Neuromuscular Disorders
Volume 22, Issue 1 , Pages 13-15, January 2012

Amyloidosis and exercise intolerance in ANO5 muscular dystrophy

  • Margherita Milone

      Affiliations

    • Department of Neurology, Mayo Clinic, Rochester, MN, USA
    • Corresponding Author InformationCorresponding author. Address: Department of Neurology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Tel.: +1 507 538 1037.
    • ,
  • Teerin Liewluck

      Affiliations

    • Department of Neurology, Mayo Clinic, Rochester, MN, USA
    • ,
  • Thomas L. Winder

      Affiliations

    • PreventionGenetics, Marshfield, WI, USA
    • ,
  • Paolo T. Pianosi

      Affiliations

    • Department of Pediatrics, Mayo Clinic, Rochester, MN, USA

Received 10 May 2011; received in revised form 1 July 2011; accepted 8 July 2011. published online 22 September 2011.

Article Outline

Abstract 

Anoctamin 5 and dysferlin mutations can result in myopathies with similar clinical phenotype. Amyloid deposits can occur in the muscle of patients with dysferlinopathy. We describe a 53-year-old woman with exercise intolerance since childhood, recurrent rhabdomyolysis and late-onset weakness. Muscle biopsy showed amyloid deposits within the blood vessel walls and around muscle fibers. Mutation analysis identified two pathogenic heterozygous mutations in anoctamin 5 and no mutations in dysferlin. To our knowledge this is the first report of muscle amyloidosis in anoctamin 5 muscular dystrophy. This finding suggests that patients with amyloid in muscle should be screened for anoctamin 5 muscular dystrophy.

Keywords: ANO5, Amyloidosis, Muscular dystrophy, Anoctaminopathy, Anoctamin 5

 

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1. Introduction 

Anoctamin 5 (ANO5) is a putative calcium-activated chloride channel mostly expressed in the human skeletal and the cardiac muscle as well as in the bone [1]. In the muscle it is expressed in intracellular membranes and it is up-regulated during myocytes differentiation [2], [3]. ANO5 consists of eight transmembrane domains and a conserved DUF590 (Domain of Unknown Function 590) [3]. Dominant mutations in ANO5 cause gnathodiaphyseal dysplasia, a disease characterized by bone fragility, sclerosis of tubular bones and cemento-osseus lesions [1]. Recessive mutations in ANO5 result in limb girdle muscular dystrophy (LGMD) 2L associated with asymmetric atrophy of the quadriceps femoris and the biceps brachii or in distal myopathy resembling dysferlinopathy with Miyoshi phenotype [4], [5], [6].

Patients with ANO5 mutations may complain of myalgia and muscle tightness during exercise or at rest [5], and occasionally they can have myoglobinuria [6].

Amyloid deposition can occur within the interstitium or blood vessel walls of the skeletal muscle of patients with dysferlinopathy. The amyloid deposits were first observed in association with dysferlin mutations clustered in the N-terminal and were shown to contain dysferlin [7]. Later, similar pathological findings were reported in patients with dysferlin mutations scattered from the N- to the C-terminal regions of the protein and not in other LGMDs, leading to the conclusion that the amyloid deposits in the muscle are unique and specific to dysferlinopathy [8].

We report a patient with ANO5 mutations presenting with a long history of exercise intolerance and amyloid deposits in the muscle, similar to those observed in dysferlinopathy.

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2. Case report 

A 53-year-old woman presented with exercise-induced myalgia and fatigue and hammer toes since childhood. She learned to limit her physical activity to avoid the myalgias which were affecting mainly the lower extremities. She had no exercise-induced muscle cramps or history of complications from exposure to general anesthesia. At age 38, due to persistent fatigue after flu, her CK value was checked for the first time and found to be 12,000U/L. A few years later she was found to have proximal leg weakness. In addition, she was experiencing exercise-induced exacerbation of the baseline weakness to the point that at times she was unable to climb small steps and required hospitalization because of severe weakness. She is of Irish and Polish descendent on the paternal and maternal sides, respectively. There is no parental consanguinity. Her parents are asymptomatic, but the mother was found to have a mild intermittent hyperCKemia while on statin, not resolving with discontinuation of the drug. The patient’s neurological examination revealed a mild symmetric weakness of the proximal limb muscles and the foot dorsiflexor muscles, inability to walk on toes and diffuse muscle atrophy, more pronounced in the left calf muscles.

CK values were 455U/L (normal 38–176U/L) at the time of her first visit at the Mayo Clinic and 1107U/L a week later. In the preceding years, the CK values had been fluctuating between 444 and 22,000U/L, with the highest values being detected after intense physical activity and accompanied by elevated serum myoglobin. Resting lactate, plasma acylcarnitine profile, urine organic acids, TSH, HIV antibodies, serum and urine protein electrophoresis and immunofixation, serum kappa and lambda free light chains, creatinine and urinalysis were normal or negative. EMG study showed mild myopathic changes with rare fibrillation potentials only in the gastrocnemius and no associated peripheral neuropathy. Electrocardiogram and echocardiogram were normal. CT of chest, abdomen and pelvis revealed no organomegaly. Abdominal subcutaneous fat aspirate showed no amyloid.

Muscle biopsy of the vastus lateralis showed rare necrotic muscle fibers and congophilic deposits in many intramuscular blood vessel walls and within the interstitium, by Congo red stain (Fig. 1A–C). Immunostain for dysferlin and caveolin-3 was normal. Dysferlin reactivity was observed in the blood vessel walls, independently from the presence of amyloid deposits (Fig. 1D). Mass spectrometry was attempted on captured muscle amyloid deposits but failed to subtype the amyloid. Biochemical assay of glycolytic enzymes, carnitine palmytoil transferase 2, coenzyme Q10 and respiratory chain complexes performed on fresh muscle tissue by a commercial laboratory (Robert Guthrie Biochemical & Molecular Genetics Laboratory, Buffalo NY) was normal.

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  • Fig. 1. 

    Patient’s biopsy of the vastus lateralis muscle. Congo red stained sections visualized under rhodamine optics (A and B) and light microscopy (C) reveal amyloid deposits within the blood vessel walls (A, B, C; arrows) and around muscle fibers (B; asterisk). Immunoreactivity for dysferlin was observed in blood vessel walls independently from the presence of amyloid deposits (D).

Sequencing of all exons and flanking noncoding regions of ANO5 (NCBI RefSeq NM_213599.2) detected two known heterozygous mutations: the common c.191dupA in exon 5 (p.Asn64Lysfs∗15) and the missense mutation c.2018A>G in exon 18 which is predicted to result in p.Tyr673Cys (Fig. 2; Electronic supplementary material). The patient’s mother was found to be a carrier of the c.191dupA mutation, while the father harbors the c.2018A>G mutation. Sequencing of all exons and flanking noncoding regions of ryanodine receptor 1 (RYR1) and transthyretin (TTR) genes revealed no mutations. DYSF, Fukutin-related protein (FKRP), lamin A/C (LMNA) gene sequencing, performed in a commercial laboratory (AthenaDiagnostics, Worcester, MA) prior to the patient’s evaluation at the Mayo Clinic, was also normal.

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3. Discussion 

We identified a patient with muscular dystrophy caused by compound heterozygous ANO5 mutations who presented with a long history of exercise intolerance since childhood and recurrent rhabdomyolysis, mimicking a metabolic myopathy, and showing amyloid deposits in muscle. The two detected ANO5 mutations have been previously reported [4], [5], [6], and a founder effect has been suggested for c.191dupA [6].

Although myalgia, “calf tightness during running” or unrelated to exercise in adult patients [5], “reduced sporting performance in the late teens” and myoglobinuria [6] have been reported in ANO5 muscular dystrophy, our patient’s clinical history underlines that ANO5 mutations can mimic a metabolic myopathy with onset as early as childhood.

The detection of amyloid deposits in the muscle of our patient with ANO5 muscular dystrophy increases the previously observed similarity between anoctaminopathy and dysferlinopathy. These two muscular dystrophies can share not only the clinical phenotype [4], [5], including the “pseudometabolic” phenotype [9], but also the pathological feature of amyloid deposits, raising the possibility of interaction between the sarcolemmal dysferlin and the intracellular membrane-associated anoctamin 5 [3]. The currently available ANO5 antibodies preclude the testing on the expression of ANO5 in the muscle of affected patients [4]. Therefore, one cannot assess if the mutant ANO5 or a fragment of ANO5 folded in β-pleated sheet is a constituent of the amyloid. At the present time, it is also unknown if the amyloidogenic properties belong to any of two specific ANO5 mutations detected in our patient or also to other mutations, as Congo red stain was not performed or not reported in the ANO5 dystrophy patients published to date [4], [5], [6]. It would be of interest to search for amyloid in other ANO5 dystrophy patients, and, in particular, in the two previously reported patients harboring the c.191dupA and p.Tyr673Cys mutations [6]. It would also have been of interest to search for amyloid deposition in the muscle of the patient’s parents to see if any of the two detected ANO5 mutations has amyloidogenic properties, but we did not think that a muscle biopsy was ethically justified in the asymptomatic parents. The amyloid deposits in the muscle of our patient demonstrate that this pathological finding is not unique to dysferlinopathy among muscular dystrophies. Therefore, its presence in the muscle cannot be considered a diagnostic criterion for dysferlinopathy, as previously suggested [8]. As in dysferlinopathy, dysferlin immunoreactivity was detected not only in the walls of the blood vessels displaying amyloid deposits, but also in the walls of blood vessels without amyloid deposits. The latter finding is in agreement with the previously reported dysferlin expression in the human and rodent blood vessels [10]. Our patient did not have any evidence for systemic amyloidosis, as indicated by the lack of immunoglobulin light-chain, lack of amyloid in abdominal fat aspirate, absence of organomegaly, and absent other organ dysfunction apart from the myopathy. Additionally, TTR sequencing was normal.

The asymptomatic mother’s mild intermittent hyperCKemia, persistent despite the discontinuation of the statin, is of interest and raises the possibility that the hyperCKemia may signal a manifested carrier status of the ANO5 c.191dupA mutation.

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Acknowledgments 

This study was supported in part by the Mayo Clinic CTSA through NIH/NCRR grant number UL1 RR024150. We thank the patient and her family for their cooperation in the study.

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Appendix A. Supplementary data 

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  • Fig. 2. 

    Schematic representation of human ANO5 and location of the disease-causing mutations. ANO5 has eight transmembrane domains and intracellular N- and C-terminals. The resulting phenotypes are indicated in parentheses next to each mutation (dMD=distal muscular dystrophy; GDD=gnathodiaphyseal dysplasia; LGMD2L=limb girdle muscular dystrophy 2L). The arrows indicate the mutations detected in our patient.

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References 

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PII: S0960-8966(11)01309-5

doi:10.1016/j.nmd.2011.07.005

Neuromuscular Disorders
Volume 22, Issue 1 , Pages 13-15, January 2012

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