Heart Center, The First Hospital of Lanzhou University, Lanzhou, Gansu, China.
Gansu Key Laboratory of Cardiovascular Diseases, The First Hospital of Lanzhou University, Lanzhou, Gansu, China.
Mol Genet Genomic Med. 2023 Sep;11(9):e2216. doi: 10.1002/mgg3.2216. Epub 2023 Jun 8.
Danon disease is characterized by the failure of lysosomal biogenesis, maturation, and function due to a deficiency of lysosomal membrane structural protein (LAMP2).
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.
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.
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.
丹农病的特征是由于溶酶体膜结构蛋白（LAMP2）缺乏，溶酶体发生生物发生、成熟和功能障碍。
本报告描述了一名女性患者，其表现为突然晕厥和肥厚型心肌病表型。我们通过全外显子组测序鉴定了患者的致病性突变，然后通过一系列分子生物学和遗传学方法对突变进行鉴定和功能分析。
心脏磁共振（CMR）、心电图（ECG）和实验室检查的提示性发现提示丹农病，基因检测结果证实了这一点。患者携带一种新的从头突变，位于起始密码子的 LAMP2 c.2T>C。对患者外周血白细胞进行定量聚合酶链反应（qPCR）和 Western blot（WB）分析显示 LAMP2 杂合不足的证据。软件预测的新起始密码子标记为绿色荧光蛋白，随后进行荧光显微镜和 Western blot 显示，原始起始密码子下游的第一个 ATG 成为新的翻译起始密码子。由 alphafold2 预测的突变蛋白的三维结构显示，它仅由六个氨基酸组成，无法形成功能性多肽或蛋白质。通过双荧光自噬指示系统评估，突变型 LAMP2 c.2T>C 的过表达导致蛋白功能丧失。突变被确认为无效，AR 实验和测序结果证实 28%的突变 X 染色体仍保持活性。
我们提出了与 LAMP2 杂合不足相关的突变的可能机制：（1）携带突变的失活 X 染色体没有明显偏斜。然而，它在 mRNA 水平和突变转录本的表达比例上降低；（2）鉴定出的突变是无效的，活性突变转录本不能翻译成正常的 LAMP2 蛋白。LAMP2 中的杂合不足和 X 染色体失活模式是导致该女性患者丹农病早发的关键因素。
