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.

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.

Myocardial Strain and Association With Clinical Outcomes in Danon Disease: A Model for Monitoring Progression of Genetic Cardiomyopathies.

Background Myocardial strain can identify subclinical left ventricular dysfunction in various cardiac diseases, but its association with clinical outcomes in genetic cardiomyopathies remains unknown. Herein, we assessed myocardial strain in patients with Danon disease (DD), a rare X-linked autophagic disorder that causes severe cardiac manifestations. Methods and Results Echocardiographic images were reviewed and used to calculate myocardial strain from a retrospective, international registry of patients with DD. Regression analyses were performed to evaluate for an association of global longitudinal strain (GLS) and ejection fraction with the composite outcome (death, ventricular assist device, heart transplantation, and implantable cardioverter defibrillator for secondary prevention). A total of 22 patients with DD (male 14 [63.6%], median age 16.5 years) had sufficient echocardiograms for analysis. Absolute GLS was reduced with a mean of 12.2% with an apical-sparing pattern observed. Univariable regression for GLS and composite outcome showed an odds ratio of 1.32 (95% CI, 1.02-1.71) with P=0.03. For receiver operating characteristic analysis, the areas under the curve for GLS and ejection fraction were 0.810 (P=0.02) and 0.605 (P=0.44), respectively. An absolute GLS cutoff of 10.0% yielded a true positive rate of 85.7% and false positive rate of 13.3%. Conclusions In this cohort of patients with DD, GLS may be a useful assessment of myocardial function and may predict clinical outcomes. This study highlights the potential use of myocardial strain phenotyping to monitor disease progression and potentially to predict clinical outcomes in DD and other genetic cardiomyopathies.

Application of Patient-Specific iPSCs for Modelling and Treatment of X-Linked Cardiomyopathies.

Inherited cardiomyopathies are among the major causes of heart failure and associated with significant mortality and morbidity. Currently, over 70 genes have been linked to the etiology of various forms of cardiomyopathy, some of which are X-linked. Due to the lack of appropriate cell and animal models, it has been difficult to model these X-linked cardiomyopathies. With the advancement of induced pluripotent stem cell (iPSC) technology, the ability to generate iPSC lines from patients with X-linked cardiomyopathy has facilitated in vitro modelling and drug testing for the condition. Nonetheless, due to the mosaicism of the X-chromosome inactivation, disease phenotypes of X-linked cardiomyopathy in heterozygous females are also usually more heterogeneous, with a broad spectrum of presentation. Recent advancements in iPSC procedures have enabled the isolation of cells with different lyonisation to generate isogenic disease and control cell lines. In this review, we will summarise the current strategies and examples of using an iPSC-based model to study different types of X-linked cardiomyopathy. The potential application of isogenic iPSC lines derived from a female patient with heterozygous Danon disease and drug screening will be demonstrated by our preliminary data. The limitations of an iPSC-derived cardiomyocyte-based platform will also be addressed.

Clinical Profile of Cardiac Involvement in Danon Disease: A Multicenter European Registry.

BACKGROUND: The X-linked Danon disease manifests by severe cardiomyopathy, myopathy, and neuropsychiatric problems. We designed this registry to generate a comprehensive picture of clinical presentations and outcome of patients with Danon disease in cardiomyopathy centers throughout Europe. METHODS: Clinical and genetic data were collected in 16 cardiology centers from 8 European countries. RESULTS: The cohort comprised 30 male and 27 female patients. The age at diagnosis was birth to 42 years in men and 2 to 65 in women. Cardiac involvement was observed in 96%. Extracardiac manifestations were prominent in men but not in women. Left ventricular (LV) hypertrophy was reported in 73% of male and 74% of female patients. LV systolic dysfunction was reported in 40% of men (who had LV ejection fraction, 34±11%) and 59% of women (LV ejection fraction, 28±13%). The risk of arrhythmia and heart failure was comparable among sexes. The age of first heart failure hospitalization was lower in men (18±6 versus 28±17 years; P<0.003). Heart failure was the leading cause of death (10 of 17; 59%), and LV systolic dysfunction predicted an adverse outcome. Eight men and 8 women (28%) underwent heart transplantation or received an LV assist device. Our cohort suggests better prognosis of female compared with male heart transplant recipients. CONCLUSIONS: Danon disease presents earlier in men than in women and runs a malignant course in both sexes, due to cardiac complications. Cardiomyopathy features, heart failure and arrhythmia, are similar among the sexes. Clinical diagnosis and management is extremely challenging in women due to phenotypic diversity and the absence of extracardiac manifestations.

Review: Danon disease: Review of natural history and recent advances.

Danon disease is a severe multisystem disorder clinically characterized by hypertrophic cardiomyopathy, skeletal myopathy and mental retardation in male patients, and by a milder phenotype (predominantly involving cardiac muscle) in female patients. The disease is inherited as an X-linked dominant trait. The primary deficiency of lysosome-associated membrane protein-2 (LAMP-2) causes disruption of autophagy, leading to an impaired fusion of lysosomes to autophagosomes and biogenesis of lysosomes. We surveyed over 500 Danon disease patients reported in the literature from the first description to the present, in order to summarize the clinical, pathological and molecular data and treatment perspectives. An early molecular diagnosis is of crucial importance for genetic counselling and for therapeutic interventions: in male patients, the prognosis is poor due to rapid progression towards heart failure, and only heart transplantation modifies the disease course.

Prevalence and clinical characteristics of Danon disease among patients with left ventricular hypertrophy and concomitant electrocardiographic preexcitation.

BACKGROUND: Cardiac involvement in Danon disease typically manifests as left ventricular hypertrophy (LVH) and ventricular preexcitation. This study aimed to identify patients with Danon disease among patients with LVH and concurrent electrocardiographic preexcitation. METHODS: Electrocardiographic preexcitation was identified in 10 of 197 patients with unexplained LVH in whom genetic testing was performed using next-generation sequencing. RESULTS: Three (3/10, 30%) patients with Danon disease were found in association with different mutations in the gene of lysosome-associated membrane protein 2 (LAMP2). Compared to seven patients without Danon disease, these three patients presented with distinctive clinical phenotypes, including onset at an earlier age (20 ± 2 years vs. 53 ± 9 years, p < 0.001), more neurological involvements (100% vs. 0, p = 0.008), higher electrocardiographic voltages (10 ± 1 mV vs. 5 ± 1 mV, p < 0.001), wider QRS complexes (163 ± 5 ms vs. 115 ± 20 ms, p = 0.006), less common asymmetric hypertrophy (0% vs. 86%, p = 0.033), and more frequent elevation of three serum enzymes (creatine kinase, aspartate aminotransferase, and lactate dehydrogenase). Intracellular vacuoles accumulation with deficiencies of LAMP2 protein was found in both cardiac and skeletal myocytes of patients with Danon disease. CONCLUSION: In patients with coexistent LVH and ventricular preexcitation, Danon disease is common with distinctive clinical presentations. Comprehensive assessment of these resemble patients can provide valuable findings for early identification and clinical decision making of patients with Danon disease.

A family with Danon disease caused by a splice site mutation in LAMP2 that generates a truncated protein.

BACKGROUND: Danon disease is an X-linked dominant hereditary condition caused by mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2), leading to failure of lysosome binding to autophagosomes, accumulation of glycogen in the heart, and abnormal cardiac function. METHODS: We describe identification of a mutation in LAMP2, c.741+1G>T, in a family with Danon disease by whole exome sequencing. RESULTS: Pathology examination of patient skeletal muscle biopsy showed myogenic damage and autophagic vacuoles with sarcolemmal features (AVSF). Numerous autophagic vacuoles accumulated in muscle cells were detected by electron microscopy, indicating abnormal autophagy function. CONCLUSION: The mutation did not result in loss of mRNA exons; rather, a 6-nucleotide (two-codon) insertion, where the latter was a stop codon, leading to early termination of LAMP2 protein translation. The resulting truncated protein lacks an important transmembrane domain, which will impair lysosome/autophagosome fusion, damage autophagy function, and result in the clinical manifestations of Danon disease.

LAMP-2B regulates human cardiomyocyte function by mediating autophagosome-lysosome fusion.

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome-lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome-lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B-mediated autophagy to treat this patient population.

A Nationwide Survey on Danon Disease in Japan.

Danon disease, an X-linked dominant cardioskeletal myopathy, is caused by primary deficiency of lysosome-associated membrane protein-2 (LAMP-2). To clarify the clinicopathological features and management, we performed the first nationwide, questionnaire-based survey on Danon disease in Japan. A total of 39 patients (17 males, 22 females) from 20 families were identified in the analysis. All patients had cardiomyopathy. Of the 21 patients who died, 20 (95%) died of cardiac failure or sudden cardiac arrest. Most patients had hypertrophic cardiomyopathy. Wolf⁻Parkinson⁻White syndrome was present at a comparatively high incidence (54% in males, 22% in females). Only one female patient received a heart transplant, which is the most effective therapy. Histopathologically, all male patients showed autophagic vacuoles with sarcolemmal features in muscle. Half of the probands showed de novo mutations. Male patients showed completely absent LAMP-2 expression in muscle. In contrast, female patients showed decreased LAMP-2 expression, which is suggested to reflect LAMP-2 haploinsufficiency due to a heterozygous null mutation. In conclusion, Danon disease is an extremely rare muscular disorder in Japan. Cardiomyopathy is the most significant prognostic factor and the main cause of death. Our findings suggest that the present survey can extend our understanding of the clinical features of this rare disease.

Characterisation of Lamp2-deficient rats for potential new animal model of Danon disease.

Danon disease (DD) is caused by the absence or malfunction of lysosomal-associated membrane protein 2 (LAMP2). Although Lamp2-deficient mice and DD patients have similar characteristics, these mice have clear limitations and are clinically inconsistent. The aim of our paper is to outline the characteristics of Lamp2-deficient rats and to contrast this model with currently available DD mouse models. The baseline levels of some serum enzymes were elevated in Lamp2y/- rats along with hypercholesterolemia and hyperglycaemia at 8 weeks. Echocardiography showed that IVSd (1.500 ± 0.071 vs. 2.200 ± 1.147, P < 0.01) and LVPWd (1.575 ± 0.063 vs. 1.850 ± 0.029, P < 0.01) were significantly increased, and GCS (-13.20 ± 0.4814 vs. -6.954 ± 0.665) and GRS (21.42 ± 1.807 vs. 7.788 ± 1.140) were sharply decreased. Meanwhile, substantial myocyte disruption, hypertrophic muscle fibres, interstitial fibrosis and microvascular hyperplasia could be observed in the heart tissue. Lamp2y/- rats also displayed abnormal behaviours in the open field and fear conditioning tests. Notably, Lamp2y/- rats manifested other system dysfunctions, such as retinopathy, chronic kidney injury and sterility. Based on these results, Lamp2-deficient rats exhibited greater similarity to DD patients in terms of onset and multisystem lesions than did mouse models, and these rats could be used as a valuable animal model for DD.

Heterogeneity in a large pedigree with Danon disease: Implications for pathogenesis and management.

BACKGROUND: Danon disease is an X-linked disturbance of autophagy manifesting with cognitive impairment and disordered heart and skeletal muscle. After a period of relative stability, patients deteriorate rapidly and may quickly become ineligible for elective heart transplantation - the only life-saving therapy. METHODS: We report a large pedigree with diverse manifestations of Danon disease in hemizygotes and female heterozygotes. RESULTS: Malignant cardiac arrhythmias requiring amiodarone treatment induced thyroid disease in two patients; intractable thyrotoxicosis, which enhances autophagy, caused the death of a 21year-old man. Our patients also had striking elevation of serum troponin I during the accelerated phase of their illness (p<0.01) and rising concentrations heralded cardiac decompensation. We argue for changes to cardiac transplantation eligibility criteria. CONCLUSION: Danon disease causes hypertrophic cardiomyopathy - here we propose a common pathophysiological basis for the metabolic and structural effects of this descriptive class of heart disorders. We also contend that troponin I may have prognostic value and merits exploration for clinical decision-making including health warning bracelets. Rapamycin (Sirolimus®), an approved immunosuppressant which also influences autophagy, may prove beneficial. In the interim, while new treatments are developed, a revaluation of cardiac transplantation eligibility criteria is warranted.

Characteristics of induced pluripotent stem cells from clinically divergent female monozygotic twins with Danon disease.

RATIONALE: Induced pluripotent stem cells (iPSCs) have been generated from patients with various forms of disease, including Danon disease (DD); however, few reports exist regarding disease-specific iPSCs derived from clinically divergent monozygotic twins. OBJECTIVE: We examined the characteristics of iPSCs and iPSC-derived cardiomyocytes (iPSC-CMs) generated from clinically divergent monozygotic female twins with DD. METHODS AND RESULTS: We generated iPSCs derived from T-cells isolated from clinically divergent, 18-year-old female twins with DD harboring a mutation in LAMP2 at the intron 6 splice site (IVS6+1_4delGTGA). Two divergent populations of iPSCs could prepare from each twin despite of their clinical divergence: one with wild-type LAMP2 expression (WT-iPSCs) and a second with mutant LAMP2 expression (MT-iPSCs). The iPSCs were differentiated into iPSC-CMs and then autophagy failure was observed only in MT-iPSC-CMs by electron microscopy, tandem fluorescent-tagged LC3 analysis, and LC3-II western blotting. Under these conditions, X-chromosome inactivation (XCI) was determined by PCR for the (CAG)n repeat in the androgen receptor gene, revealing an extremely skewed XCI pattern with the inactivated paternal wild-type and maternal mutant X-chromosomes in MT-iPSCs and WT-iPSCs, respectively, from each twin. CONCLUSION: Regardless of their clinical differences, we successfully established two sets of iPSC lines that expressed either wild-type or mutant LAMP2 allele from each monozygotic twin with DD, of which only the populations expressing mutant LAMP2 showed autophagic failure.

Human-Induced Pluripotent Stem Cell-Based Modeling of Cardiac Storage Disorders.

PURPOSE OF REVIEW: The aim of this study is to review the published human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) models of cardiac storage disorders and to evaluate the limitations and future applications of this technology. RECENT FINDINGS: Several cardiac storage disorders (CSDs) have been modeled using patient-specific hiPSC-CMs, including Anderson-Fabry disease, Danon disease, and Pompe disease. These models have shown that patient-specific hiPSC-CMs faithfully recapitulate key phenotypic features of CSDs and respond predictably to pharmacologic manipulation. hiPSC-CMs generated from patients with CSDs are representative models of the patient disease state and can be used as an in vitro system for the study of human cardiomyocytes. While these models suffer from several limitations, they are likely to play an important role in future mechanistic studies of cardiac storage disorders and the development of targeted therapeutics for these diseases.

Autophagy dysregulation in Danon disease.

The autophagy-lysosome system is critical for muscle homeostasis and defects in lysosomal function result in a number of inherited muscle diseases, generally referred to as autophagic vacuolar myopathies (AVMs). Among them, Danon Disease (DD) and glycogen storage disease type II (GSDII) are due to primary lysosomal protein defects. DD is characterized by mutations in the lysosome-associated membrane protein 2 (LAMP2) gene. The DD mouse model suggests that inefficient lysosome biogenesis/maturation and impairment of autophagosome-lysosome fusion contribute to the pathogenesis of muscle wasting. To define the role of autophagy in human disease, we analyzed the muscle biopsies of DD patients and monitored autophagy and several autophagy regulators like transcription factor EB (TFEB), a master player in lysosomal biogenesis, and vacuolar protein sorting 15 (VPS15), a critical factor for autophagosome and endosome biogenesis and trafficking. Furthermore, to clarify whether the mechanisms involved are shared by other AVMs, we extended our mechanistic study to a group of adult GSDII patients. Our data show that, similar to GSDII, DD patients display an autophagy block that correlates with the severity of the disease. Both DD and GSDII show accumulation and altered localization of VPS15 in autophagy-incompetent fibers. However, TFEB displays a different pattern between these two lysosomal storage diseases. Although in DD TFEB and downstream targets are activated, in GSDII patients TFEB is inhibited. These findings suggest that these regulatory factors may have an active role in the pathogenesis of these diseases. Therapeutic approaches targeted to normalize these factors and restore the autophagic flux in these patients should therefore be considered.

A Critical Evaluation of Liver Pathology in Humans with Danon Disease and Experimental Correlates in a Rat Model of LAMP-2 Deficiency.

Danon disease is a genetic deficiency in lysosome-associated membrane protein 2 (LAMP-2), a highly glycosylated constituent of the lysosomal membrane and characterized by a cardiomyopathy, skeletal muscle myopathy, and cognitive impairment. Patients, however, often manifest hepatic abnormalities, but liver function has not been well evaluated and the syndrome is relatively uncommon. Hence, we have taken advantage of a rat that has been deleted of LAMP-2 to study the relative role of LAMP-2 on liver function. Interestingly, rats deficient in LAMP-2 develop a striking increase in serum alkaline phosphatase (ALP) and a decrease in bile flow compared with wild-type littermates. Importantly and by ultrastructural analysis, deficient rats manifest dilated canaliculi that lack microvilli with evidence of bile-containing bodies. Moreover, following bile duct ligation, LAMP-2-deficient rats develop rapid and severe evidence of advanced cholestasis, with an increase in serum bilirubin, as early as 6 h later. In wild-type control rats, multidrug resistance-associated protein 2 (Mrp2) normally concentrates at the bile canalicular membranes to secrete conjugated bilirubin into bile. However, in LAMP-2y/- rats, Mrp2 was detected in hepatocytes compared with other canalicular proteins including P-glycoproteins, dipeptidyl peptidase IV (CD26), and aminopeptidase (CD13). Our data further suggest that LAMP-2 interacts with the membrane cytoskeletal proteins radixin and F-actin in determining the localization of integral membrane proteins.

Ischemic stroke due to hypoperfusion in a patient with a previously unrecognized Danon disease.

Danon disease, an X-linked multisystemic disorder, is due to deficiency of Lysosome-Associated Membrane Protein 2 (LAMP2). It is usually characterized by hypertrophic cardiomyopathy, mental retardation and skeletal myopathy, sometimes also with atypical features. A 20-year-old man with cognitive impairment was admitted to the Emergency Room because of a sudden chest pain. ECG showed Wolff-Parkinson-White syndrome; echocardiography revealed hypertrophic cardiomyopathy, and, shortly after, he experienced a cardiac arrest followed by an occipital ischemic stroke. On neurological examination, he complained of visual loss, and diffuse muscle wasting and weakness were also unexpectedly noted. Electromyography evidenced a myopathic pattern and a peripheral neuropathy. A muscle biopsy disclosed vacuolar myopathy with glycogen storage; immunohistochemical studies demonstrated a LAMP-2 deficiency. LAMP2 molecular analysis identified a "de novo" mutation (p. Q353X). This patient with a neglected Danon disease, experienced an unusual complication as a stroke due to cerebral hypoperfusion after cardiac arrest caused by WPW syndrome.

A novel LAMP2 mutation associated with severe cardiac hypertrophy and microvascular remodeling in a female with Danon disease: a case report and literature review.

BACKGROUND: Danon disease (DD) is a rare disorder characterized by cardiomyopathy, intellectual disability, and proximal myopathy. It is caused by mutations in the LAMP2 gene on X chromosome. Female patients most often present with late-onset cardiomyopathy and slow disease progression, but early-onset cases with unfavorable prognosis have been reported. CASE REPORT: We describe the clinical, pathological, and molecular features of a novel LAMP2 c.453delT mutation in a female patient with severe hypertrophic cardiomyopathy, Wolff Parkinson White (WPW) syndrome and rapid progression to heart failure, requiring heart transplant. Immunohistochemical analysis of LAMP2 in the explanted heart revealed a mosaic pattern of distribution, with discrete clusters of either stained or unstained cardiac myocytes, the latter being more frequent in the septum. These findings paralleled X chromosome inactivation within the myocardium. Interestingly, multiple foci of microscarring were found on histology in the Left Ventricle (LV) free wall and septum, in a close spatial relationship with remodeling and severe stenosis of intramural coronary arterioles. CONCLUSIONS: Our findings suggest that several features may contribute to the early and severe cardiac phenotype in female DD patients. The type of mutation may account for the early disease onset, while both the inhomogeneous distribution of LAMP2 loss and the presence of microvascular remodeling may be determinant in the rapid progression to heart failure.

Danon disease - dysregulation of autophagy in a multisystem disorder with cardiomyopathy.

Danon disease is a rare, severe X-linked form of cardiomyopathy caused by deficiency of lysosome-associated membrane protein 2 (LAMP-2). Other clinical manifestations include skeletal myopathy, cognitive defects and visual problems. Although individuals with Danon disease have been clinically described since the early 1980s, the underlying molecular mechanisms involved in pathological progression remain poorly understood. LAMP-2 is known to be involved in autophagy, and a characteristic accumulation of autophagic vacuoles in the affected tissues further supports the idea that autophagy is disrupted in this disease. The LAMP2 gene is alternatively spliced to form three splice isoforms, which are thought to play different autophagy-related cellular roles. This Commentary explores findings from genetic, histological, functional and tissue expression studies that suggest that the specific loss of the LAMP-2B isoform, which is likely to be involved in macroautophagy, plays a crucial role in causing the Danon phenotype. We also compare findings from mouse and cellular models, which have allowed for further molecular characterization but have also shown phenotypic differences that warrant attention. Overall, there is a need to better functionally characterize the LAMP-2B isoform in order to rationally explore more effective therapeutic options for individuals with Danon disease.