- Email: [email protected]
Inflammation and response to steroid treatment in limb-girdle muscular dystrophy 2I
ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
11 (2007) 353 – 357
Official Journal of the European Paediatric Neurology Society
Inflammation and response to steroid treatment in limb-girdle muscular dystrophy 2I ˚ hlandera, A.-R. Moslemib, A. Oldforsb, M. Tuliniusa N. Darina,, A.-K. Kroksmarka, A.-C. A a
Department of Pediatrics, Sahlgrenska University Hospital, Go¨teborg, Sweden Department of Pathology, Sahlgrenska University Hospital, Go¨teborg, Sweden
b
art i cle info
ab st rac t
Article history:
Limb-girdle muscular dystrophy (LGMD) type 2I, caused by mutations in the fukutin-related
Received 16 November 2006
protein gene (FKRP), is one of the most common forms of LGMD in childhood. We describe
Accepted 28 February 2007
two patients with LGMD2I and a Duchenne-like phenotype. In addition to the common
Keywords:
Clinical onset was triggered by viral upper respiratory tract infections. In addition to the
Limb-girdle muscular dystrophy 2I
common dystrophic pattern with a weak immune histochemical staining for a-dystrogly-
FKRP
can, muscle biopsy showed inflammatory changes. This was especially striking in one of
Inflammation
the patients with up-regulation of MHC class 1 antigen, suggestive of myositis. Both
Steroid treatment
patients showed a good clinical response to treatment with prednisolone, which was
L276I mutation, both patients had a new mutation in FKRP, L169P and P89L, respectively.
initiated at daily dosage of 0.35 mg/kg/day. Our results provide evidence for an inflammatory involvement in the pathological expression of LGMD2I and open up the possibility that this disorder could be treatable with corticosteroids. & 2007 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness of variable distribution and severity. The primary pathological mechanisms behind many of these disorders is the loss of linkage between the extracellular matrix and the actin cytoskeleton.1 Duchenne muscular dystrophy (DMD) is the most frequent and best studied of the muscular dystrophies. There is accumulating evidence from randomized controlled studies that corticosteroid treatment, at least in the short-term, improves muscle strength and function in DMD.2 In recent years, it has become increasingly recognized that inflammation is both an early and important pathophysiological event in this disorder.3 The efficiency of corticosteroid treatment in other muscular dystrophies is not known. Inflammatory infiltrates have been found in Facioscapulohumeral muscular dystrophy and limb-girdle muscular dystrophy (LGMD) 2B.4,5
The LGMD phenotype is defined by predominant involvement of the pelvic and shoulder girdle muscles. At least 15 genes, 6 autosomal dominant (LGMD1A-F) and 10 autosomal recessive (LGMD2A-J), responsible for LGMD have been mapped.6 LGMD type 2I is one of the most common forms.7 It is caused by mutations in the fukutin-related protein gene (FKRP), a putative glycosyl transferase, required for posttranslational modification of dystroglycan.8 The pattern of muscle involvement, which frequently includes calf hypertrophy as well as cardiac and respiratory dysfunction, resembles the dystrophinopathies.9 The spectrum of variability associated with mutations in FKRP is wider than in other LGMDs and adult asymptomatic carriers have been repeatedly described, suggesting that other mechanisms than the genotype modify the phenotype in this disorder.10 We present two patients with LGMD2I caused by two new mutations in the FKRP. They both had a clinical onset triggered by viral upper respiratory tract infection (URTI),
Corresponding author. The Queen Silvia Children’s Hospital, S-416 85 Goteborg, Sweden. Tel.: +46 31 3438213; fax: +46 31 257960. ¨
E-mail address: [email protected] (N. Darin). 1090-3798/$ - see front matter & 2007 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2007.02.018
ARTICLE IN PRESS 354
E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
inflammatory changes on muscle biopsy and responded clinically to corticosteroid treatment.
2.
Patients and methods
The patients were followed with regular controls at the Neuromuscular Unit of the Queen Silvia Children’s Hospital in Gothenburg, Sweden.
2.1.
Measurement procedures
Isometric muscle strength was measured with an electronic handheld dynamometer according to the procedures developed by Scott et al.11 An isometric contraction of at least 2–3 s was required and the peak force in Newton was recorded. The best of three values obtained on the nondominant side was compared with reference values.12 Motor function was assessed by using a scale designed by Scott et al.11 In all, 20 movements were assessed, including head lifting, rolling, sitting up, sitting, getting off a chair, standing, standing on the heels, standing on the toes, standing on one leg and ascending and descending stairs. The performance is scored according to a three-point scale, 0 (unable), 1 (needs self-reinforcement) and 2 (succeeds). The maximum score attainable is 40. A percentage of the total motor function score was calculated and 46-m walking time was recorded.
2.2.
Immunohistochemistry
Immuno-histochemical staining of a-dystroglycan was performed on 10-mm-thick cryostat sections. The sections were fixed in icecold 99.9% ethanol and acetic acid, 1:1, for 1 min and blocked in 1% bovine serum albumine (BSA) before they were incubated for 1 h at 37 1C with monoclonal antibodies directed against a-dystroglycan. This anti-a-dystroglycan clone VIA4-1 (Upstate, Lake Placid, NY) skall ersa¨ttas av (Upstate, Temecula, CA) was diluted 1:1000 in 1% BSA. A biotinylated horse-anti-mouse antibody (Vector Lab, Novocastra, Newcastle, UK) was used as a secondary antibody. It was diluted 1:300 in 1% BSA, and applied for 1 h at room temperature followed by incubation in ABComplex K 355 HRP (Dako, Copenhagen, Denmark). The sections were visualized in DAB+ Chromogen K5007 (Dako, Copenhagen, Denmark). All dilutions and washings were made in phosphate-buffered saline (PBS). All sections were compared with control samples from nonaffected muscles, immunostained simultaneously. The samples were then analyzed in lightmicroscope.
2.3.
DNA analysis
Total DNA was extracted from 15 10 mm fresh frozen skeletal muscle tissue, using a DNA extraction kit (Dneasy Tissue Kit, Qiagen, Hilden, Germany). The samples from the patients were screened for the 826 C4A mutation in FKRP by PCR and restriction fragment length polymorphism (RFLP) as follows; 50–100 ng of the DNA and
11 (2007) 353 – 357
20 pmol of each primer (F40795 and R41128-4112) were added to Reddy Mix PCR Master Mix (Abgene, Epsom, UK). Thermal cycling was performed in Applied Biosystems 9600 Thermal Cycler. After an initial preheating step for 5 min at 94 1C, a touchdown (TD) PCR was performed, consisting of denaturation at 94 1C for 30 s, annealing at 65 1C for 30 s, extension at 72 1C for 1 min with an 1 1C temperature decrement per cycle during the first 10 cycles. The subsequent cycles (40 cycles) each consisted of 94 1C for 30 s, 55 1C for 30 s and 72 1C for 1 min. The amplified fragments were digested with 10 u Bfa I (Biolabs, New England). The entire exon 4 coding sequence was analyzed as follows; a 1488 bp sequence containing the coding sequence was amplified by PCR using 20 pmol 1F (40436–40454) and 5R (42035–42015) primers. The PCR conditions were the same as described above. The purified PCR products were then subjected for direct sequencing using following primers: 1F (40436–40454), 3F (40875–40895), 4R (41694–41677) and 5R (42035–42015) (GenBank, accession number: AC008622). To compare the amino acid sequences of human FKRP with homologues from other species, sequences were accessed through public domains /www.ncbi.nlm.nih.gov/entrez/ query.fcgiS and aligned with Expassy proteomics server /http://ca.expasy.org/S.
3.
Results
3.1.
Patient 1
This boy had normal early motor development and started to walk unsupported at 1 year of age. At the same time, he had a febrile URTI of viral origin and transiently became weak in his neck muscles with loss of head control. At 2 years of age, within a week after another URTI, he once again lost head control and developed a waddling gait. On this occasion, clinical examination showed generalized muscle weakness. The serum creatine kinase level was increased to a maximum of 196 mkat/L (reference valueo2.5). Muscle biopsies were performed from the anterior tibial and lateral vastus muscles at 2 and 14 years of age. They showed variability in fiber size, interstitial fibrosis, occasional inflammatory infiltrates, no MHC class 1 upregulation and a-dystroglycan deficiency. Genetic investigations identified the 826C4A (Leu276Ile) and a new 506T4C (Leu169Pro) mutations in the FKRP gene (Fig. 1). At 16 years of age, when prednisolone treatment was started, he had mild facial involvement, winging of the scapulae, scoliosis and pseudohypertrophic calves. He could walk short distances indoors without support. He could not rise from the floor and only rose to standing with great difficulties from a chair. There was a rapid deterioration over the 2 years prior to prednisolone treatment (Fig. 2(A)). After a few months of treatment, he walked easier and faster. After 6 months of treatment, there was no further deterioration and he showed improvement of muscle strength, motor function tests and time tests. Still after nearly 4 years of
ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
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11 (200 7) 353 – 3 57
Fig. 1 – Muscle biopsy of patient 2 demonstrating interstitial inflammatory cell infiltration (a) consisting mainly of CD8+ T-cells (b), MHC class I upregulation (c) and deficiency of a-dystroglycan (d). (a) Haematoxylin-eosin. (b) Immunostaining of CD8 (T cell, CD8, Mouse anti-human; Dako, Glostrup, Denmark) (c) Immunostaining of MHC class I (HLA-ABC antigen Mouse anti-human; Dako, Glostrup, Denmark). (d) and (e) Immunostaining of a-dystroglycan (anti a-dystroglycan; Upstate Cell Signalling Solutions; Temecula, CA) in patient (d) and control (e). Sequence chromatograms of FKRP showing the identified mutations (f–h).
100 Prednisolone 0.32 0.37 (mg/kg/day)
0.36
0.34 0.34
100
110
90
80
100
80
70
90 80
50
70
40
60
30
110 100 90
60 %
60
120 Prednisolone 0.32 0.30 0.27 0.27 0.17 0.07 0.06 (mg/kg/day)
70 Time (s)
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20
50
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50
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-10
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Time (s)
Isometric muscle strength in knee extensors (%) Motor function according to Scott (%) 46 m walk test (s)
30 -40 -30 -20 -10 0 10 20 30 40 50 60 70 80 Start Time (months)
Fig. 2 – Results from repetitive testing of patient 1 (a) and patient 2 (b) at regular controls performed at the Neuromuscular Unit of the Queen Silvia Children’s Hospital. Isometric muscle strength in the knee extensors was measured with a hand-held dynamometer and compared with reference values.12 Motor function was assessed using a scale designed by Scott et al.11 A percentage of the total motor function score was calculated and 46 meter walking time was recorded.
follow-up, many tested parameters were at the same level as at onset of treatment. At 17 years of age, a dilated cardiomyopathy was diagnosed and he developed multiple
vertebral fractures. He was treated with three infusions of zoledronic acid (Zometa), with good effect and complete pain relief after 3 months.
ARTICLE IN PRESS 356
3.2.
E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
Patient 2
This boy had a normal early motor development. At 16 months of age, during a viral URTI, he developed ptosis, lost control of his neck and the ability to walk with support. He recovered within 2 months and started to walk unsupported at 19 months of age but he never learned to run properly. The serum creatine kinase level was increased to a maximum of 200 mkat/L. Muscle biopsies were performed from the lateral vastus muscle at 2 and 9 years of age. They showed variability in fiber size, necrotic and regeneratating fibers, interstitial fibrosis, multiple inflammatory cell infiltrates, MHC class 1 upregulation and a-dystroglycan deficiency (Fig. 1). Genetic investigations identified the 826C4A (Leu276Ile) and a new 266C4T (Pro89Leu) mutations in the FKRP gene (Fig. 1). At 10 years of age, when prednisolone treatment started, he had great difficulties rising from the ground after a fall and rising from sitting to standing and could only do this with support. He could still walk short distances unaided but was in need of a wheel chair for longer distances. There was a rapid deterioration over the 2 years prior to treatment (Fig. 2(B)). After 4 months of treatment, he could rise from the floor easier and without support; stand on one leg longer, and swim longer distances. Upon testing, there was a clear improvement on muscle strength, motor function and time tests. The effects were stable until 2 years after onset of treatment when there was a clear deterioration. This was associated with a reduction of the prednisolone dosage to 0.17 mg/kg body weight because of concern about an arrested growth in height. After 4 years of treatment, the dosage of prednisolone was only 0.07 mg/kg body weight. At this moment, upon a trial to withdraw the medication completely, he immediately deteriorated and lost the ability to rise from sitting to a standing position. When the treatment was reintroduced at the same dosage, the boy rapidly regained the same ability. A dilated cardiomyopathy was diagnosed at 14 years of age.
11 (2007) 353 – 357
to proline at aminoacid position 169 while patient 2 had a 266C4T mutation, which changed a highly conserved proline to leucine at position 89 (Fig. 1). Homozygosity for L276I seems to confer a mild clinical phenotype, while compound heterozygosity for this mutation seems to be associated with a more severe phenotype.9 In fact, there is a very strong phenotypic resemblance between LGMD2I and the dystrophinopathies18 and this has been suggested to indicate the possibility that patients with LGMD2I may respond to corticosteroid treatment in a similar way as those with DMD.19 Both our patients presented with a Duchenne-like phenotype and were initially diagnosed as DMD and the choices to initiate treatment with prednisolone were done before the final diagnosis of LGMD2I. The most effective prednisolone regime in DMD is considered to be 0.75 mg/kg/day.2 Our treatment regime with 0.35 mg/kg/day of prednisolone was based on a Swedish study.20 Both patients developed dilated cardiomyopathy during the treatment. This is also a very frequent clinical manifestation in untreated patients with LGMD2I 9 and we consider it more likely a consequence of the natural course of the disease than a side effect in these two patients. We present evidence for an immunological involvement in the pathological expression of LGMD2I. Firstly, the onset was triggered by viral URTIs. This is similar to a recent description of two siblings with LGMD2I who presented with a suspected acute virus-associated myositis.21 Secondly, muscle biopsy showed inflammatory changes. This was especially pronounced in patient 2, with up-regulation of MHC class 1 antigen, suggestive of myositis. Thirdly, there was a clinical response to corticosteroid treatment in both patients. It will be important to study further how the immune system is involved in this disorder. Randomized controlled studies will be important to investigate the potential benefits and side effects of corticosteroid treatment in LGMD2I. R E F E R E N C E S
4.
Discussion
The clinical spectrum due to mutations in the FKRP gene is extremely wide. Mutations were initially described in congenital muscular dystrophy (MDC1C)13 and LGMD2I of variable severity,14 and have since then been found in patients with CMD, mental retardation and brain abnormalities including cerebellar cysts,15 muscle–eye–brain disease and Walker–Warburg syndrome16 and in adult asymptomatic carriers.10 There is in fact a significant clinical intrafamilial variability, suggesting that additional factors play a significant role in determining disease severity.9,10 Although the function of FKRP has not been determined, it is thought to be a glycosyltransferase.8 In accordance with this, MDC1C and LGMD2I are associated with a secondary deficiency in glycosylation of a-dystroglycan, a component of the dystrophin glycoprotein complex.13,17 Both our patients were compound heterozygotes for the common 826C4A (Leu276Ile) mutation and in addition had a previously unreported missense mutation. Patient 1 had a 506T4C mutation, which changed a mildly conserved leucine
1. Emery AE. The muscular dystrophies. Lancet 2002;359:687–95. 2. Manzur AY, Kuntzer T, Pike M, et al. Glucocorticoid corticosteroids for Duchenne muscular dystrophy. In: The Cochrane database of systematic reviews, issue 2, art no CD003725. New York: Wiley; 2004. 3. Chen YW, Nagaraju K, Bakay M, et al. Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy. Neurology 2005;65:826–34. 4. Arahata K, Ishihara T, Fukunaga H, et al. Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): immunocytochemical and genetic analyses. Muscle Nerve 1995;2:S56–66. 5. McNally EM, Ly CT, Rosenmann H, et al. Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation. Am J Med Genet 2000;91:305–12. 6. Laval SH, Bushby KM. Limb-girdle muscular dystrophies— from genetics to molecular pathology. Neuropathol Appl Neurobiol 2004;30:91–105. 7. Walter MC, Petersen JA, Stucka R, et al. FKRP (826C4A) frequently causes limb-girdle muscular dystrophy in German patients. J Med Genet 2004;41:e50.
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8. Esapa CT, Benson MA, Schroder JE, et al. Functional requirements for fukutin-related protein in the Golgi apparatus. Hum Mol Genet 2002;11:3319–31. 9. Mercuri E, Brockington M, Straub V, et al. Phenotypic spectrum associated with mutations in the fukutin-related protein gene. Ann Neurol 2003;53:537–42. 10. de Paula F, Vieira N, Starling A, et al. Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum. Eur J Hum Genet 2003;11:923–30. 11. Scott OM, Hyde SA, Goddard C, et al. Quantitation of muscle function in children: a prospective study in Duchenne muscular dystrophy. Muscle Nerve 1982;5:291–301. 12. Backman E, Odenrick P, Henriksson KG, et al. Isometric muscle force and anthropometric values in normal children aged between 3.5 and 15 years. Scand J Rehabil Med 1989;21:105–14. 13. Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 2001;69:1198–209. 14. Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10:2851–9.
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15. Topaloglu H, Brockington M, Yuva Y, et al. FKRP gene mutations cause congenital muscular dystrophy, mental retardation, and cerebellar cysts. Neurology 2003;60:988–92. 16. Beltran-Valero de Bernabe D, Voit T, Longman C, et al. Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker–Warburg syndrome. J Med Genet 2004;41:e61. 17. Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 2004;164:727–37. 18. Schwartz M, Hertz JM, Sveen ML, et al. LGMD2I presenting with a characteristic Duchenne or Becker muscular dystrophy phenotype. Neurology 2005;64:1635–7. 19. Griggs RC, Bushby K. Continued need for caution in the diagnosis of Duchenne muscular dystrophy. Neurology 2005;64:1498–9. 20. Backman E, Henriksson KG. Low-dose prednisolone treatment in Duchenne and Becker muscular dystrophy. Neuromuscul Disord 1995;5:233–41. 21. von der Hagen M, Kaindl AM, Koehler K, et al. Limb girdle muscular dystrophy type 2I caused by a novel missense mutation in the FKRP gene presenting as acute virusassociated myositis in infancy. Eur J Pediatr 2006;165:62–3.
11 (2007) 353 – 357
Official Journal of the European Paediatric Neurology Society
Inflammation and response to steroid treatment in limb-girdle muscular dystrophy 2I ˚ hlandera, A.-R. Moslemib, A. Oldforsb, M. Tuliniusa N. Darina,, A.-K. Kroksmarka, A.-C. A a
Department of Pediatrics, Sahlgrenska University Hospital, Go¨teborg, Sweden Department of Pathology, Sahlgrenska University Hospital, Go¨teborg, Sweden
b
art i cle info
ab st rac t
Article history:
Limb-girdle muscular dystrophy (LGMD) type 2I, caused by mutations in the fukutin-related
Received 16 November 2006
protein gene (FKRP), is one of the most common forms of LGMD in childhood. We describe
Accepted 28 February 2007
two patients with LGMD2I and a Duchenne-like phenotype. In addition to the common
Keywords:
Clinical onset was triggered by viral upper respiratory tract infections. In addition to the
Limb-girdle muscular dystrophy 2I
common dystrophic pattern with a weak immune histochemical staining for a-dystrogly-
FKRP
can, muscle biopsy showed inflammatory changes. This was especially striking in one of
Inflammation
the patients with up-regulation of MHC class 1 antigen, suggestive of myositis. Both
Steroid treatment
patients showed a good clinical response to treatment with prednisolone, which was
L276I mutation, both patients had a new mutation in FKRP, L169P and P89L, respectively.
initiated at daily dosage of 0.35 mg/kg/day. Our results provide evidence for an inflammatory involvement in the pathological expression of LGMD2I and open up the possibility that this disorder could be treatable with corticosteroids. & 2007 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
Muscular dystrophies are inherited myogenic disorders characterized by progressive muscle wasting and weakness of variable distribution and severity. The primary pathological mechanisms behind many of these disorders is the loss of linkage between the extracellular matrix and the actin cytoskeleton.1 Duchenne muscular dystrophy (DMD) is the most frequent and best studied of the muscular dystrophies. There is accumulating evidence from randomized controlled studies that corticosteroid treatment, at least in the short-term, improves muscle strength and function in DMD.2 In recent years, it has become increasingly recognized that inflammation is both an early and important pathophysiological event in this disorder.3 The efficiency of corticosteroid treatment in other muscular dystrophies is not known. Inflammatory infiltrates have been found in Facioscapulohumeral muscular dystrophy and limb-girdle muscular dystrophy (LGMD) 2B.4,5
The LGMD phenotype is defined by predominant involvement of the pelvic and shoulder girdle muscles. At least 15 genes, 6 autosomal dominant (LGMD1A-F) and 10 autosomal recessive (LGMD2A-J), responsible for LGMD have been mapped.6 LGMD type 2I is one of the most common forms.7 It is caused by mutations in the fukutin-related protein gene (FKRP), a putative glycosyl transferase, required for posttranslational modification of dystroglycan.8 The pattern of muscle involvement, which frequently includes calf hypertrophy as well as cardiac and respiratory dysfunction, resembles the dystrophinopathies.9 The spectrum of variability associated with mutations in FKRP is wider than in other LGMDs and adult asymptomatic carriers have been repeatedly described, suggesting that other mechanisms than the genotype modify the phenotype in this disorder.10 We present two patients with LGMD2I caused by two new mutations in the FKRP. They both had a clinical onset triggered by viral upper respiratory tract infection (URTI),
Corresponding author. The Queen Silvia Children’s Hospital, S-416 85 Goteborg, Sweden. Tel.: +46 31 3438213; fax: +46 31 257960. ¨
E-mail address: [email protected] (N. Darin). 1090-3798/$ - see front matter & 2007 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpn.2007.02.018
ARTICLE IN PRESS 354
E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
inflammatory changes on muscle biopsy and responded clinically to corticosteroid treatment.
2.
Patients and methods
The patients were followed with regular controls at the Neuromuscular Unit of the Queen Silvia Children’s Hospital in Gothenburg, Sweden.
2.1.
Measurement procedures
Isometric muscle strength was measured with an electronic handheld dynamometer according to the procedures developed by Scott et al.11 An isometric contraction of at least 2–3 s was required and the peak force in Newton was recorded. The best of three values obtained on the nondominant side was compared with reference values.12 Motor function was assessed by using a scale designed by Scott et al.11 In all, 20 movements were assessed, including head lifting, rolling, sitting up, sitting, getting off a chair, standing, standing on the heels, standing on the toes, standing on one leg and ascending and descending stairs. The performance is scored according to a three-point scale, 0 (unable), 1 (needs self-reinforcement) and 2 (succeeds). The maximum score attainable is 40. A percentage of the total motor function score was calculated and 46-m walking time was recorded.
2.2.
Immunohistochemistry
Immuno-histochemical staining of a-dystroglycan was performed on 10-mm-thick cryostat sections. The sections were fixed in icecold 99.9% ethanol and acetic acid, 1:1, for 1 min and blocked in 1% bovine serum albumine (BSA) before they were incubated for 1 h at 37 1C with monoclonal antibodies directed against a-dystroglycan. This anti-a-dystroglycan clone VIA4-1 (Upstate, Lake Placid, NY) skall ersa¨ttas av (Upstate, Temecula, CA) was diluted 1:1000 in 1% BSA. A biotinylated horse-anti-mouse antibody (Vector Lab, Novocastra, Newcastle, UK) was used as a secondary antibody. It was diluted 1:300 in 1% BSA, and applied for 1 h at room temperature followed by incubation in ABComplex K 355 HRP (Dako, Copenhagen, Denmark). The sections were visualized in DAB+ Chromogen K5007 (Dako, Copenhagen, Denmark). All dilutions and washings were made in phosphate-buffered saline (PBS). All sections were compared with control samples from nonaffected muscles, immunostained simultaneously. The samples were then analyzed in lightmicroscope.
2.3.
DNA analysis
Total DNA was extracted from 15 10 mm fresh frozen skeletal muscle tissue, using a DNA extraction kit (Dneasy Tissue Kit, Qiagen, Hilden, Germany). The samples from the patients were screened for the 826 C4A mutation in FKRP by PCR and restriction fragment length polymorphism (RFLP) as follows; 50–100 ng of the DNA and
11 (2007) 353 – 357
20 pmol of each primer (F40795 and R41128-4112) were added to Reddy Mix PCR Master Mix (Abgene, Epsom, UK). Thermal cycling was performed in Applied Biosystems 9600 Thermal Cycler. After an initial preheating step for 5 min at 94 1C, a touchdown (TD) PCR was performed, consisting of denaturation at 94 1C for 30 s, annealing at 65 1C for 30 s, extension at 72 1C for 1 min with an 1 1C temperature decrement per cycle during the first 10 cycles. The subsequent cycles (40 cycles) each consisted of 94 1C for 30 s, 55 1C for 30 s and 72 1C for 1 min. The amplified fragments were digested with 10 u Bfa I (Biolabs, New England). The entire exon 4 coding sequence was analyzed as follows; a 1488 bp sequence containing the coding sequence was amplified by PCR using 20 pmol 1F (40436–40454) and 5R (42035–42015) primers. The PCR conditions were the same as described above. The purified PCR products were then subjected for direct sequencing using following primers: 1F (40436–40454), 3F (40875–40895), 4R (41694–41677) and 5R (42035–42015) (GenBank, accession number: AC008622). To compare the amino acid sequences of human FKRP with homologues from other species, sequences were accessed through public domains /www.ncbi.nlm.nih.gov/entrez/ query.fcgiS and aligned with Expassy proteomics server /http://ca.expasy.org/S.
3.
Results
3.1.
Patient 1
This boy had normal early motor development and started to walk unsupported at 1 year of age. At the same time, he had a febrile URTI of viral origin and transiently became weak in his neck muscles with loss of head control. At 2 years of age, within a week after another URTI, he once again lost head control and developed a waddling gait. On this occasion, clinical examination showed generalized muscle weakness. The serum creatine kinase level was increased to a maximum of 196 mkat/L (reference valueo2.5). Muscle biopsies were performed from the anterior tibial and lateral vastus muscles at 2 and 14 years of age. They showed variability in fiber size, interstitial fibrosis, occasional inflammatory infiltrates, no MHC class 1 upregulation and a-dystroglycan deficiency. Genetic investigations identified the 826C4A (Leu276Ile) and a new 506T4C (Leu169Pro) mutations in the FKRP gene (Fig. 1). At 16 years of age, when prednisolone treatment was started, he had mild facial involvement, winging of the scapulae, scoliosis and pseudohypertrophic calves. He could walk short distances indoors without support. He could not rise from the floor and only rose to standing with great difficulties from a chair. There was a rapid deterioration over the 2 years prior to prednisolone treatment (Fig. 2(A)). After a few months of treatment, he walked easier and faster. After 6 months of treatment, there was no further deterioration and he showed improvement of muscle strength, motor function tests and time tests. Still after nearly 4 years of
ARTICLE IN PRESS E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
355
11 (200 7) 353 – 3 57
Fig. 1 – Muscle biopsy of patient 2 demonstrating interstitial inflammatory cell infiltration (a) consisting mainly of CD8+ T-cells (b), MHC class I upregulation (c) and deficiency of a-dystroglycan (d). (a) Haematoxylin-eosin. (b) Immunostaining of CD8 (T cell, CD8, Mouse anti-human; Dako, Glostrup, Denmark) (c) Immunostaining of MHC class I (HLA-ABC antigen Mouse anti-human; Dako, Glostrup, Denmark). (d) and (e) Immunostaining of a-dystroglycan (anti a-dystroglycan; Upstate Cell Signalling Solutions; Temecula, CA) in patient (d) and control (e). Sequence chromatograms of FKRP showing the identified mutations (f–h).
100 Prednisolone 0.32 0.37 (mg/kg/day)
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Fig. 2 – Results from repetitive testing of patient 1 (a) and patient 2 (b) at regular controls performed at the Neuromuscular Unit of the Queen Silvia Children’s Hospital. Isometric muscle strength in the knee extensors was measured with a hand-held dynamometer and compared with reference values.12 Motor function was assessed using a scale designed by Scott et al.11 A percentage of the total motor function score was calculated and 46 meter walking time was recorded.
follow-up, many tested parameters were at the same level as at onset of treatment. At 17 years of age, a dilated cardiomyopathy was diagnosed and he developed multiple
vertebral fractures. He was treated with three infusions of zoledronic acid (Zometa), with good effect and complete pain relief after 3 months.
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3.2.
E U R O P E A N J O U R N A L O F PA E D I AT R I C N E U R O L O G Y
Patient 2
This boy had a normal early motor development. At 16 months of age, during a viral URTI, he developed ptosis, lost control of his neck and the ability to walk with support. He recovered within 2 months and started to walk unsupported at 19 months of age but he never learned to run properly. The serum creatine kinase level was increased to a maximum of 200 mkat/L. Muscle biopsies were performed from the lateral vastus muscle at 2 and 9 years of age. They showed variability in fiber size, necrotic and regeneratating fibers, interstitial fibrosis, multiple inflammatory cell infiltrates, MHC class 1 upregulation and a-dystroglycan deficiency (Fig. 1). Genetic investigations identified the 826C4A (Leu276Ile) and a new 266C4T (Pro89Leu) mutations in the FKRP gene (Fig. 1). At 10 years of age, when prednisolone treatment started, he had great difficulties rising from the ground after a fall and rising from sitting to standing and could only do this with support. He could still walk short distances unaided but was in need of a wheel chair for longer distances. There was a rapid deterioration over the 2 years prior to treatment (Fig. 2(B)). After 4 months of treatment, he could rise from the floor easier and without support; stand on one leg longer, and swim longer distances. Upon testing, there was a clear improvement on muscle strength, motor function and time tests. The effects were stable until 2 years after onset of treatment when there was a clear deterioration. This was associated with a reduction of the prednisolone dosage to 0.17 mg/kg body weight because of concern about an arrested growth in height. After 4 years of treatment, the dosage of prednisolone was only 0.07 mg/kg body weight. At this moment, upon a trial to withdraw the medication completely, he immediately deteriorated and lost the ability to rise from sitting to a standing position. When the treatment was reintroduced at the same dosage, the boy rapidly regained the same ability. A dilated cardiomyopathy was diagnosed at 14 years of age.
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to proline at aminoacid position 169 while patient 2 had a 266C4T mutation, which changed a highly conserved proline to leucine at position 89 (Fig. 1). Homozygosity for L276I seems to confer a mild clinical phenotype, while compound heterozygosity for this mutation seems to be associated with a more severe phenotype.9 In fact, there is a very strong phenotypic resemblance between LGMD2I and the dystrophinopathies18 and this has been suggested to indicate the possibility that patients with LGMD2I may respond to corticosteroid treatment in a similar way as those with DMD.19 Both our patients presented with a Duchenne-like phenotype and were initially diagnosed as DMD and the choices to initiate treatment with prednisolone were done before the final diagnosis of LGMD2I. The most effective prednisolone regime in DMD is considered to be 0.75 mg/kg/day.2 Our treatment regime with 0.35 mg/kg/day of prednisolone was based on a Swedish study.20 Both patients developed dilated cardiomyopathy during the treatment. This is also a very frequent clinical manifestation in untreated patients with LGMD2I 9 and we consider it more likely a consequence of the natural course of the disease than a side effect in these two patients. We present evidence for an immunological involvement in the pathological expression of LGMD2I. Firstly, the onset was triggered by viral URTIs. This is similar to a recent description of two siblings with LGMD2I who presented with a suspected acute virus-associated myositis.21 Secondly, muscle biopsy showed inflammatory changes. This was especially pronounced in patient 2, with up-regulation of MHC class 1 antigen, suggestive of myositis. Thirdly, there was a clinical response to corticosteroid treatment in both patients. It will be important to study further how the immune system is involved in this disorder. Randomized controlled studies will be important to investigate the potential benefits and side effects of corticosteroid treatment in LGMD2I. R E F E R E N C E S
4.
Discussion
The clinical spectrum due to mutations in the FKRP gene is extremely wide. Mutations were initially described in congenital muscular dystrophy (MDC1C)13 and LGMD2I of variable severity,14 and have since then been found in patients with CMD, mental retardation and brain abnormalities including cerebellar cysts,15 muscle–eye–brain disease and Walker–Warburg syndrome16 and in adult asymptomatic carriers.10 There is in fact a significant clinical intrafamilial variability, suggesting that additional factors play a significant role in determining disease severity.9,10 Although the function of FKRP has not been determined, it is thought to be a glycosyltransferase.8 In accordance with this, MDC1C and LGMD2I are associated with a secondary deficiency in glycosylation of a-dystroglycan, a component of the dystrophin glycoprotein complex.13,17 Both our patients were compound heterozygotes for the common 826C4A (Leu276Ile) mutation and in addition had a previously unreported missense mutation. Patient 1 had a 506T4C mutation, which changed a mildly conserved leucine
1. Emery AE. The muscular dystrophies. Lancet 2002;359:687–95. 2. Manzur AY, Kuntzer T, Pike M, et al. Glucocorticoid corticosteroids for Duchenne muscular dystrophy. In: The Cochrane database of systematic reviews, issue 2, art no CD003725. New York: Wiley; 2004. 3. Chen YW, Nagaraju K, Bakay M, et al. Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy. Neurology 2005;65:826–34. 4. Arahata K, Ishihara T, Fukunaga H, et al. Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): immunocytochemical and genetic analyses. Muscle Nerve 1995;2:S56–66. 5. McNally EM, Ly CT, Rosenmann H, et al. Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation. Am J Med Genet 2000;91:305–12. 6. Laval SH, Bushby KM. Limb-girdle muscular dystrophies— from genetics to molecular pathology. Neuropathol Appl Neurobiol 2004;30:91–105. 7. Walter MC, Petersen JA, Stucka R, et al. FKRP (826C4A) frequently causes limb-girdle muscular dystrophy in German patients. J Med Genet 2004;41:e50.
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8. Esapa CT, Benson MA, Schroder JE, et al. Functional requirements for fukutin-related protein in the Golgi apparatus. Hum Mol Genet 2002;11:3319–31. 9. Mercuri E, Brockington M, Straub V, et al. Phenotypic spectrum associated with mutations in the fukutin-related protein gene. Ann Neurol 2003;53:537–42. 10. de Paula F, Vieira N, Starling A, et al. Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum. Eur J Hum Genet 2003;11:923–30. 11. Scott OM, Hyde SA, Goddard C, et al. Quantitation of muscle function in children: a prospective study in Duchenne muscular dystrophy. Muscle Nerve 1982;5:291–301. 12. Backman E, Odenrick P, Henriksson KG, et al. Isometric muscle force and anthropometric values in normal children aged between 3.5 and 15 years. Scand J Rehabil Med 1989;21:105–14. 13. Brockington M, Blake DJ, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan. Am J Hum Genet 2001;69:1198–209. 14. Brockington M, Yuva Y, Prandini P, et al. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10:2851–9.
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15. Topaloglu H, Brockington M, Yuva Y, et al. FKRP gene mutations cause congenital muscular dystrophy, mental retardation, and cerebellar cysts. Neurology 2003;60:988–92. 16. Beltran-Valero de Bernabe D, Voit T, Longman C, et al. Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker–Warburg syndrome. J Med Genet 2004;41:e61. 17. Brown SC, Torelli S, Brockington M, et al. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies. Am J Pathol 2004;164:727–37. 18. Schwartz M, Hertz JM, Sveen ML, et al. LGMD2I presenting with a characteristic Duchenne or Becker muscular dystrophy phenotype. Neurology 2005;64:1635–7. 19. Griggs RC, Bushby K. Continued need for caution in the diagnosis of Duchenne muscular dystrophy. Neurology 2005;64:1498–9. 20. Backman E, Henriksson KG. Low-dose prednisolone treatment in Duchenne and Becker muscular dystrophy. Neuromuscul Disord 1995;5:233–41. 21. von der Hagen M, Kaindl AM, Koehler K, et al. Limb girdle muscular dystrophy type 2I caused by a novel missense mutation in the FKRP gene presenting as acute virusassociated myositis in infancy. Eur J Pediatr 2006;165:62–3.