Mechanistic insights into NFIX frameshift mutations in Malan syndrome: proteasomal degradation-mediated haploinsufficiency
Year: 2025
Type: article
Abstract: Objective To investigated the pathogenic mechanism of NFIX frameshift mutations in Malan syndrome. Methods Reviewed the clinical diagnosis and treatment processes of the Malan syndrome proband, analyzing the relationship between NFIX frameshift mutation genotypes and clinical phenotypes, and the inheritance pattern. To analyzed the functional domain where the mutation was located and the conservation of the mutated amino acid residue, thereby elucidating the potential impact of the mutation on the protein. Validated effects on pre-mRNA splicing using RDDC SC , SpliceAI, and FF databases. Assessed variant pathogenicity via MutationTaster, PolyPhen-2, and VarCards. Constructed wild-type/mutant plasmids, transfected to HEK293T cells, and quantified NFIX mRNA and protein expression levels via qPCR and Western blot. Analyzed degradation pathways using ubiquitin-proteasome inhibitor MG132 and autophagy-lysosome inhibitor Chloroquine (CQ). Results The proband exhibited intellectual disability, distinctive facial features, ocular abnormalities, scoliosis, and primary infertility. A de novo mutation in NFIX (c.164delC, p.Ala55Glyfs*2) associated with these phenotypes was identified. Neither the proband’s father nor his mother was found to have this mutation. Parental testing confirmed de novo inheritance. The amino acid at position 55 was highly conserved and had been Alanine in 5 species. Results from databases including RDDC SC , SpliceAI, and FF indicated that the NFIX c.164delC p.Ala55Glyfs*2 mutation did not affect splicing function. Predictions by MutationTaster and PolyPhen-2 classified the c.707G>A p.Arg236Gln mutation as “damaging,” suggesting an altered amino acid sequence, frameshift mutation, NMD, and potential modification of protein characteristics. Quantitative real-time PCR (qPCR) analysis detected comparable mRNA levels between mutant and wild-type strains. In contrast, Western blotting revealed significantly diminished protein expression in the mutant ( P < 0.05), suggesting post-transcriptional regulation effects. Results from protein degradation pathway analysis demonstrated that the truncated protein generated after mutation was degraded via the ubiquitin-proteasome pathway. Conclusion The NFIX c.164delC p.Ala55Glyfs*2 frameshift mutation did not significantly affect mRNA expression levels, but induced protein degradation via the ubiquitin-proteasome pathway, resulting in haploinsufficiency and ultimately causing Malan syndrome.
Source: Frontiers in Genetics
Institution: Jingchu University of Technology
Language: English
Topic: Genomics and Rare Diseases
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,801
Awards: G4417158568, G762269078, G8694036849
OpenAlex ID: W4415974898