Pediatric Pathology

Danon Disease (LAMP2 Deficiency)

Glenn P. Taylor
Hospital for Sick Children
Toronto, Ontario, Canada


Clinical History
A 15 year-old boy was seen by a neurologist for the complaint of leg weakness that was characterized as episodes of his "knees giving away". This had developed in the past 2 or 3 months. Prior, he had no health problems. Examination revealed mild to moderate proximal muscle weakness, but no other significant findings. Family review disclosed that the boy's mother had developed dilated cardiomyopathy in her late thirties and had recently received an implantable cardioverter-defibrillator. This information prompted the boy's urgent referral to a cardiologist. An echocardiogram showed marked concentric hypertrophic cardiomyopathy and a large pericardial effusion. He was admitted to hospital for pericardiocentesis and additional investigations. Endomyocardial biopsies and biopsy of vastus lateralis muscle were performed. The virtual slide and other images are from the endomyocardial biopsy.

Slide 1
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Figure 1
Intermediate power image, elastic trichrome stain. The cardiac myocytes have cytoplasmic vacuoles of varying size, most appearing empty.
Figure 2
High power image, hematoxylin and eosin stain. The high power image shows myocyte hypertrophic change with "blocky" enlarged nuclei, in addition to the cytoplasmic vacuoles.
Figure 3
High power image, PAS stain. Abundant cytoplasmic glycogen is demonstrated, but many vacuoles have irregular or absent PAS positive contents.

Figure 4
High power image, PAS with diastase predigestion. The large majority of the PAS staining is removed by predigestion with diastase. However, some lipofuscin-like granules remain.
Figure 5
Intermediate power electron micrograph. Ultrastructural examination shows membrane-bound vacuoles having heterogeneous contents.
Figure 6
High power electron micrograph. High power demonstrates the autophagosome-like appearance of the cytoplasmic vacuoles.

Introduction:
Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: The endomyocardial biopsy shows vacuolation of the cardiac myocytes. The vacuoles are of variable size. Many, but not all, have positive staining with periodic acid Schiff, and some have PAS staining resistant to diastase predigestion. Ultrastructural examination demonstrates the vacuoles to be membrane bound and to have heterogeneous content including glycogen, mitochondrial debris and membrane profiles.

Differential Diagnoses:
The ultrastructural appearance of the cardiac myocyte membrane-bound inclusions is characteristic of Danon disease.

Final Diagnosis:
Danon disease (LAMP2 deficiency).

Clinical Follow-up
Additional review of the family medical history disclosed that the maternal grandmother died from heart disease at 38 years of age. The boy's mother received heart transplantation for her dilated cardiomyopathy at the age of 42 years. The boy's proximal weakness did not significantly progress over the 3 years following his diagnosis, but he was less physically active and he had occasional palpitations with exertion. At 19 years of age he presented to hospital with shortness of breath, fatigue, cough and recent onset of orthopnea. An implantable cardioverter-defibrillator was inserted and he was treated for heart failure. Two weeks after that admission he represented to hospital in fulminant cardiac failure. He initially improved after insertion of an intra-aortic balloon pump, but 6 days from admission he had a sudden cardiac arrest. He could not be resuscitated. His heart at autopsy was 900 g and showed marked concentric hypertrophy.

Discussion

Danon Disease
Danon disease, also known as lysosomal glycogen storage disease without acid maltase deficiency, glycogen storage disease type IIb and X-linked vacuolar cardiomyopathy and myopathy, but more properly called LAMP2 deficiency, is a rare, X-linked inherited disorder characterized by cardiomyopathy, skeletal muscle myopathy and impaired intellectual development [1]. Hypertrophic cardiomyopathy is the predominant clinical manifestation with the other features variable in severity. Affected boys clinically present during the later years of the first or in the second decades, but rare presentations in boys as young as 2 or 3 years of age have been documented [2]. The heterozygous carrier females may be asymptomatic or present later, in the 4th or 5th decades, most often with a dilated cardiomyopathy. Various cardiac dysrhythmias, including Wolff-Parkinson-White syndrome, often complicate the cardiomyopathies. The cardiac manifestations of Danon disease are progressive with most males succumbing to heart failure or sudden cardiac death within 10 years of initial presentation. Indeed, the natural history of Danon disease, to paraphrase Maron et al, represents "one of the most lethal cardiomyopathies in young and usually male patients." [3] The only effective treatment is heart transplantation.

Danon disease was originally thought to be a glycogen storage disorder based on the ultrastructural finding of membrane-bound glycogen accumulations within cardiac and skeletal muscle [1]. The disease differed from Pompe disease by having normal acid maltase activity and was consequently designated as glycogen storage disease type IIb [4]. However, recognition that the characteristic myocyte vacuoles of Danon disease had other constituents besides glycogen, such as membrane profiles, sarcomere remnants and mitochondrial debris, led to the concept that they represented autophagic rather than storage vacuoles. The discovery by Nishino et al that Danon disease was due to a deficiency of lysosomal-associated membrane protein-2 (LAMP2) has led to the current understanding that the manifestations of Danon disease result from abnormalities in lysosome-autophagosome/phagosome interaction (see below) [5, 6].

Although cardiomyopathy is the predominant consequence of Danon disease, the disorder is systemic, with other organ systems affected in varying degrees of severity [7]. Skeletal myopathy is usually mild and may be clinically unapparent. When symptomatic, it tends to present as a proximal weakness that remains stable or is only slowly progressive. However, elevated serum creatine kinase level is universal in males. Intellectual impairment occurs in approximately 70% of males, but is uncommon in heterozygous females. The mental impairment is generally mild. Hepatomegaly, splenomegaly and a pigment retinopathy are other occasional manifestations [8].

Marked hypertrophic cardiomyopathy, with left ventricle wall thickness up to 6 cm, is the hallmark gross pathology of Danon disease [3]. However, in symptomatic heterozygous females the cardiomyopathy is more heterogeneous and often has a dilated morphology [9]. "Transformation" or "burn-out" over the course of years of hypertrophic cardiac morphology to dilated morphology has been clinically documented in some males and females. Autopsy hearts may exceed 1000 g and show concentric or asymmetric left ventricle wall thickening. The cardiac gross phenotypic similarity of Danon disease to hypertrophic cardiomyopathy due to sarcomere gene mutations is to a degree carried to microscopic similarities of myofiber disarray, intramural small vessel dysplasia and replacement fibrosis [3]. However, Danon disease is histologically distinguished by the presence of myocyte vacuolar change. The cytoplasmic vacuoles vary in diameter from 2 µm to 30 µm and may be granular or homogenous and basophilic, eosinophilic or "cleared" on hematoxylin and eosin stain [1]. They have variable positivity for periodic acid Schiff staining. The vacuoles on ultrastructural examination contain glycogen, often accompanied by increased cytosol non membrane-bound glycogen, lipid, membrane profiles and degraded organelles such as mitochondria and sarcomere constituents [10]. However, glycogen may be a minor component of the vacuolar content. The vacuoles accumulate with age and are most numerous in cardiac myocytes. They are variably demonstrated in skeletal muscle . In younger children skeletal myocyte vacuolar change may not be apparent on light microscopy and may be inconspicuous on electron microscopy [11]. Thus, negative skeletal muscle biopsies may not rule out a diagnosis of Danon disease in a boy with hypertrophic cardiomyopathy. Immunohistochemical staining by immunoperoxidase or immunofluorescence technique can demonstrate in cardiac or skeletal muscle biopsies the absence of LAMP2 in Danon disease. Ultrastructural examination of skin biopsy has been proposed as a less invasive diagnostic approach, but tissues other than muscle generally have very low expression of vacuolar change - endomyocardial biopsy remains the "gold standard" for morphologic diagnosis [12]. The generalized LAMP2 deficiency in males may allow diagnosis by leukocyte protein immunoblot [13]. Diagnosis of carrier females, especially if asymptomatic, optimally requires molecular genetic analysis.

LAMP2
The gene for lysosome-associated membrane protein-2 is at locus Xq24. LAMP2 and the similar LAMP1 (gene locus 13q34) are glycoproteins that account for approximately 50% of the protein compliment of the lysosomal membrane. Disease due to LAMP1 deficiency is not characterized. LAMP2 deficiency, generally as a result of a null mutation, causes Danon disease. The variable severity of the disease in females relates to "unfavorable lyonization" of the X chromosome. LAMP2 is a polypeptide of about 40 kD that has 16 to 20 N-linked saccharides attached to the core. It has small cytosol external surface, transmembrane and large luminal surface domains [14]. Its function, with LAMP1, was thought to be to protect the lysosome membrane from the hydrolytic enzymes within the lysosome lumen. More recently, however, it appears that the clinically crucial roles of LAMP2 are in facilitating fusion of the lysosome with the phagocytic or autophagic vacuole and in maturation of the phagosome to the contents-degraded form. In part this relates to the mechanism of microtubular transport of lysosomes and phagosomes to the perinuclear region where fusion of the two occurs. Loss of LAMP2 function impairs lysosome fusion with phagosomes, preventing degradation of the vacuoles and thereby promoting their accumulation within the cell [5, 14, 15].

Childhood Hypertrophic Cardiomyopathy
Excluding secondary causes of cardiac hypertrophy, such as hemodynamic obstructions, familial hypertrophic cardiomyopathy due to mutations in sarcomere genes, affecting 1 person in 500, is the most common cause of hypertrophic cardiomyopathy in older children and young adults [16].There are many other genetic diseases that may have hypertrophic cardiomyopathy as a component of the condition. These are especially a consideration in infant or early childhood presentations. However, for most the cardiac manifestations are superseded by neurological and other system involvement and diagnosis of the cardiomyopathy is not an issue. Diagnosis becomes more of a challenge for those few genetic conditions that have hypertrophic cardiomyopathy as a predominant or potentially predominant feature. These include:

Hypertrophic Cardiomyopathy in Genetic Disorders [17]

Predominant* Potentially Predominant
Danon Disease Fabry Disease
PRKAG2 Mutation
(GSD IX, Cardiac phosphorylase kinase deficiency) 		GSD II (Pompe Disease)
GSD III (Cori Disease)
GSD IV (Andersen Disease)
* excluding familial hypertrophic cardiomyopathy due to sarcomere gene mutations Mitochondriopathy
(e.g. SCO2 deficiency)

Noonan Syndrome
LEOPARD Syndrome

Together these conditions account for less than 5% of cases of "primary" hypertrophic cardiomyopathy that present in older childhood or adolescence. Danon disease is the most common, identified in up to 4% of patients that have childhood or adolescent onset of hypertrophic cardiomyopathy [18, 19]. In a group of 24 probands selected for left ventricular hypertrophy accompanied by electrocardiographic finding of ventricular pre-excitation (Wolff-Parkinson-White syndrome), Danon disease was identified in 4 [18].

The other predominant cardiac-specific disorder is caused by PRKAG2 gene (locus 7q36) mutations. The gene encodes for an adenosine monophosphate-activated protein kinase involved in modulating glucose uptake and glycolysis [20]. Mutations cause marked ventricular hypertrophy without myofiber disarray. Myocytes have cytoplasmic vacuoles containing glycogen [21]. Older patients may have vacuoles with periodic acid Schiff positive, diastase-resistant contents, similar to those of myocyte basophilic degeneration [20]. Accumulation of the glycogen leads to hypertrophic cardiomyopathy and eventual heart failure. Wolff-Parkinson-White syndrome (WPW) frequently accompanies the hypertrophic cardiomyopathy. Some families with PRKAG2 mutation have WPW without significant cardiac hypertrophy [22]. Clinical presentation is usually in the third or fourth decades, although presentation in childhood, including infancy, occurs. The disorder was formerly known as a variant of glycogen storage disease type IX and as cardiac phosphorylase kinase deficiency [23]. Diagnosis, like with Danon disease, is made by skeletal muscle or endomyocardial biopsy or by molecular genetic analysis.

References:
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