Polyglucosan body myopathy caused by defective ubiquitin ligase RBCK1



Glycogen storage diseases are important causes of myopathy and cardiomyopathy. We describe 10 patients from 8 families with childhood or juvenile onset of myopathy, 8 of whom also had rapidly progressive cardiomyopathy, requiring heart transplant in 4. The patients were homozygous or compound heterozygous for missense or truncating mutations in RBCK1, which encodes for a ubiquitin ligase, and had extensive polyglucosan accumulation in skeletal muscle and in the heart in cases of cardiomyopathy. We conclude that RBCK1 deficiency is a frequent cause of polyglucosan storage myopathy associated with progressive muscle weakness and cardiomyopathy. Ann Neurol 2013;74:914–919

Glycogen storage diseases (GSDs) are rare disorders caused by defects in various enzymes involved in glycogen metabolism. Muscle, including cardiac muscle, and liver are particularly affected due to the large quantity and fast turnover of glycogen in these tissues.[1, 2] Glycogenoses are therefore important in the differential diagnosis of hypertrophic cardiomyopathy.[3] Some of the GSDs are characterized by accumulation of polyglucosan, which unlike normal glycogen is resistant to digestion by alpha-amylase and contains abnormally long and poorly branched glucosyl chains. Such diseases include branching enzyme deficiency (GSD IV, Andersen disease), phosphofructokinase deficiency (GSD VII, Tarui disease), AMP-activated protein kinase deficiency (due to mutations in PRKAG2), and Lafora disease (some forms of which are due to mutations in NHLRC1, encoding the E3 ubiquitin ligase malin).[4]

Two sisters with adolescent onset of progressive proximal muscle weakness and later onset of cardiomyopathy showed striking accumulation of polyglucosan in muscle, but gene defects known to be associated with polyglucosan accumulation were excluded. Exome sequencing demonstrated that both sisters were compound heterozygous for mutations in the gene coding for the E3 ubiquitin ligase RBCK1 (HOIL-1). Screening for mutations in additional patients of various ethnic backgrounds with cardiomyopathy/myopathy and polyglucosan bodies revealed 8 additional cases from 7 unrelated families. These findings demonstrate that different E3 ubiquitin ligases are involved in glycogen metabolism and that mutations in RBCK1 should be considered in patients with cardiomyopathy.

Patients and Methods

Altogether, 32 individuals with polyglucosan body myopathy were investigated. We identified 10 individuals, 15 to 50 years of age, from 8 families of various ethnic backgrounds who were either homozygous or compound heterozygous for mutations in RBCK1 (Fig 1). Informed consent was obtained from the patients. The study was approved by the regional ethical review board in Gothenburg, Sweden.

Figure 1.

Pedigrees and RBCK1 mutations in 8 families (A–H). Filled circles and squares indicate affected individuals. +/+ = homozygous for mutation; +/− = heterozygous; −/− = homozygous for wild type. Accession NM_031229 and University of California Santa Cruz (UCSG) assembly Hg 19 gives c.727G>T: p.Glu243*: chr20:400,346, c.1160A>G: p.Asn387Ser: chr20:408,087, c. 896_899delAGTG: p.Glu299Valfs*18: chr20:401,654-57, c.722delC: p.Ala241Glyfs*34: chr20:400,341, c.52G>C: p.Ala18Pro: chr20:390,554, c.727_728insGGCG: p.Glu243Glyfs*114: chr20:400,346-47, c.ex1_ex4del: chr20:367,384–399,180, c.1054C>T: p.Arg352*: chr20:407,981, c.917+3_917+4insG: p.Arg298Argfs*40: chr20:401,678-79, and c.494delG: p.Arg165Argfs*111: chr20:400,024.

All patients underwent open biopsy of the deltoid or of the quadriceps muscle. Morphologic and histochemical analyses of fresh-frozen muscle tissue and frozen or formalin-fixed myocardium of heart explants were performed as described previously.[5, 6]

Whole exome sequencing was performed in the 2 affected sisters of Family A (AII:2 and AII:3) and in their healthy mother (AI:1; Supplementary Methods). Analyses of genomic DNA and mRNA in all patients were performed as described in the Supplementary Material.


Clinical Findings

All patients experienced proximal leg muscle weakness starting in childhood or adolescence (range = 4–17 years of age; see Fig 1 and Table). Muscle weakness was slowly progressive over several years, leading to difficulties in ambulation. Upper extremities and facial muscles were relatively spared. Ptosis was found in 4 patients. Three had scoliosis.

Table 1. Clinical Findings
  1. a

    See also Schoser et al.[6]

  2. b

    See also de la Blanchardière et al.[23]

  3. CK = creatine kinase; DCM = dilated cardiomyopathy; F = female; M = male; ND = not determined.

Age, yr50472419291920 (expired)263215 (expired)
Age at onset, yr121665ChildhoodChildhood412179
Initial symptomsLeg weaknessLeg weaknessLeg weaknessDifficulty runningDifficulty runningNDDifficulty runningLeg weaknessLeg weaknessDifficulty running
Walking abilityWheelchairWalks with aidWalks without aidWalks without aidNDWalks without aidWheelchairNDMinimally restrictedWalked without aid
Facial muscle weakness/ptosisNo/noNo/noNo/mildNo/noYes/noNo/NDNo/mildNo/mildYes/noNo/mild
Serum CK×2Normal×5×6NormalNDNormal×1.5NormalNormal
Pulmonary vital capacity, l [%]1.8 [56]2.6 [76]1.5 [52]1.6 [66]2.6 [71]ND2.0 [40]2.8 [54]3.64 [87.9]ND
Ejection fraction35–40%ND<20%18%25%15%23%15%ND28%
Heart transplantationNoNoAge 14 yearsAge 13 yearsNoNoNoAge 20 yearsNoAge 15 years

All 6 patients who were homozygous or compound heterozygous for truncating mutations developed dilated cardiomyopathy with onset in adolescence. The cardiomyopathy was rapidly progressive. Because of cardiac failure, 4 patients were transplanted at 13, 14, 15, and 29 years of age, and 1 succumbed due to cardiorespiratory failure at 20 years of age. Patients with missense mutations had an apparently milder clinical expression of the disease, with less muscle weakness and either later onset cardiomyopathy or no apparent cardiac involvement.

Liver involvement with storage of polyglucosan was identified in 2 patients (DII:1 and FII:2), 1 of whom had liver enlargement, slightly raised levels of alanine aminotransferase (ALT), and a coagulation defect (DII:1). Three additional patients had slightly elevated levels of ALT, but polyglucosan storage in the liver was not documented (BIII:1, BIII:2, and HII:1).

Four patients had growth retardation (BIII:1, BIII:2, EII:3, and HII:1). One patient (EII:3) had frequent episodes of pharyngitis and lymphadenopathy in childhood in addition to granulomatous tonsillitis and enteritis, psoriasis, and osteoporosis, but immunological investigations did not reveal any dysfunction. One patient had type 1 diabetes (AII:2), 1 had sarcoidosis at age 20 years (AII:3), and 1 had gluten intolerance (GII:3), but otherwise there were no signs of severe immunodeficiency or autoimmunity.

Morphologic Analysis of Muscle and Heart

Morphological investigations by light and electron microscopy of muscle and heart revealed characteristic alterations. In skeletal muscle, approximately 50% of the fibers were typically depleted of normal glycogen but had inclusions consisting of periodic acid-Schiff (PAS)-positive material that was incompletely digested by alpha-amylase (Fig 2). The storage material was partially resistant to proteinase K treatment, was ubiquitinated, and contained the ubiquitin-binding protein sequestosome-1 (p62), indicating that it had been marked for proteosomal degradation (Supplementary Fig 1). By electron microscopy, the storage material had granular and partly fibrillar structure and variable electron density, characteristic features of polyglucosan. Cardiac muscle revealed hypertrophy and alpha-amylase–resistant PAS-positive inclusions, with the ultrastructural characteristics of polyglucosan in virtually every cardiomyocyte.

Figure 2.

Characteristic morphological alterations of (A–H) skeletal muscle and (I, J) myocardium. Staining of skeletal muscle cryostat sections with periodic acid-Schiff (PAS) reagent demonstrates that numerous fibers (arrows) lack the normal intermyofibrillar glycogen but show accumulation of PAS-positive material (A). Unlike normal glycogen, the storage material is not removed by amylase treatment (B, arrows) and is ubiquitinated as shown by immunohistochemistry with an antiubiquitin antibody (NCL-UBIQ; Novocastra Laboratories, Newcastle Upon Tyne, UK; C, arrows). Electron microscopy demonstrates that the accumulated polyglucosan is different from normal glycogen and consists of partly filamentous material (D, arrows). Muscle biopsies from Patients HII:1 (E), DII:1 (F), EII:3 (G), and CII:3 (H) demonstrate similar abnormally accumulated PAS-positive material in fibers that are typically depleted of glycogen. A paraffin section from the myocardium (Patient BIII:1) stained with PAS demonstrates PAS-positive material in all cardiomyocytes (I, arrows), with ultrastructural characteristics of polyglucosan (J).

Molecular Genetics

Whole exome sequencing of Family A (Patients AII:2 and AII:3 and their healthy mother AI:1) revealed that both affected sisters harbored 2 mutations in the gene encoding heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1, Hugo Gene Nomenclature Committee–approved gene symbol: RBCK1). Both sisters carried a missense mutation (p.Asn387Ser) in 1 allele and a premature stop codon (p.Glu243*) in the other allele. The allele harboring the missense mutation was the only allele detected in cDNA (see Supplementary Methods). Screening for mutations in RBCK1 in 32 additional patients with muscle weakness and polyglucosan storage identified 8 additional cases from 7 different families. Each family had unique mutations (see Fig 1). Five (B, C, E, F, and H) carried truncating mutations, including premature stop codons, frameshifting deletions, or insertions. Family D harbored an N-terminal missense mutation (p.Ala18Pro) and Family G a single base insertion in the donor splice site of intron 7 (p.Arg298Argfs*40) that resulted in a new splice site in exon 7 and a frameshift. None of the mutations in RBCK1 was reported in public databases as a polymorphism. Affected individuals were either homozygous or compound heterozygous for these mutations.


We have identified mutations in RBCK1, encoding an E3 ubiquitin ligase, as the cause of a GSD with cardiomyopathy and skeletal myopathy.

RBCK1 interacts with a phosphatase encoded by the gene EYA1,[7] which is involved in myogenesis and is enriched in nuclei of fast-twitch glycolytic muscle fibers.[8] Mutations in EYA1 leads to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies.[9, 10] When the ortholog for RBCK1 was knocked down in zebrafish, it led to a BOR syndrome–like phenotype.[7] RBCK1 also interacts with the iron regulatory protein 2 (IRP2) and controls iron homeostasis by degrading the oxidized form of IRP2.[11]

Together with HOIL-1L interacting protein (HOIP) and Sharpin, RBCK1 forms the linear ubiquitin assembly complex LUBAC,[12, 13] which was identified as a critical regulator of the canonical nuclear factor κ B (NFκB) pathway through the ubiquitination of IKBKG (inhibitor of NFκB kinase subunit γ; NEMO).[14] NF-κB has an important role in the regulation of the immune system.[15] A study of primary hepatocytes isolated from Rbck1 (HOIL-1) knockout mice reported impaired tumor necrosis factor-α (TNFα)-induced NF-κB activation. However, the TNFα-induced NF-κB activation was not completely abolished in the Rbck1 knockout mice and—in contrast to Sharpin knockout mice that have chronic dermatitis and immunodeficiency—Rbck1 knockouts did not show any overt phenotype.[16]

A recent report by Boisson et al[17] described 2 young siblings and 1 unrelated child with failure to thrive, chronic autoinflammation, and recurrent episodes of sepsis associated with loss-of-function mutations in RBCK1. Two of the children died from sepsis at ages 8 and 3.5 years, and 1 child, who had an allogeneic bone marrow transplantation at 13 months of age, died from sudden respiratory distress at 4 years of age. Similar to our patients, inclusions of polyglucosan were identified in skeletal muscle, heart, and liver.

None of our 10 patients with RBCK1 mutations had recurrent episodes of sepsis or apparent chronic autoinflammation, but instead they suffered from cardiomyopathy and/or skeletal myopathy. One patient (EII:3) had frequent episodes of laryngitis from 4 to 6 years of age, requiring tonsillectomy. He also had psoriasis and a nonspecific granulomatous disease that may indicate immunologic disturbance. However, immunological investigations did not reveal any dysfunction.

Thus, RBCK1 deficiency may cause either an immune disorder or myopathy/cardiomyopathy. This clinical diversity may be explained by the nature and location of the RBCK1 mutations. The 3 mutations described by Boisson et al and associated with immune dysfunction were loss-of-function mutations located in the N-terminal region of RBCK1. With the exception of Families A and E, all our patients were homozygous for mutations in the middle or C-terminal part of RBCK1 (Supplementary Fig 2). The expressed allele of RBCK1 in the compound heterozygous patients in Family A (II:2 and II:3) harbored a C-terminal missense mutation. Patient EII:3 was the only patient in our cohort with a possible but unconfirmed immune dysfunction. He was compound heterozygous for 2 loss-of-function mutations. One of those was the same 31.799bp deletion described in 2 siblings by Boisson et al. The deletion includes the N-terminal part of RBCK1 and the expressed regions of TRIB3, a protein known to induce NF-κB activation.[18] In addition to the loss-of function mutations in RBCK1, a monoallelic expression of TRIB3 may further perturb the NF-κB pathway.

Further comparative studies of the expression of Sharpin and HOIP in cell lines from patients with either myopathy/cardiomyopathy or immunodeficiency may shed light on the phenotypic variability associated with different RBCK1 mutations.

Polyglucosan accumulation is typically observed in branching enzyme deficiency, including a variant referred to as adult polyglucosan body disease, in some cases of phosphofructokinase deficiency, and in Lafora disease.[2] A dominantly inherited myopathy with polyglucosan bodies in horses is associated with a heterozygous missense mutation in GYS1 (glycogen synthase).[19] The pathogenesis of polyglucosan accumulation is not known, but various mechanisms may be involved. There is evidence that imbalance between the activities of glycogen synthase and branching enzyme results in the production of polyglucosan bodies.[19, 20] Malin (NHLRC1), an E3 ubiquitin ligase, has been associated with Lafora disease.[21] How malin deficiency causes formation of polyglucosan has not yet been established.[22] How RBCK1 is involved in glycogen metabolism remains unknown, but the results from this study clearly demonstrate that defective RBCK1 may cause extensive accumulation of polyglucosan that is associated with muscle weakness and cardiomyopathy.


Supported by a grant from the Swedish Research Council (7122; A.O.).

We thank Drs B. Eymard, D. Duboc, M. Fardeau, S. Weis, and J. Weis for the clinical investigations of the patients; Dr A. R. Moslemi for technical assistance; and the patients' families for their support.

Potential Conflicts of Interest

P.L.: board membership, grants/grants pending, speaking fees, Genzyme. C.L.: grants/grants pending, Genzyme. A.G.E.: grants/grants pending, NIH. S.D.: Editor, MedLink Neurology.