrial respiratory function. Early riboflavin treatment may have therapeutic value for patients with AIFM1 variants.

Research Significance

This study provides new scientific evidence for mitochondrial diseases caused by AIFM1 gene variants, emphasizes the importance of early riboflavin treatment, and provides a reference for future clinical treatments.