Identification and functional analysis of a novel de novo missense mutation located in the initiation codon of LAMP2 associated with early onset female Danon disease

Abstract Background Danon disease is characterized by the failure of lysosomal biogenesis, maturation, and function due to a deficiency of lysosomal membrane structural protein ( LAMP2 ). Methods The current report describes a female patient with a sudden syncope and hypertrophic cardiomyopathy phenotype. We identified the pathogenic mutations in patients by whole‐exon sequencing, followed by a series of molecular biology and genetic approaches to identify and functional analysis of the mutations. Results Suggestive findings by cardiac magnetic resonance (CMR), electrocardiogram (ECG), and laboratory examination suggested Danon disease which was confirmed by genetic testing. The patient carried a novel de novo mutation, LAMP2 c.2T>C located at the initiation codon. The quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis of peripheral blood leukocytes from the patients revealed evidence of LAMP2 haploinsufficiency. Labeling of the new initiation codon predicted by the software with green fluorescent protein followed by fluorescence microscopy and Western blotting showed that the first ATG downstream from the original initiation codon became the new translational initiation codon. The three‐dimensional structure of the mutated protein predicted by alphafold2 revealed that it consisted of only six amino acids and failed to form a functional polypeptide or protein. Overexpression of the mutated LAMP2 c.2T>C showed a loss of function of the protein, as assessed by the dual‐fluorescence autophagy indicator system. The mutation was confirmed to be null, AR experiments and sequencing results confirmed that 28% of the mutant X chromosome remained active. Conclusion We propose possible mechanisms of mutations associated with haploinsufficiency of LAMP2 : (1) The inactivation X chromosome carrying the mutation was not significantly skewed. However, it decreased in the mRNA level and the expression ratio of the mutant transcripts; (2) The identified mutation is null, and the active mutant transcript fails to translate into the normal LAMP2 proteins. The presence of haploinsufficiency in LAMP2 and the X chromosome inactivation pattern were crucial factors contributing to the early onset of Danon disease in this female patient.