PMC8655779_01
The clinical and genetic characteristics of the pedigree. (A) The 12-lead electrocardiogram (ECG) of the proband revealed ventricular tachycardia and intraventricular block. (B) The family tree of the cardiac-only DD pedigree is included in this report. Male and female are indicated by squares and circles, respectively. The black filled symbol represents the clinical affected individual. The gray-filled symbol represents the phenotype unknown individual. The arrow shows the proband. -/+ represents heterozygous LAMP2 c.741+2T>C variant. -/- represents hemizygous LAMP2 c.741+2T>C variant. +/+ represents a wild type. (C) The Sanger sequencing of the family. Colored blocks show the evolutionary conservation of the cluster across multiple species.
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F1
TnI, CK, lactic acid, liver and kidney function, levels of serum electrolytes, blood glucose, and lipids) of the mother were all in the normal range. The rest of his relatives did not show obvious anomalies during physical or laboratory examinations (Figure 1B). After filtering and Sanger sequencing validation, we identified a pathogenic hemizygous 3' splice site mutation, c.741+2T>C, in intron 5 of the LAMP2 gene (Figure 1C), according to the ACMG guideline. The mutation was also identified in the mother (heterozygous) but was not identified in the rest relatives or the extra 800 unrelated Chinese healthy controls (Figure 1). Furthermore, the mutation was in an evolutionary highly conserved region across multiple species (Figure 1C).
PMC8655779_02
The clinical and genetic characteristics of the pedigree. (A) The 12-lead electrocardiogram (ECG) of the proband revealed ventricular tachycardia and intraventricular block. (B) The family tree of the cardiac-only DD pedigree is included in this report. Male and female are indicated by squares and circles, respectively. The black filled symbol represents the clinical affected individual. The gray-filled symbol represents the phenotype unknown individual. The arrow shows the proband. -/+ represents heterozygous LAMP2 c.741+2T>C variant. -/- represents hemizygous LAMP2 c.741+2T>C variant. +/+ represents a wild type. (C) The Sanger sequencing of the family. Colored blocks show the evolutionary conservation of the cluster across multiple species.
fcvm-08-763240-g0001.jpg
PMC8655779_02_fcvm-08-763240-g0001.jpg
F1
TnI, CK, lactic acid, liver and kidney function, levels of serum electrolytes, blood glucose, and lipids) of the mother were all in the normal range. The rest of his relatives did not show obvious anomalies during physical or laboratory examinations (Figure 1B). After filtering and Sanger sequencing validation, we identified a pathogenic hemizygous 3' splice site mutation, c.741+2T>C, in intron 5 of the LAMP2 gene (Figure 1C), according to the ACMG guideline. The mutation was also identified in the mother (heterozygous) but was not identified in the rest relatives or the extra 800 unrelated Chinese healthy controls (Figure 1). Furthermore, the mutation was in an evolutionary highly conserved region across multiple species (Figure 1C).
The minigene assay of the mutation. (A) The construction of the LAMP2-PET01 minigene plasmid. (B) Electrophoresis of the cDNA products. Total RNA was extracted after 48 h the minigene plasmid was transfected and reverse transcribed into cDNA. Mut represents cDNA from mutant plasmid treated. WT represents cDNA from wild-type plasmid treated. Con, represents blank control without any cDNA. (C) Sanger sequencing of the cDNA products. Sequencing identified extra 6-bp preservation of intron 5 at the junction between exons 5 and exon SA during transcriptional processing of the mRNA, which creates a stop codon.
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F2
We compared the RT-PCR products of wild-type and mutant mRNA by electrophoresis and found the mutant cDNA was a little larger than the wild-type cDNA (Figure 2B). The inserted nucleotides had the same sequence as the 5'-end of intron 5, which implied the preservation of intron 5 at the junction between exons 5 and 6 during transcriptional processing of the mRNA (Figure 2C). The inserted sequence encoded an in-frame stop codon, which predicted a premature termination of the nascent polypeptide (Figure 2C).
In-silico structural modeling of the identified mutation. (A) Schematic diagram of the wild-type LAMP2 protein and the mutant truncated LAMP2 protein. (B) Protein structural modeling of the wild-type and mutant LAMP2 protein.
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F3
The mutation truncated the LAMP2 protein to 248-amino-acid residues, which lacks the transmembrane domain and the cytoplasmic domain, therefore, fail to function properly (Figure 3).