Frameshift mutation of LAMP2:c.667delT in a 17-year-old male with hypertrophic cardiomyopathy and dyslexia: a novel pathogenic variant for Danon disease

INTRODUCTION Danon disease is a rare X-linked dominant disorder caused by pathogenic mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene.[1] Clinical features include hypertrophic cardiomyopathy (HCM), skeletal myopathy, intellectual disability and manifestations of retinal and hepatic systems.[2,3,4] Males are usually more severely affected compared to females.[5] We report a case of Danon disease caused by a mutation that has not been reported in the current literature. CASE PRESENTATION A 17-year-old male with underlying dyslexia presented to the emergency department with central chest discomfort for 3 days, with no palpitations, syncope or symptoms suggestive of ischaemia, cardiac failure or pulmonary causes. There was no personal or family history of cardiovascular, muscular and pulmonary disease. The patient was alert and normotensive with a regular heart rhythm of 75 bpm, and he was saturating comfortably at 100% under room air. He had a displaced apex beat, normal heart sounds and no signs of heart failure. Muscle bulk, reflexes, power, sensation and gait were normal, and examination of the respiratory system was unremarkable. Electrocardiogram (ECG) showed left bundle branch block (LBBB) evidenced by broad QRS complex, deep S-wave in V1 and monomorphic R-wave at V5–V6 [Figure 1a]. Echocardiogram revealed severe concentric left ventricular hypertrophy (LVH) (intervernicular septum in diastole: 2.1 cm, left ventricle posterior wall in diastole: 2.1 cm, left ventricular in diastole mass index: 326.66 g/m2), systolic anterior motion of the mitral valve with non-significant left ventricular outflow obstruction measuring 6 mmHg (peak gradient) at rest, Valsalva gradient of 11 mmHg and diastolic dysfunction of restrictive pattern (echocardiographic early to late diastolic transmitral flow velocity [E/A]: 1.93) with elevated left ventricular filling pressure (average E/e': 37, septal e': 2 cm/s, lateral e': 2 cm/s). The left atrium was normal in size (15 mL/m2).Figure 1: ((a) ECG shows left bundle branch block on 2.5 mm/mV. (b) Cardiac MR images show thickened left ventricular wall at the basal anterior aspect.Chest X-ray showed borderline cardiomegaly with no signs of pulmonary congestion or pneumothorax. Laboratory tests revealed raised high-sensitivity troponin T, creatine kinase (CK), lactate dehydrogenase (LDH) and transaminases to <5 times the upper limit of normal [Table 1]. Based on echocardiogram findings and non-evolutionary ECG and troponin changes, HCM was diagnosed.Table 1: Laboratory test results.Subsequent cardiac magnetic resonance imaging (MRI) reported left ventricular ejection fraction of 52%, maximum left ventricular wall thickness of 34 mm at the basal anterior aspect [Figure 1b], and diffuse intramyocardial and epicardial enhancements at the anterior, lateral and inferior walls on gadolinium study. Exercise stress test was normal — no arrhythmias were detected up to Stage 3 of the Bruce protocol (stopped at 6 min 38 s due to fatigue, target heart rate 92%, metabolic equivalents 7.0). Notably, three of the patient's brothers had HCM on echocardiogram screening. The patient and his brothers attended special education school, two of whom are on the National Persons with Disability (Intellectual) register. The patient's mother did not report any delay in the patient's developmental milestones otherwise, and second-degree relatives on both maternal and paternal sides were cognitively able. Both parents were well, and parental union was non-consanguineous. The patient's family was subsequently counselled to perform whole exome sequencing to identify sequence variations. Only the proband and his mother consented. Whole exome sequencing was performed using SureSelect Human All Exon Kit (Agilent, Santa Clara, CA, USA) on NovaSeq 6000 (Illumina, San Diego, CA, USA). The sequencing reads were aligned to hg38/GRCh38 using Burrow-Wheeler Aligner. Duplicates were removed by Picard version 1.57 (http://picard.sourceforge.net/). The average sequencing depth was targeted for 100×, and data with Q30 were used for analysis. Genome analysis toolkit (https://software.broadinstitute.org/gatk/) was employed to identify single nucleotide variation and indels. Variant annotation and interpretation were conducted by ANNOtate VARiation,[6] and variants were searched in the database of single nucleotide polymorphisms (http://www.ncbi.nlm.nih.gov/SNP/) and 1000 Genomes Project database (http://www. 1000genomes.org/). Functional annotation and pathogenicity of mutations were predicted by SNPeffect.[7] We also searched disease and phenotype databases such as OMIM (http://www.omim.org), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar), HGMD (http://www.hgmd.org), PubMed (http://www.ncbi.nlm.nih.gov/pubmed), ClinGen (http://www.clinicalgenenome.org) and Orphanet (https://www.orpha.net). Variants with minor allele frequency of <0.05 were selected for interpretation based on the evidence of pathogenicity, clinical synopsis and inheritance mode of associated disease. The patient's mother was found to be heterozygous for a novel variant (LAMP2:c.667delT) that has high-impact functional changes based on SNPeffect. It is, therefore, deduced that LAMP2:c.667delT detected in the proband was inherited through an X-linked dominant fashion. Translation of LAMP2:c.667delT resulted in an amino acid change from tyrosine (Y) to isoleucine (I), producing a truncated protein with several stop codons. The homology models of LAMP-2 proteins were prepared using Iterative Threading ASSEmbly Refinement (I-TASSER).[8] The top threading template for LAMP2:c.667delT and wild-type models were based on Protein Data Bank accession number 5gv3A, with an identity = 0.53, normalised Z-score = 2.95 and coverage = 0.67. The refined protein structures were submitted to PROCHECK programs to check the stereochemical quality of protein structures to assess the quality of the stereochemistry of the modelled protein structures.[9] Ramachandran plot analysis showed that both wild-type and LAMP2:c.667delT structures had quality scores of 95.4% and 97.7%, respectively. The deletion of a single-nucleotide T at position c.667 resulted in a truncated amino acid from p.222 onwards [Figure 2]. Refined 3D protein models were then visualised using the PyMOL software.[10]Figure 2: Predicted secondary structures based on homology modelling using Iterative Threading ASSEmbly Refinement show (a) structure for the wild-type LAMP-2 protein, (b) the mutant LAMP-2 c.667delT, and (c) superimposed LAMP-2 wild type and LAMP-2 c.667delT mutant generated using PyMOL.DISCUSSION Danon disease is a rare X-linked dominant disorder caused by a mutation in the LAMP2 gene. It has been reported in 1.0% of patients with HCM,[11] 1.5% of patients with unexplained LVH, 33.3% of patients with concomitant LVH and ventricular pre-excitation,[12] and 12.0% of females aged < 40 years with non-ischaemic acute heart failure.[13] The LAMP2 gene encodes proteins involved in the fusion of autophagic vacuoles and lysosomes to facilitate degradation of cellular material.[14] Mutations cause reduced or absent LAMP-2 expression, resulting in impaired fusion and accumulation of autophagic vacuoles in myocytes.[4] To date, over 100 pathogenic LAMP2 mutations have been reported in ClinVar.[15] Herein, we identified a novel frameshift mutation (LAMP2:c.667delT) in both the proband and his mother, which caused truncated LAMP2 protein. While most forms are familial, de novo mutations have also been reported.[11] The clinical spectrum of Danon disease is broad and varied. Hemizygous males usually present more severely and earlier in adolescence,[16] where 10% of them progress to end-stage heart failure requiring heart transplantation within the first two decades of life.[5,11] Heterozygous females are usually phenotypically mild or asymptomatic, progress slower and present later in adulthood,[13,16] although severe cases requiring heart transplantation have been reported, possibly due to skewed X-inactivation.[13,17] Cardiac presentations, such as chest pain, syncope, palpitations and LVH with ventricular pre-excitation, are common in both genders,[12,16,18] while proximal myopathy and cognitive impairment are more common in males.[2,5,13,16] Troponin immunoassays are not only cardio-specific, but are also raised in non-ischaemic pathologies, including LVH,[19] due to increased number of contractile units and proteins.[20] Dynamic troponin concentrations such as the 0/1- and 0/2-h algorithms have, therefore, become prerequisites in diagnosing myocardial infarction to improve sensitivity.[21,22] Unexplained elevation of transaminases, CK and LDH distinguishes Danon disease from HCM and glycogen-associated cardiomyopathy.[18] Males with Danon disease are more commonly affected and show higher degrees of elevation compared to females and probands with mosaicism.[16,18] In the absence of liver pathology, alanine transaminase may be raised due to accumulation of autophagic vacuoles in the hepatocytes, leading to vein fibrosis, fatty changes and nuclear vacuolisation.[4,16] Aspartate transaminase is less liver specific as isoenzymes are also found in the skeletal muscles and heart, all of which are affected in Danon disease. Similarly, LDH isoenzymes are found in the liver, skeletal muscle, heart, kidney and red blood cells.[23] Plasma CK predominantly exists in its CK-MM form and does not increase in LVH, in contrast to troponin.[20] Therefore, it is likely that the patient has liver involvement and biochemical abnormalities that are skeletal in source, although liver biopsy and enzyme isotyping are required for confirmation. Management of Danon disease focuses on controlling disease progression and genetic counselling. Pacing is used to treat 35%–50% of male patients with complete heart block, and 60% of patients with arrhythmias benefit from cardioversion or implantable cardioverter defibrillators.[2,16] Ultimately, heart transplantation is required for end-stage heart failure and to improve survival.[5,16,24] Myopathy and cognitive impairments are managed with rehabilitative therapies,[16] and LAMP2B gene therapy has been shown to improve biochemical abnormalities, cardiac function and survival in murine studies.[25] With genetic testing becoming more accessible, Danon disease should be considered in young men with moderate-to-severe cardiac hypertrophy, unexplained biochemical abnormalities and unusual clinical features. Identifying Danon disease is important as its disease course, prognosis and genetic counselling differ from those of HCM. Family members should also be screened to initiate counselling and case-specific therapy. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.