Loss-of-function SLC25A20 mutation causes carnitine-acylcarnitine translocase deficiency by reducing SLC25A20 protein stability.

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Autosomal-recessive carnitine-acylcarnitine translocase deficiency (CACTD) is a rare disorder of long-chain fatty acid oxidation caused by variants in the SLC25A20 gene. Under fasting conditions, most newborns with severe CACTD experience sudden cardiac arrest and hypotonia, often leading to premature death due to rapid disease progression. Understanding of genetic factors and pathogenic mechanisms in CACTD is essential for its diagnosis, treatment, and prevention. Whole-exome sequencing was carried out on the CACTD patients. Bioinformatics analysis predicted the pathogenicity and three-dimensional structure of SLC25A20. Quantitative PCR was employed to detect changes in SLC25A20, CPT1A and CPT2 mRNA levels. The expression and stability of the mutant protein were assessed via Western blot. Additionally, the subcellular localization of the mutant protein was observed using immunofluorescence. We identified novel pathogenic compound heterozygous variants of SLC25A20 (c.476 T > C and c.199-10 T > G) in CACTD families, with patients exhibiting an abnormal carnitine spectrum. In vitro functional studies demonstrated that the c.476 T > C and c.199-10 T > G variants decreased the protein stability of SLC25A20, reduced CPT1A and CPT2 mRNA expression, and caused protein aggregation of SLC25A20. We propose that the decreased stability of the SLC25A20 variants c.476 T > C and c.199-10 T > G has the potential to lead to the development of CACTD by affecting the mitochondrial shuttle of acylcarnitine and carnitine, thereby inhibiting the β-oxidation pathway. Therefore, we believe this novel combination of variants (c.199-10 T > G and c.476 T > C) are loss-of-function variants. Our findings provide valuable data on CACTD pathogenesis and genotype-phenotype correlations.