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Brody syndrome: A clinically heterogeneous entity distinct from Brody disease
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Neuromuscular Disorders 22 (2012) 944–954 www.elsevier.com/locate/nmd
Brody syndrome: A clinically heterogeneous entity distinct from Brody disease A review of literature and a cross-sectional clinical study in 17 patients N.C. Voermans a,⇑, A.E. Laan a,c, A. Oosterhof b, T.H. van Kuppevelt b, G. Drost a,d, M. Lammens a,e, E.J. Kamsteeg f, C. Scotton g, F. Gualandi g, V. Guglielmi h, L. van den Heuvel i, G. Vattemi h, B.G. van Engelen a a
Neuromuscular Centre Nijmegen, Department of Neurology, Radboud University Nijmegen Medical Centre, The Netherlands b Department of Biochemistry, Radboud University Nijmegen Medical Centre, The Netherlands c Institute for Genetic and Metabolic Disease, Radboud University Nijmegen Medical Centre, The Netherlands d Department of Clinical Neurophysiology, Radboud University Nijmegen Medical Centre, The Netherlands e Department of Pathology, Radboud University Nijmegen Medical Centre, The Netherlands f Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands g Department of Medical Genetics, Radboud University Nijmegen Medical Centre, University of Ferrara, Italy h Department of Neurological, Neuropsychological, Morphological, and Movement Sciences, University of Verona, Italy i Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, The Netherlands Received 24 February 2012; received in revised form 8 March 2012; accepted 30 March 2012
Abstract Brody disease is a rare inherited myopathy due to reduced sarcoplasmic reticulum Ca2+ ATPase (SERCA)1 activity caused by mutations in ATP2A1, which causes delayed muscle relaxation and silent cramps. So far the disease has mostly been diagnosed by measurement of SERCA1 activity. Since mutation analysis became more widely available, it has appeared that not all patients with reduced SERCA1 activity indeed have ATP2A1 mutations, and a distinction between Brody disease (with ATP2A1 mutations) and Brody syndrome (without ATP2A1 mutations) was proposed. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome and detect clinical features which help to distinguish between the two. In addition, we describe the Brody syndrome phenotype in more detail. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. The results showed that Brody disease presents with an onset in the 1st decade, a generalized pattern of muscle stiffness, delayed muscle relaxation after repetitive contraction on physical examination, and autosomal recessive inheritance. Patients with Brody syndrome more often report myalgia and experience a considerable impact on daily life. Future research should focus on the possible mechanisms of reduction of SERCA activity in Brody syndrome and other genetic causes, and on evaluation of treatment options. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Brody syndrome; Brody disease; Brody myopathy; Ca ATPase; SERCA1; Delayed relaxation; Silent cramps
⇑ Corresponding author. Address: Neuromuscular Centre Nijmegen,
Department of Neurology, 935 Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31 024 3616600; fax: +31 24 3541122. E-mail address: [email protected] (N.C. Voermans). 0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.03.012
1. Introduction Brody disease is a rare inherited myopathy due to a diminished sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)1 activity caused by mutations in ATP2A1. The
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incidence of Brody disease is estimated to be 1 in 10,000,000, but this might be underestimated due to incomplete recognition [1,2]. Brody disease was initially described by Brody in 1969 [2]. He reported a patient with muscle stiffness starting at five years of age, which occurred during vigorous exercise or sudden rapid movements, and disappeared after several seconds of rest. Most muscle groups were involved. Muscle contraction was normal, but the relaxation phase became increasingly slow following repeated exercise (Table 1: Clinical key features of Brody disease). Myotonia was ruled out by the presence of electrical silence on electromyography during stiffness (“silent cramps”) [2]. Reduced SERCA1 activity results in delayed muscle relaxation due to prolonged increase of intracellular calcium concentration following contraction [3]. Fig. 1 illustrates the role of SERCA1 in muscle relaxation. Two types of SERCA predominate in muscle: SERCA1 predominates in type 2 fast-twitch skeletal muscle fibres and SERCA2 predomi-
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nates in type 1 slow-twitch skeletal and cardiac muscle fibres [9]. Brody disease is due to modifications of SERCA1 only, and accordingly no cardiac complications in Brody disease have been reported. Since SERCA1 is only present in fasttwitch muscle fibres (type 2), impaired relaxation specifically occurs after phasic exercise (alternate contraction and relaxation). In contrast, tonic activity (such as maintaining posture) requires slow-twitch motor-unit activation, which recruits predominantly type 1 fibres [4,5]. In 1996, mutations in the ATP2A1 gene were found to cause Brody disease. However genetic heterogeneity is likely considering that ATP2A1 mutations are missed in a quote of patients with recessive inheritance and have never been found in patients with an autosomal dominant pattern. In 2002, MacLennan proposed to distinguish patients with reduced SERCA1 activity and ATP2A1 mutations (Brody disease) from patients with reduced SERCA activity without ATP2A1 mutations (Brody syndrome) [6]. Brody
Table 1 Clinical key features of Brody disease and definitions used in this study. Clinical key features of Brody disease based upon the first case description of Brody in 1969 [1] Onset in childhood: “his earliest recollection of a movement disorder was falling down in a foot race at the age of 5 years because his muscles stiffened” Exercise-induced muscle stiffness: “muscle stiffness occurred during vigorous exercise or upon sudden, rapid movement,. . .disappeared after several seconds of rest” Normal muscle strength: “skeletal muscles, which were normal in contour, strength and tone” Involvement of most muscle groups: “this response to exercise was noted in the muscles of the limbs, the face and the jaws” Delayed muscle relaxation after repetitive contraction on physical examination: “skeletal muscles. . .showed a progressive slowing of relaxation with vigorous exercise” No myalgia independent of muscle stiffness: “painless unless the patient continued his efforts to contract the shortened muscle” Increase in cold: “but in cold weather the stiffness occurred more readily” “after three minutes in a room refrigerated to 5° there was a marked increase in the patient’s exercise intolerance. Repetitive contraction produced paralysis within five seconds, whereas 10–15 seconds where required at the usual room temperature” Silent cramp on electromyography: “when sustained muscle shortening was induced by exercise, insertion activity was normal and was followed by electrical silence” Definitions used in literature review and clinical study History
Physical examination
Exercise-induced muscle stiffness Muscle cramp Myalgia Pattern of involvement of muscles Effect of medication Muscle weakness Delayed muscle relaxation after repetitive contractions Delayed muscle relaxation after sustained contractions Effect of cold
Ancillary investigations
Silent cramp
Involuntary painless hardening of the muscle immediately following the onset of exercise Sustained painful shortening of the muscle Muscle pain independent of muscle stiffness or cramp Pattern of involvement of muscles in which exercise-induces stiffness is reported: this is classified as generalized when muscles in both limbs and face are involved Reduction of the exercise-induced muscle stiffness with use of medication Reduction of maximal voluntary muscle force measured by manual muscle testing scored by the MRC score Inability to immediately relax muscles after voluntary repetitive contractions with maximal effort for at least 10 s, which results in a stiffness of the muscle Inability to immediately relax muscles after voluntary sustained contraction with maximal effort for at least 10 s, which results in a stiffness of the muscle Increase of delay of muscle relaxation after voluntary repetitive contractions with maximal effort for at least 15 s in cold water Electromyography during the delayed muscle relaxation shows normal insertion activity followed by electrical silence
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Fig. 1. Excitation, contraction, and relaxation of skeletal muscle. End-plate potential in T-tubulus causes opening of the ryanodine receptor: the end-plate action potential (AP) propagates throughout the myocyte’s sarcolemma and into the T-tubule system. The T-tubule contains dihydropyridine receptors (DHPR) which are voltage-dependent calcium channels and are activated by the AP. The dihydropyridine receptors transmit the voltage-mediated signal through a mechanical linkage to the ryanodine receptors (RYR) in the sarcoplasmic reticulum (SR). Ryanodine receptors undergo a conformational change that opens their channel. Contraction: Opening of RYR causes a flow of Ca2+ from the sarcoplasmic reticulum into the cytoplasm. In this release, Ca2+ unbinds from the calcium-binding protein called calsequestrin (CSQ). Ca2+ released from the sarcoplasmic reticulum binds to Troponin C on actin filaments, which subsequently leads to the troponin complex being physically moved aside to uncover cross-bridge binding sites on the actin filament. By hydrolyzing ATP, myosin forms a cross bridges with the actin filaments, and pulls the actin toward the centre of the sarcomere resulting in contraction of the sarcomere. Relaxation: Simultaneously, the sarco/endoplasmic reticulum Ca2+-ATPase actively pumps Ca2+ back into the sarcoplasmic reticulum where Ca2+ rebinds to calsequestrin. With Ca2+ no longer bound to troponin C, the troponin complex slips back to its blocking position over the binding sites on actin. Since cross-bridge cycling is ceasing then the load on the muscle causes the inactive sarcomeres to lengthen.
syndrome patients generally suffer from (exercise-induced) muscle stiffness and delayed muscle relaxation; however, only a few muscle groups are involved, and onset is in adolescence or adulthood [6]. This was described as an expert opinion [6], is not easy to trace and not referred to in the sparse literature on Brody disease since then. Furthermore, muscle stiffness, a prominent feature of Brody syndrome, is a very frequent complaint among patients at the neuromuscular outpatients’ clinic. Therefore a more detailed investigation of the Brody syndrome phenotype is warranted. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. 2. Patients and methods 2.1. Definition of clinical key features of Brody disease Table 1 shows the clinical key features of Brody disease abstracted from the initial clinical case description of Brody in 1969 [2].
2.2. Review of literature cases and selection of literature (L) cases We reviewed all publications cited by Pubmed on ‘Brody disease’, ‘Brody syndrome’ or ‘Brody myopathy’ since the initial description in 1969 by Brody [2] until 2010 and the references of these articles, and selected all patients in whom clinical data were available (n = 32; Supplementary Table 1) [2,4,5,7–14]. We classified them as literature (“L”) cases, numbered them chronologically, and screened the case descriptions for clinical key features of Brody disease. The mode of SERCA(1) activity measurement, ATP2A1 mutations, and modes of inheritance were noted. 2.3. Clinical study and selection of study (S) patients At the outpatient department of our neuromuscular centre, which is a tertiary referral centre for neuromuscular diseases, SERCA activity is measured in case of (exerciseinduced) muscle stiffness and/or silent muscle cramps in absence of other causes. It is compared with normal values obtained in control subjects as described previously [5]. Between 1992 and 2007, SERCA activity was measured in approximately 200 patients, and reduced compound SERCA activity had been detected in 22 patients. One
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Fig. 2. SERCA1 and SERCA2 staining. SERCA1 and SERCA2 antibodies stained the SR ATPase in type 2 and type 1 fibers, respectively. Immunoreactivity for SERCA1 and SERCA2 was similar in patients’ and controls’ muscles. The images are the staining in one of the patients with Brody syndrome in this study.
patient was later diagnosed with sodium channel myotonia due to a SCN4A mutation (SERCA activity 22.6 mU/mg at age of 60 years; this is below the normal value; see Fig. 3). Two other patients were excluded because of neurological and psychiatric co-morbidity (leucencephalopathy in one, and somatisation disorder in two other patients), one patient could not be traced anymore, and one patient declined participation. Hence, 17 patients were included. Four of these patients had been described by Benders in 1994 (S6, 7, 11 and 17) [5] as well as by Poels and Wevers (S11) [9,21]. 2.4. Questionnaires All patients completed a standardized questionnaire on the Brody disease clinical key features defined in Table 1. Additional questions focused on initial and current symp-
toms, fatigue, medical history, medication use, functional impairment (graded with the modified Rankin score) [15], and use of mobility aids. 2.5. Physical examination Physical examination consisted of a neurological examination including manual muscle testing in 24 muscles (11 bilateral limb muscles and neck extensors and flexors; score 0–5; with a MRC sum score of 0–120) and dynamometry in nine muscles bilaterally as previously described (Citec hand-held dynamometer: www.citec.nl) [16,17]. To detect delayed muscle relaxation, duration of relaxation after sustained contraction (Sustained Contraction (SC), sustained forceful eyelid closure (E), and sustained forceful clenching of hands (H), both for 10 s) was measured, the latter both in room temperature and in cold water. Furthermore, the
Fig. 3. Compound SERCA activity of the patients in this study related to age. Compound SERCA activity is expressed in mU/mg protein.
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patients were asked to perform forceful repetitive elbow flexion and contraction (RC: alternate contraction and relaxation) for 10 s, with qualitative evaluation of delayed relaxation. Delay of muscle relaxation was defined as duration until complete relaxation >1 s.
nificance of the differences was calculated with the Fisher Exact test (two-sided). Data analysis was performed using Microsoft Excel and SPSS (version 16.0 for Windows) to generate descriptive statistics. 3. Results
2.6. Previously performed ancillary investigations 3.1. Features of patients in literature: L1–L32 The medical files of these patients were reviewed for results of creatine kinase measurements, nerve conduction studies, electromyography, and muscle biopsy (needle biopsy of the quadriceps femoris muscle). All these tests had been performed previously in our centre according to existing diagnostic protocols. Standard enzyme histochemical and immunohistochemical techniques had been used to exclude common myopathies [18]. 2.7. SERCA1 and SERCA2 immunohistochemical staining SERCA1 and SERCA2 immunohistochemical staining was performed on the muscle biopsies that were still available (n = 10). This was done on transverse muscle sections using fluorescence method with antibodies to SERCA1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and SERCA2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). We compared the results with controls including biopsies of vastus lateralis muscle from subjects who were found to be free of muscle disease [14,19]. 2.8. SERCA activity SERCA activity measurements have been performed in our centre since 1992. Compound SERCA activity was measured in whole muscle homogenates obtained from needle biopsies of the quadriceps femoris muscle as previously described [5]. Compound SERCA activity is considered a good estimate of SERCA1 activity, since SERCA1 is the predominant isoforms in the quadriceps femoris muscle [5]. 2.9. Mutation analysis of ATP2A1
Since the initial report of Brody in 1969 until 2010, 35 patients have been reported in the literature, of which 32 with a description of clinical features (L1–L32 in Tables 2A, 2B and Supplementary Table 1) [3–5,7–10,12–14,20– 23]. L1 represents the patient reported by Brody in 1961 [2]. Results of history, physical examination, and ancillary investigations are summarized in Table 2A. Different techniques have been used to measure SERCA1 activity or content; therefore, these quantitative data are difficult to compare. Some studies measure protein content, while others measure the activity of the SERCA1 protein [5], or both [4]. Benders et al. report the compound activity of SERCA1, SERCA2, and SERCA3. In the quadriceps muscle this consists predominantly of SERCA1 activity (83%) [5]. Furthermore, SERCA1 activity is often measured in isolated microsomal or SR fractions [4,5], while other studies report data on SERCA1 activity in whole muscle homogenates [7]. Reduction of isolated SERCA1 activity, compound SERCA activity, or calcium flow or SERCA content has been reported in all cases, with a residual SERCA1 activity ranging from 2% to 50% of normal [4,5,8,14]. Recessive mutations in the ATP2A1 gene (16p12.1–12.2) have been detected in 11 patients from seven families, including deletions and missense, frameshift or nonsense mutations. In other families with either autosomal dominant or autosomal recessive inheritance, no ATP2A1 mutations were found. SLN, the gene encoding sarcolipin was considered a prime candidate gene for Brody disease. However, mutation analysis of this gene in five Brody families did not reveal any alternations in coding, splice junction or promoter sequences [11].
Mutation analysis of the ATP2A1 gene was performed in six patients in Nijmegen in 2000 (Radboud University Nijmegen Medical Centre, The Netherlands) by checking DNA sequences of the individual exons, the splice donor and splice acceptor sites [20]. Gualandi (Ferrara University, Italy) performed mutation analysis in eleven patients in 2010 on genomic DNA as previously described [16]. Subsequently, segregation analysis was performed in seven sibs and the mother of one of the patients in whom an heterozygous unclassified variant had been detected.
3.2. Features of patients in clinical study: S1–S17
2.10. Data analysis
3.3. Questionnaires
We compared the clinical features, and results of genetic analysis and ancillary investigations of patients with Brody disease and those with Brody syndrome. The statistical sig-
Table 2A summarizes the clinical key features and Table 3 shows the additional features of the patients in this study (S1–S17). Frequent initial symptoms were: muscle
Table 2A shows the demographic features; nine females and eight males were included and the mean age was 51 years (SD 12.5 years). Ten patients were sporadic cases; and seven patients from three families were included, two of which revealed an autosomal recessive inheritance pattern. Clinical presentation of patients within these families was heterogeneous. The medical history included epilepsy (n = 1), minor stroke (n = 1), and hypothyroidism (n = 2).
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Table 2A Clinical features of patients with Brody disease and Brody syndrome in literature and in this study.
(continued on next page)
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Table 2A (continued)
L1, Brody, 1969 [1]; L9–L10, Taylor, 1988 [4]; L 21–22, Zhang, 1995 [8]; L29–L30, Novelli, 2004 [13]; L2–L4, Karpati, 1986 [2]; L11, Wevers, 1992 [5]; L23, Odermatt, 1997 [10]; L31–L32, Vattemi, 2010 [14]; L5–L8, Danon, 1988 [3]; L12–L20, Benders, 1994 [7]; L24–L28, Odermatt, 2000 [12]. L# = patient described in literature, numbered chronologically and corresponding to the numbers in Table 2. L2 and L3, siblings; L5 and L6, siblings; L9 and L10, siblings; L14 and L15, siblings; L26 and 27, siblings; L31 and L 32, siblings. S# = patient included in this study, some of which were also reported in literature before. Numbered following age, from young to old. S17, mother of S6; S8 and S10, siblings; S13,14 and 16, siblings. , (effect) absent; +, (effect) present; blank, unknown/not reported; n.p., not performed; ", mildly increased; D, dantrolene; V, verapamil; s.e., withdrawn due to side effects; n.u., never used; AD: autosomal dominant; AR: autosomal recessive; S: sporadic. MMT: manual muscle testing; CK: creatine kinase; EMG, electromyography; SC (EMG): silent cramps on electromyography; M (EMG): myopathic pattern on electromyography; M (biopsy), myopathic findings on muscle biopsy.
cramps, myalgia, muscle stiffness, and easy fatigability. Not any of the patients initially presented with delayed muscle relaxation. Frequently reported current symptoms were: exercise-induced muscle stiffness, muscle cramps, myalgia (Tables 2A and 2B) and muscle weakness (Table 3). Muscle stiffness and delayed muscle relaxation were reported most frequently in hand, legs, and toes, but not generalized. Myalgia occurred after exercise in seven patients and continuously in ten patients; predominantly in legs and arms and with a mean VAS score of 7.1 (SD 1.7). Muscle cramps occurred spontaneously in two patients in rest, after exercise in four patients, and both in rest and after exercise in eight patients, most often in fingers and toes (mean VAS score of 6.0 (SD 1.8)). Cold caused an exacerbation of muscle stiffness in 10 patients and of cramps in eight. Fourteen patients reported progression of symptoms over the years. The symptoms caused considerable impact on daily life (Table 3). Nine patients used a wheelchair, rollator or mobility scooter. Thirteen reported severe fatigue, which in some cases required daytime sleeping. Six patients were unable to work fulltime, and two of them were fully unfit for work. Thirteen patients refrained from sporting because of the severity of complaints. The effect of medication was limited: with a positve effect in only few patients of dantrolene (n = 1), verapamil (n = 2), and clonazepam (n = 3). Ten patients stopped the
use of dantrolene or verapamil because of side effects (muscle weakness, depression, and malaise) or absence of effect. Analgesics (paracetamol, ibuprofen, tramadol, naproxen) were used with only limited effect on myalgia and cramps. Two patients reported a positive effect of etoricoxib. One patient (S11) reported several episodes of exertional rhabdomyolysis with myoglobinuria, which was confirmed by elevated serum creatine kinase values up to 11,000 U/l [21]. Various other non-related symptoms were reported: palpitations (n = 3), exercise dyspnoea (n = 9), oedema (n = 4), dysphagia (n = 3), sense of slow passage of food in oesophagus (n = 4), constipation (n = 6), stress-incontinence (n = 2), urge-incontinence (n = 5), paresthesias (n = 7), numbness (n = 6), headache (n = 7), and vertigo (n = 3). 3.4. Physical examination Results of physical examination are summarized in Tables 2A and 3. Delayed muscle relaxation after sustained forceful eyelid closure and/or after forceful clenching of both hands was observed in seven patients. In contrast, delayed relaxation after repetitive contraction was not observed in any of the patients. No percussion myotonia was observed. One patient had mild muscular hypertrophy while not performing sports (S8).
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Table 2B Comparison of clinical features between patients with Brody disease and Brody syndrome.
Gen.: Genetic features CK: creatine kinase: EMG: electromyography SC (EMG): silent cramps on electromyography M (EMG): myopathic pattern on electromyography M (biopsy): myopathic findings on muscle biopsy * Fisher exact test (two-tailed).
Table 3 Additional results of questionnaire, physical examination and ancillary investigations in clinical study. Questionnaire MW MRS MA (0–5)
S1 S2 S3 S4 S5 S6 (L17) S7 (L19) S8 S9 S10 S11 (L11) S12 S13 S14 S15 S16 S17 (L20) Mean/total (%)
+ + + + + + + + + + + + + + 15 (88%)
2 2 3 3 2 3 3 3 2 2 3 1 2 3 2 2 3 2.4
+ + + + + + + + + 9 (53%)
Physical examination F
+ + + + + + + + + + + + + 13 (72%)
Genetics
Muscle MRC Muscle weakness sum weakness (MMT) score (D) (0–120)
Muscles with weakness on D (0– 17)
Delayed ATP2A1 relaxation mutation after analysis
+ + + + + + 6 (35%)
4 2 1 5 7 0 9 5 4 3 2 3 9 8 9 9 9 5
0
120 120 120 120 120 120 89 120 120 120 120 120 113 104 82 88 104 113
+ + + + + + + + + + + + + + + + 17 (94%)
SERCA1 Ancillary investigations activity Muscle biopsy
RC SC H/ E +/+ +/+ +/ +/+ +/+ +/+ +/+ 7 (41%)
Type Internal RR IH Biochemical I nuclei fibres SERA1 analysis fibres and 2 uv 0
21.5 35.3 19.4 16.4 22.3 19.4 21.2 24.7 23.3 26.3 20.6 20.5 31.7 21.5 21.9 29.6 22.6
# N N N N # N " N N # N # # N " N
N N N N N
"
+
N N
"
N N N N N # N
N + +
N N N
+
MW, Muscle weakness; MRS, Modified Rankin scale; MA, use of mobility aids (e.g. wheelchair, rollator, bicycle with electrical assistance); F, fatigue; MMT, manual muscle testing; D, dynamometry; N, normal; ", increased; #, decreased +, present; , absent; uv = unclassified variant, heterozygous R604H (c.1995G>A). Subsequent segregation analysis has revealed that this heterozygous uv was also detected in five sibs and the mother of this patient, none of whom were affected. The only affected sib had two wild type alleles. The column “Delayed relaxation after repetitive contractions (RC)” is a duplication of Table 2A but included to show the difference with “Delayed relaxation after sustained contraction (SC)”. H, hands; E, eyes.
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3.5. Previously performed ancillary investigations Results of previously performed ancillary investigations are summarized in Tables 2A and 3. One patient (S11) showed an elevated serum creatine kinase (CK = 710 U/l (normal = <220 U/l)). This patient had previously been reported to have exertional rhabdomyolysis [21]. CK increase with a history of rhabdomyolysis and myoglobinuria has been reported in only one other Brody syndrome patient (L29) [13]. In all other patients, CK was normal. Electromyography had previously been performed in all patients at the time of diagnosis; in six patients this revealed mild to moderate myopathic features, including small, polyphasic units and fast recruitment. In one of them and in another patient, electromyography was performed during a clinical cramp, which was electrically silent. Electromyography was normal in the other patients. Histological analysis of the quadriceps biopsies revealed a normal distribution of type I and type II fibres in 10 patients; type II fibres were relatively abundant in five patients, and in two patient type I fibres were more predominant (57% and 58%, respectively [normal 35–50% type I]). Mild myopathic features including increased variation in fibre diameter, increase of internal nuclei and few necrotic fibres were observed in only two patients (S8 and S11). In four patients, few ragged-red fibres were observed, which were within normal limits for their age. Oxidative phosphorylation enzyme activities and total ATP production capacity were measured in eight patients: in one of them, ATP production capacity was reduced without a reduction of the enzyme activities. 3.6. SERCA1 and SERCA2 immunohistochemical staining SERCA1 and SERCA2 antibodies stained the SERCA in type 2 and type 1 fibers, respectively. Immunoreactivity for SERCA1 and SERCA2 was similar in patients’ and controls’ muscles (Fig. 2) [14]. 3.7. SERCA activity Fig. 3 shows the compound SERCA activity of the patients in this study. The 99% confidence interval of compound SERCA activity in normal subjects is depicted by the diagonal lines. All patients in whom delayed muscle relaxation after sustained contraction was detected on physical examination had a considerably reduced SERCA activity below 23 mU/mg. 3.8. Mutation analysis of ATP2A1 Checking of DNA sequences of the individual exons, the splice donor and splice acceptor sites of the ATP2A1 gene of the first seven patients (S6, S7, S11, S13, S14, S16, S17) in 2000 revealed no mutations. Sequence analysis of one of the remaining 10 patients only revealed a previously unre-
ported heterozygous unclassified variant in exon 15: R604H (c.1995G>A) in S8. Subsequent segregation analysis in seven sibs and the mother revealed that this missense change was also present in five sibs and the proband mother, none of whom were affected. The only affected sister (S10) had two wildtype alleles. This R604H variation was therefore not considered pathogenic. No pathogenic mutations were detected in other patients. Several known polymorphisms were detected (Supplementary Table 2) as well as a synonymous change within exon 15 (Pro603Pro) occurring in heterozygosity in two unrelated patients. 3.9. Comparison of Brody disease and Brody syndrome patients Table 2B shows the differences of Brody disease and Brody syndrome patients. Patients with Brody disease more often report an onset in the 1st decade, a generalized pattern of muscle involvement, and autosomal recessive inheritance. Delayed relaxation after repetitive contraction of the elbow flexors is observed more often in Brody disease. In contrast, patients with Brody syndrome report myalgia more frequently. A history of exercise-induced muscle stiffness or muscle cramps, effect of medication, and the presence of muscle weakness or increase with cold on physical examination does not distinguish between the two disorders. 4. Discussion This literature review and cross-sectional study in 17 patients with Brody syndrome shows that Brody disease (reduced SERCA activity with ATP2A1 mutations) presents with onset in childhood; a generalized pattern of muscle involvement, autosomal recessive inheritance, and delayed relaxation after repetitive contraction on physical examination. In contrast, patients with Brody syndrome report myalgia more frequently (reduced SERCA activity without ATP2A1 mutations). Furthermore, the impact of Brody syndrome on daily functioning was greater than previously described and the effect of medication (dantrolene and verapamil) was limited mainly due to side effects. A history of exercise induced muscle stiffness did not distinguish between the two groups and may be considered aspecific. Increase of delayed relaxation in cold and presence of silent cramps on electromyography did neither. These features were described in the initial case by Brody, but have only been tested in few literature cases. Therefore, these features should be tested prospectively in a larger number of patients in order to be able to determine their predictive value. These findings thus confirm the distinction between Brody disease and Brody syndrome proposed by MacLennan and Loke [6], and we suggest to use this distinction in current clinical practice. Of note, we continue using the term Brody ‘syndrome’ as suggested by MacLennan, although strictly speaking, ‘syndrome’ refers to the association of several clinically recognizable features (signs and
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symptoms) [6], whereas Brody syndrome patients have been biochemically classified. In addition, these findings suggest that the specificity of reduced SERCA1 activity in muscle for detecting Brody disease is limited. This was also put forth by a study on reduced SERCA1 activity in muscle tissue from patients with myotonic dystrophy type 1 [23]. The findings of this study are very relevant since muscle stiffness, muscle cramps, and myalgia are frequent complaints among patients at a neuromuscular outpatients clinic and difficult to measure. Measurement of SERCA activity in these patients is a way to characterize these symptoms quantitatively, and the findings of this study might be of help to select patients for ATP2A1 mutation screening. The differences between Brody disease and Brody syndrome raise the question whether Brody syndrome is a separate entity or an epiphenomenon of other underlying diseases with a final common pathway of altered calcium transport. Several previous observations support this latter hypothesis. One of the patients was excluded for this study since a heterozygous SCN4A mutation had been detected and she had been diagnosed with sodium channel myotonia shortly after the SERCA measurement was performed. Furthermore, S11 [5,9,21] was the only patient in our series with both recurrent rhabdomyolysis (CK up to 11,000 IU/ L) and myopathic features in the muscle biopsy. Although reported with Brody disease [5,9,21], he might have another myopathy for which he is currently under investigation (so far, RYR1, FKRP, CPT2, and LPIN1 mutations have been excluded). Hypothyroidism might be another factor that influences SERCA activity. Animal studies have indeed shown that hypothyroidism induces a decrease in SERCA activity with delayed muscle relaxation [24]. Two of the patients in this study had a history of hypothyroidism, and in one of them the initial muscle symptoms started after the onset of hypothyroidism. Lastly, since SERCA1 is an ATPase channel, SERCA function might be altered in patients with mitochondrial disease and reduced ATP production. Mutations in other genes might also influence SERCA1 activity. SLN, the gene encoding sarcolipin was considered a prime candidate gene for Brody disease, but no mutations were detected in five Brody families [11]. Interestingly, patients with polymorphic ventricular tachycardia can have mutations in the cardiac specific isoform of calsequestrin, a sarcoplasmic reticulum calcium store [25]. When expressed in rat myocytes, these mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of I(Ca)-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. In analogy, mutations in the skeletal muscle specific calsequestrin isoform may be involved in Brody syndrome. Also, the function of SERCA1 points to other neighbouring proteins (for an overview see [26]), which may be involved in the aberrant muscle Ca2+ homeostasis in Brody syndrome. Finally, the fact that some patients reported prolonged stiffness in hands after tonic rather than phasic exercises raises the
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questions whether mutations in ATP2A2 (encoding SERCA2 in slow-twitch type 1 fibers) might be involved; however, mutations in this gene have so far only been associated with dermatological disease (Darier’s disease) or neuropsychiatric disorders [27]. One limitation is that in 2000 we only checked the DNA sequences of the individual exon, the splice donor and splice acceptor sites of the ATP2A1 gene, and did not screen the promoter sequence or enhancer/silencing sequences of this gene for the presence of disease-causing mutations. This means we cannot fully exclude mutations in these patients. Another limitation is that only patients with Brody syndrome were clinically investigated, whereas clinical features of Brody disease were deduced from the literature cases. A related problem is the lack of data on natural course of Brody disease, including functional impairment, since most reports in literature describe recently diagnosed patients. If ATP2A1 mutations analysis becomes more widely available and more cases of Brody disease are recognized, a more direct comparison between the two groups might be possible. In short, we have presented the result of a literature review and clinical study on features of Brody disease and syndrome, showing that Brody disease typically presents with early onset; generalized exercise-induced muscle stiffness, autosomal recessive inheritance, and delayed relaxation after repetitive contraction on physical examination. In contrast, patients with Brody syndrome report myalgia more frequently. Further research should focus on various mechanism involved in reduction of SERCA activity in Brody syndrome, e.g. hypothyroidism or mitochondrial disease, and other genetic causes. 5. Financial disclosure N.V. was supported by a clinical fellowship for neuromuscular disorders by the Prinses Beatrixfonds, the Netherlands. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2012.03.012. References [1] MacLennan DH. Ca2+ signalling and muscle disease. Eur J Biochem 2000;267:5291–7. [2] Brody IA. Muscle contracture induced by exercise. A syndrome attributable to decreased relaxing factor. N Engl J Med 1969;281: 187–92. [3] De Ruiter CJ, Wevers RA, Van Engelen BG, Verdijk PW, De HA. Muscle function in a patient with Brody’s disease. Muscle Nerve 1999;22:704–11. [4] Karpati G, Charuk J, Carpenter S, Jablecki C, Holland P. Myopathy caused by a deficiency of Ca2+-adenosine triphosphatase in sarcoplasmic reticulum (Brody’s disease). Ann Neurol 1986;20:38–49.
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[5] Benders AA, Veerkamp JH, Oosterhof A, et al. Ca2+ homeostasis in Brody’s disease. A study in skeletal muscle and cultured muscle cells and the effects of dantrolene an verapamil. J Clin Invest 1994;94: 741–8. [6] MacLennan DH, Loke JCP. Brody disease associated with defects in a calcium pump. In: Karpati G, editor. Structural and molecular basis of skeletal muscle disease. Basel: ISN Neuropath; 2002. p. 103–5. [7] Danon MJ, Karpati G, Charuk J, Holland P. Sarcoplasmic reticulum adenosine triphosphatase deficiency with probable autosomal dominant inheritance. Neurology 1988;38:812–5. [8] Taylor DJ, Brosnan MJ, Arnold DL, et al. Ca2+-ATPase deficiency in a patient with an exertional muscle pain syndrome. J Neurol Neurosurg Psychiatry 1988;51:1425–33. [9] Wevers RA, Poels PJ, Joosten EM, Steenbergen GG, Benders AA, Veerkamp JH. Ischaemic forearm testing in a patient with Ca(2+)ATPase deficiency. J Inherit Metab Dis 1992;15:423–5. [10] Zhang Y, Fujii J, Phillips MS, et al. Characterization of cDNA and genomic DNA encoding SERCA1, the Ca(2+)-ATPase of human fast-twitch skeletal muscle sarcoplasmic reticulum, and its elimination as a candidate gene for Brody disease. Genomics 1995;30: 415–24. [11] Odermatt A, Taschner PE, Scherer SW, et al. Characterization of the gene encoding human sarcolipin (SLN), a proteolipid associated with SERCA1: absence of structural mutations in five patients with Brody disease. Genomics 1997;45:541–53. [12] Odermatt A, Barton K, Khanna VK, et al. The mutation of Pro789 to Leu reduces the activity of the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) and is associated with Brody disease. Hum Genet 2000;106:482–91. [13] Novelli A, Valente EM, Bernardini L, et al. Autosomal dominant Brody disease cosegregates with a chromosomal (2;7)(p11.2;p12.1) translocation in an Italian family. Eur J Hum Genet 2004;12:579–83. [14] Vattemi G, Gualandi F, Oosterhof A, et al. Brody disease: insights into biochemical features of SERCA1 and identification of a novel mutation. J Neuropathol Exp Neurol 2010;69:246–52. [15] Rankin J. Cerebral vascular accidents in patients over the age of 60. III. Diagnosis and treatment. Scott Med J 1957;2:254–68.
[16] Peterson-Kendall F, Kendall-McCreary E, Geise-Provance P, McIntyre-Rodgers M, Romani WA. Muscles testing and function with posture and pain. Baltimore, MD, USA: Lipincott Williams & Wilkins; 2005. [17] van der Ploeg RJ, Fidler V, Oosterhuis HJ. Hand-held myometry: reference values. J Neurol Neurosurg Psychiatry 1991;54:244–7. [18] Voermans NC, van Alfen N, Pillen S, et al. Neuromuscular involvement in various types of Ehlers–Danlos syndrome. Ann Neurol 2009;65:687–97. [19] Vattemi G, Engel WK, McFerrin J, Askanas V. Endoplasmic reticulum stress and unfolded protein response in inclusion body myositis muscle. Am J Pathol 2004;164:1–7. [20] Odermatt A, Taschner PE, Khanna VK, et al. Mutations in the geneencoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase, are associated with Brody disease. Nat Genet 1996;14:191–4. [21] Poels PJ, Wevers RA, Braakhekke JP, Benders AA, Veerkamp JH, Joosten EM. Exertional rhabdomyolysis in a patient with calcium adenosine triphosphatase deficiency. J Neurol Neurosurg Psychiatry 1993;56:823–6. [22] Hiel H, Happe HK, Warr WB, Morley BJ. Regional distribution of a creatine transporter in rat auditory brainstem: an in-situ hybridization study. Hear Res 1996;98:29–37. [23] Benders AA, Wevers RA, Veerkamp JH. Ion transport in human skeletal muscle cells: disturbances in myotonic dystrophy and Brody’s disease. Acta Physiol Scand 1996;156:355–67. [24] Periasamy M, Kalyanasundaram A. SERCA pump isoforms: their role in calcium transport and disease. Muscle Nerve 2007;35:430–42. [25] di Barletta MR, Viatchenko-Karpinski S, Nori A, et al. Clinical phenotype and functional characterization of CASQ2 mutations associated with catecholaminergic polymorphic ventricular tachycardia. Circulation 2006;114:1012–9. [26] Treves S, Vukcevic M, Maj M, Thurnheer R, Mosca B, Zorzato F. Minor sarcoplasmic reticulum membrane components that modulate excitation–contraction coupling in striated muscles. J Physiol 2009;587:3071–9. [27] Jacobsen NJ, Lyons I, Hoogendoorn B, et al. ATP2A2 mutations in Darier’s disease and their relationship to neuropsychiatric phenotypes. Hum Mol Genet 1999;8:1631–6.
Neuromuscular Disorders 22 (2012) 944–954 www.elsevier.com/locate/nmd
Brody syndrome: A clinically heterogeneous entity distinct from Brody disease A review of literature and a cross-sectional clinical study in 17 patients N.C. Voermans a,⇑, A.E. Laan a,c, A. Oosterhof b, T.H. van Kuppevelt b, G. Drost a,d, M. Lammens a,e, E.J. Kamsteeg f, C. Scotton g, F. Gualandi g, V. Guglielmi h, L. van den Heuvel i, G. Vattemi h, B.G. van Engelen a a
Neuromuscular Centre Nijmegen, Department of Neurology, Radboud University Nijmegen Medical Centre, The Netherlands b Department of Biochemistry, Radboud University Nijmegen Medical Centre, The Netherlands c Institute for Genetic and Metabolic Disease, Radboud University Nijmegen Medical Centre, The Netherlands d Department of Clinical Neurophysiology, Radboud University Nijmegen Medical Centre, The Netherlands e Department of Pathology, Radboud University Nijmegen Medical Centre, The Netherlands f Department of Human Genetics, Radboud University Nijmegen Medical Centre, The Netherlands g Department of Medical Genetics, Radboud University Nijmegen Medical Centre, University of Ferrara, Italy h Department of Neurological, Neuropsychological, Morphological, and Movement Sciences, University of Verona, Italy i Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, The Netherlands Received 24 February 2012; received in revised form 8 March 2012; accepted 30 March 2012
Abstract Brody disease is a rare inherited myopathy due to reduced sarcoplasmic reticulum Ca2+ ATPase (SERCA)1 activity caused by mutations in ATP2A1, which causes delayed muscle relaxation and silent cramps. So far the disease has mostly been diagnosed by measurement of SERCA1 activity. Since mutation analysis became more widely available, it has appeared that not all patients with reduced SERCA1 activity indeed have ATP2A1 mutations, and a distinction between Brody disease (with ATP2A1 mutations) and Brody syndrome (without ATP2A1 mutations) was proposed. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome and detect clinical features which help to distinguish between the two. In addition, we describe the Brody syndrome phenotype in more detail. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. The results showed that Brody disease presents with an onset in the 1st decade, a generalized pattern of muscle stiffness, delayed muscle relaxation after repetitive contraction on physical examination, and autosomal recessive inheritance. Patients with Brody syndrome more often report myalgia and experience a considerable impact on daily life. Future research should focus on the possible mechanisms of reduction of SERCA activity in Brody syndrome and other genetic causes, and on evaluation of treatment options. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Brody syndrome; Brody disease; Brody myopathy; Ca ATPase; SERCA1; Delayed relaxation; Silent cramps
⇑ Corresponding author. Address: Neuromuscular Centre Nijmegen,
Department of Neurology, 935 Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31 024 3616600; fax: +31 24 3541122. E-mail address: [email protected] (N.C. Voermans). 0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.03.012
1. Introduction Brody disease is a rare inherited myopathy due to a diminished sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)1 activity caused by mutations in ATP2A1. The
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incidence of Brody disease is estimated to be 1 in 10,000,000, but this might be underestimated due to incomplete recognition [1,2]. Brody disease was initially described by Brody in 1969 [2]. He reported a patient with muscle stiffness starting at five years of age, which occurred during vigorous exercise or sudden rapid movements, and disappeared after several seconds of rest. Most muscle groups were involved. Muscle contraction was normal, but the relaxation phase became increasingly slow following repeated exercise (Table 1: Clinical key features of Brody disease). Myotonia was ruled out by the presence of electrical silence on electromyography during stiffness (“silent cramps”) [2]. Reduced SERCA1 activity results in delayed muscle relaxation due to prolonged increase of intracellular calcium concentration following contraction [3]. Fig. 1 illustrates the role of SERCA1 in muscle relaxation. Two types of SERCA predominate in muscle: SERCA1 predominates in type 2 fast-twitch skeletal muscle fibres and SERCA2 predomi-
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nates in type 1 slow-twitch skeletal and cardiac muscle fibres [9]. Brody disease is due to modifications of SERCA1 only, and accordingly no cardiac complications in Brody disease have been reported. Since SERCA1 is only present in fasttwitch muscle fibres (type 2), impaired relaxation specifically occurs after phasic exercise (alternate contraction and relaxation). In contrast, tonic activity (such as maintaining posture) requires slow-twitch motor-unit activation, which recruits predominantly type 1 fibres [4,5]. In 1996, mutations in the ATP2A1 gene were found to cause Brody disease. However genetic heterogeneity is likely considering that ATP2A1 mutations are missed in a quote of patients with recessive inheritance and have never been found in patients with an autosomal dominant pattern. In 2002, MacLennan proposed to distinguish patients with reduced SERCA1 activity and ATP2A1 mutations (Brody disease) from patients with reduced SERCA activity without ATP2A1 mutations (Brody syndrome) [6]. Brody
Table 1 Clinical key features of Brody disease and definitions used in this study. Clinical key features of Brody disease based upon the first case description of Brody in 1969 [1] Onset in childhood: “his earliest recollection of a movement disorder was falling down in a foot race at the age of 5 years because his muscles stiffened” Exercise-induced muscle stiffness: “muscle stiffness occurred during vigorous exercise or upon sudden, rapid movement,. . .disappeared after several seconds of rest” Normal muscle strength: “skeletal muscles, which were normal in contour, strength and tone” Involvement of most muscle groups: “this response to exercise was noted in the muscles of the limbs, the face and the jaws” Delayed muscle relaxation after repetitive contraction on physical examination: “skeletal muscles. . .showed a progressive slowing of relaxation with vigorous exercise” No myalgia independent of muscle stiffness: “painless unless the patient continued his efforts to contract the shortened muscle” Increase in cold: “but in cold weather the stiffness occurred more readily” “after three minutes in a room refrigerated to 5° there was a marked increase in the patient’s exercise intolerance. Repetitive contraction produced paralysis within five seconds, whereas 10–15 seconds where required at the usual room temperature” Silent cramp on electromyography: “when sustained muscle shortening was induced by exercise, insertion activity was normal and was followed by electrical silence” Definitions used in literature review and clinical study History
Physical examination
Exercise-induced muscle stiffness Muscle cramp Myalgia Pattern of involvement of muscles Effect of medication Muscle weakness Delayed muscle relaxation after repetitive contractions Delayed muscle relaxation after sustained contractions Effect of cold
Ancillary investigations
Silent cramp
Involuntary painless hardening of the muscle immediately following the onset of exercise Sustained painful shortening of the muscle Muscle pain independent of muscle stiffness or cramp Pattern of involvement of muscles in which exercise-induces stiffness is reported: this is classified as generalized when muscles in both limbs and face are involved Reduction of the exercise-induced muscle stiffness with use of medication Reduction of maximal voluntary muscle force measured by manual muscle testing scored by the MRC score Inability to immediately relax muscles after voluntary repetitive contractions with maximal effort for at least 10 s, which results in a stiffness of the muscle Inability to immediately relax muscles after voluntary sustained contraction with maximal effort for at least 10 s, which results in a stiffness of the muscle Increase of delay of muscle relaxation after voluntary repetitive contractions with maximal effort for at least 15 s in cold water Electromyography during the delayed muscle relaxation shows normal insertion activity followed by electrical silence
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Fig. 1. Excitation, contraction, and relaxation of skeletal muscle. End-plate potential in T-tubulus causes opening of the ryanodine receptor: the end-plate action potential (AP) propagates throughout the myocyte’s sarcolemma and into the T-tubule system. The T-tubule contains dihydropyridine receptors (DHPR) which are voltage-dependent calcium channels and are activated by the AP. The dihydropyridine receptors transmit the voltage-mediated signal through a mechanical linkage to the ryanodine receptors (RYR) in the sarcoplasmic reticulum (SR). Ryanodine receptors undergo a conformational change that opens their channel. Contraction: Opening of RYR causes a flow of Ca2+ from the sarcoplasmic reticulum into the cytoplasm. In this release, Ca2+ unbinds from the calcium-binding protein called calsequestrin (CSQ). Ca2+ released from the sarcoplasmic reticulum binds to Troponin C on actin filaments, which subsequently leads to the troponin complex being physically moved aside to uncover cross-bridge binding sites on the actin filament. By hydrolyzing ATP, myosin forms a cross bridges with the actin filaments, and pulls the actin toward the centre of the sarcomere resulting in contraction of the sarcomere. Relaxation: Simultaneously, the sarco/endoplasmic reticulum Ca2+-ATPase actively pumps Ca2+ back into the sarcoplasmic reticulum where Ca2+ rebinds to calsequestrin. With Ca2+ no longer bound to troponin C, the troponin complex slips back to its blocking position over the binding sites on actin. Since cross-bridge cycling is ceasing then the load on the muscle causes the inactive sarcomeres to lengthen.
syndrome patients generally suffer from (exercise-induced) muscle stiffness and delayed muscle relaxation; however, only a few muscle groups are involved, and onset is in adolescence or adulthood [6]. This was described as an expert opinion [6], is not easy to trace and not referred to in the sparse literature on Brody disease since then. Furthermore, muscle stiffness, a prominent feature of Brody syndrome, is a very frequent complaint among patients at the neuromuscular outpatients’ clinic. Therefore a more detailed investigation of the Brody syndrome phenotype is warranted. We aim to compare the clinical features of patients with Brody disease and those with Brody syndrome. We therefore performed a literature review on clinical features of both Brody disease and Brody syndrome and a cross-sectional clinical study consisting of questionnaires, physical examination, and a review of medical files in 17 Brody syndrome patients in our centre. 2. Patients and methods 2.1. Definition of clinical key features of Brody disease Table 1 shows the clinical key features of Brody disease abstracted from the initial clinical case description of Brody in 1969 [2].
2.2. Review of literature cases and selection of literature (L) cases We reviewed all publications cited by Pubmed on ‘Brody disease’, ‘Brody syndrome’ or ‘Brody myopathy’ since the initial description in 1969 by Brody [2] until 2010 and the references of these articles, and selected all patients in whom clinical data were available (n = 32; Supplementary Table 1) [2,4,5,7–14]. We classified them as literature (“L”) cases, numbered them chronologically, and screened the case descriptions for clinical key features of Brody disease. The mode of SERCA(1) activity measurement, ATP2A1 mutations, and modes of inheritance were noted. 2.3. Clinical study and selection of study (S) patients At the outpatient department of our neuromuscular centre, which is a tertiary referral centre for neuromuscular diseases, SERCA activity is measured in case of (exerciseinduced) muscle stiffness and/or silent muscle cramps in absence of other causes. It is compared with normal values obtained in control subjects as described previously [5]. Between 1992 and 2007, SERCA activity was measured in approximately 200 patients, and reduced compound SERCA activity had been detected in 22 patients. One
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Fig. 2. SERCA1 and SERCA2 staining. SERCA1 and SERCA2 antibodies stained the SR ATPase in type 2 and type 1 fibers, respectively. Immunoreactivity for SERCA1 and SERCA2 was similar in patients’ and controls’ muscles. The images are the staining in one of the patients with Brody syndrome in this study.
patient was later diagnosed with sodium channel myotonia due to a SCN4A mutation (SERCA activity 22.6 mU/mg at age of 60 years; this is below the normal value; see Fig. 3). Two other patients were excluded because of neurological and psychiatric co-morbidity (leucencephalopathy in one, and somatisation disorder in two other patients), one patient could not be traced anymore, and one patient declined participation. Hence, 17 patients were included. Four of these patients had been described by Benders in 1994 (S6, 7, 11 and 17) [5] as well as by Poels and Wevers (S11) [9,21]. 2.4. Questionnaires All patients completed a standardized questionnaire on the Brody disease clinical key features defined in Table 1. Additional questions focused on initial and current symp-
toms, fatigue, medical history, medication use, functional impairment (graded with the modified Rankin score) [15], and use of mobility aids. 2.5. Physical examination Physical examination consisted of a neurological examination including manual muscle testing in 24 muscles (11 bilateral limb muscles and neck extensors and flexors; score 0–5; with a MRC sum score of 0–120) and dynamometry in nine muscles bilaterally as previously described (Citec hand-held dynamometer: www.citec.nl) [16,17]. To detect delayed muscle relaxation, duration of relaxation after sustained contraction (Sustained Contraction (SC), sustained forceful eyelid closure (E), and sustained forceful clenching of hands (H), both for 10 s) was measured, the latter both in room temperature and in cold water. Furthermore, the
Fig. 3. Compound SERCA activity of the patients in this study related to age. Compound SERCA activity is expressed in mU/mg protein.
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patients were asked to perform forceful repetitive elbow flexion and contraction (RC: alternate contraction and relaxation) for 10 s, with qualitative evaluation of delayed relaxation. Delay of muscle relaxation was defined as duration until complete relaxation >1 s.
nificance of the differences was calculated with the Fisher Exact test (two-sided). Data analysis was performed using Microsoft Excel and SPSS (version 16.0 for Windows) to generate descriptive statistics. 3. Results
2.6. Previously performed ancillary investigations 3.1. Features of patients in literature: L1–L32 The medical files of these patients were reviewed for results of creatine kinase measurements, nerve conduction studies, electromyography, and muscle biopsy (needle biopsy of the quadriceps femoris muscle). All these tests had been performed previously in our centre according to existing diagnostic protocols. Standard enzyme histochemical and immunohistochemical techniques had been used to exclude common myopathies [18]. 2.7. SERCA1 and SERCA2 immunohistochemical staining SERCA1 and SERCA2 immunohistochemical staining was performed on the muscle biopsies that were still available (n = 10). This was done on transverse muscle sections using fluorescence method with antibodies to SERCA1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and SERCA2 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). We compared the results with controls including biopsies of vastus lateralis muscle from subjects who were found to be free of muscle disease [14,19]. 2.8. SERCA activity SERCA activity measurements have been performed in our centre since 1992. Compound SERCA activity was measured in whole muscle homogenates obtained from needle biopsies of the quadriceps femoris muscle as previously described [5]. Compound SERCA activity is considered a good estimate of SERCA1 activity, since SERCA1 is the predominant isoforms in the quadriceps femoris muscle [5]. 2.9. Mutation analysis of ATP2A1
Since the initial report of Brody in 1969 until 2010, 35 patients have been reported in the literature, of which 32 with a description of clinical features (L1–L32 in Tables 2A, 2B and Supplementary Table 1) [3–5,7–10,12–14,20– 23]. L1 represents the patient reported by Brody in 1961 [2]. Results of history, physical examination, and ancillary investigations are summarized in Table 2A. Different techniques have been used to measure SERCA1 activity or content; therefore, these quantitative data are difficult to compare. Some studies measure protein content, while others measure the activity of the SERCA1 protein [5], or both [4]. Benders et al. report the compound activity of SERCA1, SERCA2, and SERCA3. In the quadriceps muscle this consists predominantly of SERCA1 activity (83%) [5]. Furthermore, SERCA1 activity is often measured in isolated microsomal or SR fractions [4,5], while other studies report data on SERCA1 activity in whole muscle homogenates [7]. Reduction of isolated SERCA1 activity, compound SERCA activity, or calcium flow or SERCA content has been reported in all cases, with a residual SERCA1 activity ranging from 2% to 50% of normal [4,5,8,14]. Recessive mutations in the ATP2A1 gene (16p12.1–12.2) have been detected in 11 patients from seven families, including deletions and missense, frameshift or nonsense mutations. In other families with either autosomal dominant or autosomal recessive inheritance, no ATP2A1 mutations were found. SLN, the gene encoding sarcolipin was considered a prime candidate gene for Brody disease. However, mutation analysis of this gene in five Brody families did not reveal any alternations in coding, splice junction or promoter sequences [11].
Mutation analysis of the ATP2A1 gene was performed in six patients in Nijmegen in 2000 (Radboud University Nijmegen Medical Centre, The Netherlands) by checking DNA sequences of the individual exons, the splice donor and splice acceptor sites [20]. Gualandi (Ferrara University, Italy) performed mutation analysis in eleven patients in 2010 on genomic DNA as previously described [16]. Subsequently, segregation analysis was performed in seven sibs and the mother of one of the patients in whom an heterozygous unclassified variant had been detected.
3.2. Features of patients in clinical study: S1–S17
2.10. Data analysis
3.3. Questionnaires
We compared the clinical features, and results of genetic analysis and ancillary investigations of patients with Brody disease and those with Brody syndrome. The statistical sig-
Table 2A summarizes the clinical key features and Table 3 shows the additional features of the patients in this study (S1–S17). Frequent initial symptoms were: muscle
Table 2A shows the demographic features; nine females and eight males were included and the mean age was 51 years (SD 12.5 years). Ten patients were sporadic cases; and seven patients from three families were included, two of which revealed an autosomal recessive inheritance pattern. Clinical presentation of patients within these families was heterogeneous. The medical history included epilepsy (n = 1), minor stroke (n = 1), and hypothyroidism (n = 2).
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Table 2A Clinical features of patients with Brody disease and Brody syndrome in literature and in this study.
(continued on next page)
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Table 2A (continued)
L1, Brody, 1969 [1]; L9–L10, Taylor, 1988 [4]; L 21–22, Zhang, 1995 [8]; L29–L30, Novelli, 2004 [13]; L2–L4, Karpati, 1986 [2]; L11, Wevers, 1992 [5]; L23, Odermatt, 1997 [10]; L31–L32, Vattemi, 2010 [14]; L5–L8, Danon, 1988 [3]; L12–L20, Benders, 1994 [7]; L24–L28, Odermatt, 2000 [12]. L# = patient described in literature, numbered chronologically and corresponding to the numbers in Table 2. L2 and L3, siblings; L5 and L6, siblings; L9 and L10, siblings; L14 and L15, siblings; L26 and 27, siblings; L31 and L 32, siblings. S# = patient included in this study, some of which were also reported in literature before. Numbered following age, from young to old. S17, mother of S6; S8 and S10, siblings; S13,14 and 16, siblings. , (effect) absent; +, (effect) present; blank, unknown/not reported; n.p., not performed; ", mildly increased; D, dantrolene; V, verapamil; s.e., withdrawn due to side effects; n.u., never used; AD: autosomal dominant; AR: autosomal recessive; S: sporadic. MMT: manual muscle testing; CK: creatine kinase; EMG, electromyography; SC (EMG): silent cramps on electromyography; M (EMG): myopathic pattern on electromyography; M (biopsy), myopathic findings on muscle biopsy.
cramps, myalgia, muscle stiffness, and easy fatigability. Not any of the patients initially presented with delayed muscle relaxation. Frequently reported current symptoms were: exercise-induced muscle stiffness, muscle cramps, myalgia (Tables 2A and 2B) and muscle weakness (Table 3). Muscle stiffness and delayed muscle relaxation were reported most frequently in hand, legs, and toes, but not generalized. Myalgia occurred after exercise in seven patients and continuously in ten patients; predominantly in legs and arms and with a mean VAS score of 7.1 (SD 1.7). Muscle cramps occurred spontaneously in two patients in rest, after exercise in four patients, and both in rest and after exercise in eight patients, most often in fingers and toes (mean VAS score of 6.0 (SD 1.8)). Cold caused an exacerbation of muscle stiffness in 10 patients and of cramps in eight. Fourteen patients reported progression of symptoms over the years. The symptoms caused considerable impact on daily life (Table 3). Nine patients used a wheelchair, rollator or mobility scooter. Thirteen reported severe fatigue, which in some cases required daytime sleeping. Six patients were unable to work fulltime, and two of them were fully unfit for work. Thirteen patients refrained from sporting because of the severity of complaints. The effect of medication was limited: with a positve effect in only few patients of dantrolene (n = 1), verapamil (n = 2), and clonazepam (n = 3). Ten patients stopped the
use of dantrolene or verapamil because of side effects (muscle weakness, depression, and malaise) or absence of effect. Analgesics (paracetamol, ibuprofen, tramadol, naproxen) were used with only limited effect on myalgia and cramps. Two patients reported a positive effect of etoricoxib. One patient (S11) reported several episodes of exertional rhabdomyolysis with myoglobinuria, which was confirmed by elevated serum creatine kinase values up to 11,000 U/l [21]. Various other non-related symptoms were reported: palpitations (n = 3), exercise dyspnoea (n = 9), oedema (n = 4), dysphagia (n = 3), sense of slow passage of food in oesophagus (n = 4), constipation (n = 6), stress-incontinence (n = 2), urge-incontinence (n = 5), paresthesias (n = 7), numbness (n = 6), headache (n = 7), and vertigo (n = 3). 3.4. Physical examination Results of physical examination are summarized in Tables 2A and 3. Delayed muscle relaxation after sustained forceful eyelid closure and/or after forceful clenching of both hands was observed in seven patients. In contrast, delayed relaxation after repetitive contraction was not observed in any of the patients. No percussion myotonia was observed. One patient had mild muscular hypertrophy while not performing sports (S8).
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Table 2B Comparison of clinical features between patients with Brody disease and Brody syndrome.
Gen.: Genetic features CK: creatine kinase: EMG: electromyography SC (EMG): silent cramps on electromyography M (EMG): myopathic pattern on electromyography M (biopsy): myopathic findings on muscle biopsy * Fisher exact test (two-tailed).
Table 3 Additional results of questionnaire, physical examination and ancillary investigations in clinical study. Questionnaire MW MRS MA (0–5)
S1 S2 S3 S4 S5 S6 (L17) S7 (L19) S8 S9 S10 S11 (L11) S12 S13 S14 S15 S16 S17 (L20) Mean/total (%)
+ + + + + + + + + + + + + + 15 (88%)
2 2 3 3 2 3 3 3 2 2 3 1 2 3 2 2 3 2.4
+ + + + + + + + + 9 (53%)
Physical examination F
+ + + + + + + + + + + + + 13 (72%)
Genetics
Muscle MRC Muscle weakness sum weakness (MMT) score (D) (0–120)
Muscles with weakness on D (0– 17)
Delayed ATP2A1 relaxation mutation after analysis
+ + + + + + 6 (35%)
4 2 1 5 7 0 9 5 4 3 2 3 9 8 9 9 9 5
0
120 120 120 120 120 120 89 120 120 120 120 120 113 104 82 88 104 113
+ + + + + + + + + + + + + + + + 17 (94%)
SERCA1 Ancillary investigations activity Muscle biopsy
RC SC H/ E +/+ +/+ +/ +/+ +/+ +/+ +/+ 7 (41%)
Type Internal RR IH Biochemical I nuclei fibres SERA1 analysis fibres and 2 uv 0
21.5 35.3 19.4 16.4 22.3 19.4 21.2 24.7 23.3 26.3 20.6 20.5 31.7 21.5 21.9 29.6 22.6
# N N N N # N " N N # N # # N " N
N N N N N
"
+
N N
"
N N N N N # N
N + +
N N N
+
MW, Muscle weakness; MRS, Modified Rankin scale; MA, use of mobility aids (e.g. wheelchair, rollator, bicycle with electrical assistance); F, fatigue; MMT, manual muscle testing; D, dynamometry; N, normal; ", increased; #, decreased +, present; , absent; uv = unclassified variant, heterozygous R604H (c.1995G>A). Subsequent segregation analysis has revealed that this heterozygous uv was also detected in five sibs and the mother of this patient, none of whom were affected. The only affected sib had two wild type alleles. The column “Delayed relaxation after repetitive contractions (RC)” is a duplication of Table 2A but included to show the difference with “Delayed relaxation after sustained contraction (SC)”. H, hands; E, eyes.
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3.5. Previously performed ancillary investigations Results of previously performed ancillary investigations are summarized in Tables 2A and 3. One patient (S11) showed an elevated serum creatine kinase (CK = 710 U/l (normal = <220 U/l)). This patient had previously been reported to have exertional rhabdomyolysis [21]. CK increase with a history of rhabdomyolysis and myoglobinuria has been reported in only one other Brody syndrome patient (L29) [13]. In all other patients, CK was normal. Electromyography had previously been performed in all patients at the time of diagnosis; in six patients this revealed mild to moderate myopathic features, including small, polyphasic units and fast recruitment. In one of them and in another patient, electromyography was performed during a clinical cramp, which was electrically silent. Electromyography was normal in the other patients. Histological analysis of the quadriceps biopsies revealed a normal distribution of type I and type II fibres in 10 patients; type II fibres were relatively abundant in five patients, and in two patient type I fibres were more predominant (57% and 58%, respectively [normal 35–50% type I]). Mild myopathic features including increased variation in fibre diameter, increase of internal nuclei and few necrotic fibres were observed in only two patients (S8 and S11). In four patients, few ragged-red fibres were observed, which were within normal limits for their age. Oxidative phosphorylation enzyme activities and total ATP production capacity were measured in eight patients: in one of them, ATP production capacity was reduced without a reduction of the enzyme activities. 3.6. SERCA1 and SERCA2 immunohistochemical staining SERCA1 and SERCA2 antibodies stained the SERCA in type 2 and type 1 fibers, respectively. Immunoreactivity for SERCA1 and SERCA2 was similar in patients’ and controls’ muscles (Fig. 2) [14]. 3.7. SERCA activity Fig. 3 shows the compound SERCA activity of the patients in this study. The 99% confidence interval of compound SERCA activity in normal subjects is depicted by the diagonal lines. All patients in whom delayed muscle relaxation after sustained contraction was detected on physical examination had a considerably reduced SERCA activity below 23 mU/mg. 3.8. Mutation analysis of ATP2A1 Checking of DNA sequences of the individual exons, the splice donor and splice acceptor sites of the ATP2A1 gene of the first seven patients (S6, S7, S11, S13, S14, S16, S17) in 2000 revealed no mutations. Sequence analysis of one of the remaining 10 patients only revealed a previously unre-
ported heterozygous unclassified variant in exon 15: R604H (c.1995G>A) in S8. Subsequent segregation analysis in seven sibs and the mother revealed that this missense change was also present in five sibs and the proband mother, none of whom were affected. The only affected sister (S10) had two wildtype alleles. This R604H variation was therefore not considered pathogenic. No pathogenic mutations were detected in other patients. Several known polymorphisms were detected (Supplementary Table 2) as well as a synonymous change within exon 15 (Pro603Pro) occurring in heterozygosity in two unrelated patients. 3.9. Comparison of Brody disease and Brody syndrome patients Table 2B shows the differences of Brody disease and Brody syndrome patients. Patients with Brody disease more often report an onset in the 1st decade, a generalized pattern of muscle involvement, and autosomal recessive inheritance. Delayed relaxation after repetitive contraction of the elbow flexors is observed more often in Brody disease. In contrast, patients with Brody syndrome report myalgia more frequently. A history of exercise-induced muscle stiffness or muscle cramps, effect of medication, and the presence of muscle weakness or increase with cold on physical examination does not distinguish between the two disorders. 4. Discussion This literature review and cross-sectional study in 17 patients with Brody syndrome shows that Brody disease (reduced SERCA activity with ATP2A1 mutations) presents with onset in childhood; a generalized pattern of muscle involvement, autosomal recessive inheritance, and delayed relaxation after repetitive contraction on physical examination. In contrast, patients with Brody syndrome report myalgia more frequently (reduced SERCA activity without ATP2A1 mutations). Furthermore, the impact of Brody syndrome on daily functioning was greater than previously described and the effect of medication (dantrolene and verapamil) was limited mainly due to side effects. A history of exercise induced muscle stiffness did not distinguish between the two groups and may be considered aspecific. Increase of delayed relaxation in cold and presence of silent cramps on electromyography did neither. These features were described in the initial case by Brody, but have only been tested in few literature cases. Therefore, these features should be tested prospectively in a larger number of patients in order to be able to determine their predictive value. These findings thus confirm the distinction between Brody disease and Brody syndrome proposed by MacLennan and Loke [6], and we suggest to use this distinction in current clinical practice. Of note, we continue using the term Brody ‘syndrome’ as suggested by MacLennan, although strictly speaking, ‘syndrome’ refers to the association of several clinically recognizable features (signs and
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symptoms) [6], whereas Brody syndrome patients have been biochemically classified. In addition, these findings suggest that the specificity of reduced SERCA1 activity in muscle for detecting Brody disease is limited. This was also put forth by a study on reduced SERCA1 activity in muscle tissue from patients with myotonic dystrophy type 1 [23]. The findings of this study are very relevant since muscle stiffness, muscle cramps, and myalgia are frequent complaints among patients at a neuromuscular outpatients clinic and difficult to measure. Measurement of SERCA activity in these patients is a way to characterize these symptoms quantitatively, and the findings of this study might be of help to select patients for ATP2A1 mutation screening. The differences between Brody disease and Brody syndrome raise the question whether Brody syndrome is a separate entity or an epiphenomenon of other underlying diseases with a final common pathway of altered calcium transport. Several previous observations support this latter hypothesis. One of the patients was excluded for this study since a heterozygous SCN4A mutation had been detected and she had been diagnosed with sodium channel myotonia shortly after the SERCA measurement was performed. Furthermore, S11 [5,9,21] was the only patient in our series with both recurrent rhabdomyolysis (CK up to 11,000 IU/ L) and myopathic features in the muscle biopsy. Although reported with Brody disease [5,9,21], he might have another myopathy for which he is currently under investigation (so far, RYR1, FKRP, CPT2, and LPIN1 mutations have been excluded). Hypothyroidism might be another factor that influences SERCA activity. Animal studies have indeed shown that hypothyroidism induces a decrease in SERCA activity with delayed muscle relaxation [24]. Two of the patients in this study had a history of hypothyroidism, and in one of them the initial muscle symptoms started after the onset of hypothyroidism. Lastly, since SERCA1 is an ATPase channel, SERCA function might be altered in patients with mitochondrial disease and reduced ATP production. Mutations in other genes might also influence SERCA1 activity. SLN, the gene encoding sarcolipin was considered a prime candidate gene for Brody disease, but no mutations were detected in five Brody families [11]. Interestingly, patients with polymorphic ventricular tachycardia can have mutations in the cardiac specific isoform of calsequestrin, a sarcoplasmic reticulum calcium store [25]. When expressed in rat myocytes, these mutants decreased the sarcoplasmic reticulum Ca2+-storing capacity and reduced the amplitude of I(Ca)-induced Ca2+ transients and of spontaneous Ca2+ sparks in permeabilized myocytes. In analogy, mutations in the skeletal muscle specific calsequestrin isoform may be involved in Brody syndrome. Also, the function of SERCA1 points to other neighbouring proteins (for an overview see [26]), which may be involved in the aberrant muscle Ca2+ homeostasis in Brody syndrome. Finally, the fact that some patients reported prolonged stiffness in hands after tonic rather than phasic exercises raises the
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questions whether mutations in ATP2A2 (encoding SERCA2 in slow-twitch type 1 fibers) might be involved; however, mutations in this gene have so far only been associated with dermatological disease (Darier’s disease) or neuropsychiatric disorders [27]. One limitation is that in 2000 we only checked the DNA sequences of the individual exon, the splice donor and splice acceptor sites of the ATP2A1 gene, and did not screen the promoter sequence or enhancer/silencing sequences of this gene for the presence of disease-causing mutations. This means we cannot fully exclude mutations in these patients. Another limitation is that only patients with Brody syndrome were clinically investigated, whereas clinical features of Brody disease were deduced from the literature cases. A related problem is the lack of data on natural course of Brody disease, including functional impairment, since most reports in literature describe recently diagnosed patients. If ATP2A1 mutations analysis becomes more widely available and more cases of Brody disease are recognized, a more direct comparison between the two groups might be possible. In short, we have presented the result of a literature review and clinical study on features of Brody disease and syndrome, showing that Brody disease typically presents with early onset; generalized exercise-induced muscle stiffness, autosomal recessive inheritance, and delayed relaxation after repetitive contraction on physical examination. In contrast, patients with Brody syndrome report myalgia more frequently. Further research should focus on various mechanism involved in reduction of SERCA activity in Brody syndrome, e.g. hypothyroidism or mitochondrial disease, and other genetic causes. 5. Financial disclosure N.V. was supported by a clinical fellowship for neuromuscular disorders by the Prinses Beatrixfonds, the Netherlands. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2012.03.012. References [1] MacLennan DH. Ca2+ signalling and muscle disease. Eur J Biochem 2000;267:5291–7. [2] Brody IA. Muscle contracture induced by exercise. A syndrome attributable to decreased relaxing factor. N Engl J Med 1969;281: 187–92. [3] De Ruiter CJ, Wevers RA, Van Engelen BG, Verdijk PW, De HA. Muscle function in a patient with Brody’s disease. Muscle Nerve 1999;22:704–11. [4] Karpati G, Charuk J, Carpenter S, Jablecki C, Holland P. Myopathy caused by a deficiency of Ca2+-adenosine triphosphatase in sarcoplasmic reticulum (Brody’s disease). Ann Neurol 1986;20:38–49.
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