The Myocardial Accumulation of Aggregated Desmin Protein in a Case of Desminopathy with a de novo DES p.R406W Mutation

Abstract

Desminopathy is a cardiac and skeletal myopathy caused by disease-causing variants in the desmin (DES) gene and represents a subgroup of myofibrillar myopathies, where cytoplasmic desmin-postive immunoreactivity is the pathological hallmark. We herein report a 28-year-old Japanese man who was initially diagnosed with sporadic hypertrophic cardiomyopathy with atrioventricular block at 9 years old and developed weakness in the soft palate and extremities. The myocardial tissue dissected during implantation of the ventricular-assisted device showed a dilated phase of hypertrophic cardiomyopathy and intracellular accumulation of proteinase K-resistant desmin aggregates. Genetic testing confirmed a de novo mutation of DES, which has already been linked to desminopathy. As the molecular diagnosis of desminopathy is challenging, particularly if patients show predominantly cardiac signs and a routine skeletal muscle biopsy is unavailable, these characteristic pathological findings of endomyocardial proteinase K-resistant desmin aggregates might aid in clinical practice.

Introduction

Desminopathy is a subgroup of myofibrillar myopathy (MFM) (1) caused by disease-causing variants in the desmin (DES) gene, where myofibril dissolution and the abnormal accumulation of degradation products cause dense desmin-immunoreactive deposition in the skeletal muscle (2). Desmin, a protein encoded by the DES gene, is a muscle-specific 53-kDa intermediate filament protein expressed in cardiac, skeletal, and smooth muscle tissues and forms type III intermediate filaments to regulate sarcomere architecture in these tissues (3). Mutations in the DES gene affect the intracellular filamentous network with the accumulation of insoluble granulo-filamentous material in these muscle cells.

Clinically, the skeletal muscle weakness often starts with the anterior leg compartment of the lower limb, spreading to the upper extremities, shoulder girdle, and truncal and facial muscles (4,5). Typical cardiac presentations include hypertrophic cardiomyopathy (HCM), restricted cardiomyopathy, and dilated cardiomyopathy (DCM) (6). Desminopathy also manifests as cardiac conduction disease, sometimes leading to sudden death (7).

More than 100 genetic disease-causing variants have been identified in the DES gene, and clinical features, including the severity of skeletal and myocardial involvement, onset age, and progression vary according to the mutation loci. Desminopathy is typically inherited as an autosomal dominant trait, and in some cases, it is caused by de novo mutations. Furthermore, while most cases exhibit a combination of cardiac and skeletal myopathy, severe myocardial impairment precedes skeletal muscle involvement in some cases, which makes the diagnosis even more complicated, as the pathological features of endocardial tissues have rarely been reported.

We herein report a patient diagnosed with desminopathy who developed severe cardiomyopathy followed by skeletal involvement and had a ventricular assist device implanted to treat his chronic heart failure. Endomyocardial tissue biopsies revealed proteinase K-resistant desmin aggregates confirming desminopathy. Whole-exome sequencing and Sanger sequencing identified a de novo heterozygous c.1216 C>T (NM_001927.4) (p.Arg406Trp) mutation in the DES gene.

Case Report

The proband was a 28-year-old Japanese man with no family history of neuromuscular disorders (Figure A). While his motor developmental milestones and function were normal, he had been diagnosed with hypertrophic cardiomyopathy at nine years old. He also had nasal speech, suggestive of subclinical dysfunction of the palatal muscles. At 20 years old, atrioventricular block (AVB) developed, which required cardiac pacemaker implantation. Annual follow-up echocardiography revealed progression of systolic dysfunction [ejection fraction (EF): 38%], suggesting conversion to the dilated phase of hypertrophic cardiomyopathy (HCM). Palatal muscle dysfunction and chronic cardiac failure raised the suspicion of cardiac and skeletal myopathy; thus, he presented to our neurology department at 27 years old.

Figure.

Clinical and pathological findings of the case. (A) Pedigree tree of the family affected by the DES R406W mutations. Open symbols indicate unaffected individuals, whereas a filled symbol with an arrow indicates the proband. (B, C) Histopathological features of the myocardial tissue. Hematoxylin and Eosin staining (B) and Azan (C) staining are shown. Bar=200 μm. (D, E) Immunohistochemical features of the myocardial tissue from the patient and control. Panel D and E show immunostaining with an anti-human desmin antibody (1: 100; Clone D33, Dako Denmark A/S, Glostrup, Denmark), without and with proteinase K pretreatment (0.2 mg/mL for 20 minutes; Dako Denmark A/S), respectively. Bar=200 μm. (F) DNA sequencing chromatogram of the parents (I-1, I-2) and the patient (II-2) shows a de novo mutation in DES.

A neurological examination showed mild muscle weakness [Medical Research Council Scale (MRC) grade 4/5]; atrophy in the neck, shoulder girdle, and distal legs; and impaired soft palate elevation associated with nasal speech. However, his tendon reflexes were normal. Laryngoscopy revealed moderate pharyngeal hyposystole. His vital capacity decreased to 77% of that of the average adult. The serum creatine kinase (CK) level was 36 U/L (normal range, 59-248 U/L). At 28 years old, his cardiac function decreased to EF 25%, and a ventricular-assisted device was implanted. Hematoxylin and eosin (H&E) and Azan staining of the dissected myocardial tissue revealed myocardial disarray and muscle fiber size variation with moderate interstitial fibrosis, which was compatible with dilated-phase HCM (Figure B, C). A histopathological examination of his myocardial tissue using previously published methods (8) showed the intracellular accumulation of proteinase K-resistant desmin aggregates in addition to the findings of dilated phase of HCM, suggestive of desmin-related cardiomyopathy (Figure D, E) (8).

Although he had no family history of neuromuscular disorders (Figure A), young-onset cardiomyopathy with skeletal muscle involvement suggested hereditary muscular disorders. Thus, whole-exome sequencing of the patient was performed to search for rare variants in the genes related to cardiomyopathy, muscular dystrophy, and myopathy (Supplementary material 1). Genomic DNA fragments containing exonic sequences were enriched using the SureSelect Human All Exon v6+UTRs kit (Agilent, Santa Clara, USA) and a HiSeq 2500 sequencer (Illumina, San Diego, USA). Short-read alignment to the human reference genome (GRCh37/hg19) was performed using the Burrows-Wheeler Aligner (BWA) v0.5.9 (9), and single-nucleotide variants (SNVs) and small insertions/deletions (indels) were called using SAMtools (v0.1.18) (10-17).

We utilized our in-house exome database of 1,194 Japanese controls to identify variants with allele frequencies <0.01 whose quality score was over 10. A total of 422 rare variants met these criteria. Of the 422 variants that met the above criteria, rare variants in the target genes (Supplementary material 1) are listed in Table, after excluding variants registered as benign or likely benign in ClinVar. While the significance of the variants in the NEB gene was unknown, a heterozygous c.1216 C>T (NM_001927.4) (p.Arg406Trp) variant identified in the DES gene had been previously reported to be pathogenic (18). This variant was confirmed by Sanger sequencing (Figure F). His parents did not have this variant, indicating that the mutation had occurred de novo. Paternity and maternity were confirmed by Sanger sequencing of 10 rare single-nucleotide variants (data not shown).

Table.

Rare Variants Revealed by Whole-exome Sequencing.

Gene		Zygosity		Variant		Pathogenicity (ClinVar)
DES		Heterozygous		c.1216C>T (p.Arg406Trp)		Pathogenic
NEB		Heterozygous		c.8588G>A (p.Ser2863Asn)		Not registered
NEB		Heterozygous		c.25639G>A (p.Gly8547Ser)		Uncertain significance

Discussion

Desminopathy is typically a cardiac and skeletal myopathy caused by autosomal dominant inheritance; our patient presented with severe cardiomyopathy with AVB followed by skeletal muscle involvement and had a de novo heterozygous c.1216C>T (p.Arg406Trp) in the DES gene (NM_001927.4). In addition to the dilated phase of HCM, myocardial tissue showed the intracellular accumulation of proteinase K-resistant desmin aggregates.

Desmin is a muscle-specific type III intermediate filament in which a highly conserved YRKLLEGEE motif at the C-terminal end of the 2 B helix plays a key role in filament assembly (11-17). Although the C-terminal end of the 2 B helix region consists of approximately 25% of the coding region, more than 20 causative mutations, which account for approximately 50% of the known causative mutations in the DES gene, have been reported in this region, supporting its functional relevance. R406 is located in this region, and seven reports investigating patients with this mutation have been published thus far (11-17). They reported 11 patients from 9 families in total, among whom 7 patients (63.6%) had a de novo R406W mutation in a parental genetic analysis, indicating that desmin position 406 is a hotspot for de novo mutations. Of the 10 patients with clinical description, cardiomyopathy, cardiac conduction disease, and skeletal myopathy were documented in 9 (90.0%), 10 (100.0%), and 8 patients (80.0%), respectively.

Our patient presented severe cardiomyopathy with AVB, combined with milder skeletal muscle involvement. A previous report included a literature review of 159 DES mutation carriers (11-17), demonstrating that both neurological and cardiological signs were present in 49% of carriers, and isolated cardiological signs in 22%. Furthermore, approximately 60% of carriers had cardiac conduction disease or arrhythmias. While they hypothesized that the variability of phenotypes associated with different mutations might be attributed to the location of the mutations, and mutations in domain 2 B tended to be associated with the isolated neurological phenotype, our case with the R460W mutation in domain 2 B showed a combination of cardiological and neurological signs. Among the 10 patients with R406W who had their clinical data, 7 had both neurological and cardiological signs (11-17). Our case showed severe cardiomyopathy followed by neurological manifestations, which was consistent with previous reports. Thus, it was intuited that there is a certain relationship between genetic mutations and the phenotype.

Histopathological features in the skeletal muscle, including cytoplasmic inclusions with desmin immunoreactivity, myofibrillar dissolution in nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) staining, and abnormal electron-dense deposition in electron microscopy (19), are well-established hallmarks for the diagnosis of desminopathy. However, there have been a limited number of reports on myocardial findings. Those reports have almost always shown the cytoplasmic accumulation of desmin protein in the endomyocardial cells of genetically confirmed cases with DES mutations. Rarely were patients reported without the accumulation of desmin, harboring DES E401D with less intense staining of desmin (16) or a Q364H mutation with a decreased desmin expression (20). However, endomyocardial staining of desmin can be observed in the advanced stage of idiopathic DCM (20) and HCM (21) without DES mutations, including in healthy subjects. A previous study on desmin-related cardiomyopathy with a pathological diagnosis reported that proteinase K pretreatment could degrade desmin protein, leaving only the aggregated desmin protein and thus improving the diagnostic accuracy (8). Our case is the first report showing desmin aggregates in myocardial tissue after proteinase K pretreatment in a genetically identified case of desminopathy.

The R406 residue contributes to the stable assembly of intermediate filaments and is highly conserved across species (22). In silico modeling of the desmin fragment predicted that the R406 and E401 residues formed an inter-helical salt-bridge to stabilize dimerization, and the R406W mutation destabilized the filament formation, leading to an abnormal salt-bridge. In vitro experiments supported this model, showing that R406W-desmin formed abnormally thick and irregular filaments resulting in its aggregation (17). Furthermore, desmin has an amyloidogenic region and forms amyloid fibrils even under physiologic conditions, and the mutated form of desmin has a stronger amyloidogenic potential than the wild type (23). These structural features may account for the aggregation-prone property of mutated desmin protein. Aggregation cores are generally proteinase K-resistant, which may be the mechanism by which proteinase K pretreatment improves the diagnostic accuracy in desminopathy.

Conclusions

We presented a case of desminopathy with a de novo DES R406W mutation. The patient presented with a dilated phase of HCM with AVB and minimal skeletal muscle involvement. Myocardial tissue revealed myocardial disarray with interstitial fibrosis, consistent with the dilated phase of HCM and proteinase K-resistant desmin accumulation, suggesting the cytoplasmic accumulation of aggregated desmin. The degree of cardiac involvement in desminopathy varies, and approximately 20% of cases show predominantly cardiac signs; the diagnosis is therefore difficult, especially in de novo cases. The cytoplasmic accumulation of aggregated desmin protein is an important clue for the diagnosis, and proteinase K pretreatment can solidify the diagnostic value of desmin staining.

This study was approved by the Institutional Review Board of the University of Tokyo Hospital (G1396). The proband and each family member provided their written informed consent for the genetic and pathological analyses.

The authors state that they have no Conflict of Interest (COI).

Financial Support

This study was supported by grants from the Japan Agency for Medical Research and Development (grant numbers JP19ek0109279 and JP20ek0109491), and JSPS KAKENHI Grant Number 22K07512.

Supplementary Material

A list of genes related to cardiomyopathy, muscular dystrophy, and myopathy

Whole-exome sequencing of the patient was performed to search for rare variants in the genes related to cardiomyopathy, muscular dystrophy, and myopathy.