JNNP Online First, published on July 26, 2010 as 10.1136/jnnp.2009.178038 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.2009.178038 on 14 June 2009. Downloaded from Research paper New aspects on patients affected by dysferlin deficient muscular dystrophy Lars Klinge,1,2 Ahmed Aboumousa,1 Michelle Eagle,1 Judith Hudson,1 Anna Sarkozy,1 Gianluca Vita,1 Richard Charlton,1 Mark Roberts,3 Volker Straub,1 Rita Barresi,1 Hanns Lochmu¨ller,1 Kate Bushby1 1University of Newcastle, ABSTRACT distal muscle groups and vice versa.46The factors Institute of Human Genetics, Mutations in the dysferlin gene lead to limb girdle responsible for these distinct patterns of presenta- International Centre for Life, muscular dystrophy 2B, Miyoshi myopathy and distal tion are unknown. Therefore, further character- Newcastle upon Tyne, UK fi 2Department of Paediatrics and anterior compartment myopathy. A cohort of 36 patients isation of patients with dysferlin de ciency may Paediatric Neurology, University affected by dysferlinopathy is described, in the first UK help to identify possible distinct features within Medical Centre, Go¨ttingen, study of clinical, genetic, pathological and biochemical this entity and might provide clues to underlying Germany 7 3 data. The diagnosis was established by reduction of pathogenetic mechanisms. Greater Manchester fi Neurosciences Centre, Salford, dysferlin in the muscle biopsy and subsequent mutational Dysferlin de cient muscular dystrophy is UK analysis of the dysferlin gene. Seventeen mutations were inherited as an autosomal recessive trait, age of novel; the majority of mutations were small deletions/ onset has been found to be usually young adult- Correspondence to insertions, and no mutational hotspots were identified. hood and the progression to be slow.4 68Overt Dr K Bushby, University of Sixty-one per cent of patients (22 patients) initially cardiac pathology appears not to be a common Newcastle, Institute of Human fi 46 Genetics, The Institute of presented with limb girdle muscular dystrophy 2B, 31% nding in most patients. However, cardiac Human Genetics, International (11 patients) with a Miyoshi phenotype, one patient with impairment in dysferlin deficiency under stress has Centre for Life, Central Parkway, proximodistal mode of onset, one patient with muscle been shown in the knockout mouse,9 and two Newcastle upon Tyne NE1 3BZ, stiffness after exercise and one patient as studies have demonstrated dilated cardiomyopathy UK; [email protected] a symptomatic carrier. A wider range of age of onset in a total of six patients with dysferlinopathy.10 11 Received 16 March 2009 was noted than previously reported, with 25% of Dysferlin is a 230 kDa transmembrane protein and Revised 27 May 2009 patients having first symptoms before the age of has been shown to be involved in the process of Accepted 31 May 2009 13 years. Independent of the initial mode of presentation, membrane repair,12 myoblast differentiation,13 in our cohort of patients the gastrocnemius muscle was T tubulogenesis14 15 and muscle regeneration,16 the most severely affected muscle leading to an inability most likely through mechanisms of membrane to stand on tiptoes, and lower limbs were affected more organisation. So far this has not been proven but severely than upper limbs. As previous anecdotal the hypothesis is based on the following findings: evidence on patients affected by dysferlinopathy the biochemical structure of dysferlin with six C2 suggests good muscle prowess before onset of domains which are calcium sensitive domains symptoms, we also investigated pre-symptomatic fitness involved in fusion and trafficking of membranes, levels of the patients. Fifty-three per cent of the patients the homology to fer-1 in C elegans and otoferlin, were very active and sporty before the onset of both proteins important for vesicle fusion, an http://jnnp.bmj.com/ symptoms which makes the clinical course of upregulation of proteins involved in vesicle traf- dysferlinopathy unusual within the different forms of ficking,17 a subsarcolemmal accumulation of vesi- muscular dystrophy and provides a challenge to cles below the surface of dysferlin deficient understanding the underlying pathomechanisms in this myofibres18 and altered T tubule morphology and disease. orientation on the ultrastructural level.15 Recent evidence also suggests a role for dysferlin in monocyte phagocytosis.19 on October 2, 2021 by guest. Protected copyright. A unique finding within the spectrum of muscular dystrophies is that the majority of INTRODUCTION patients with dysferlin deficiency appear to have Ten years ago the dysferlin gene was cloned and good muscle strength before onset of symptoms identified as the gene mutated in limb girdle leading to good performance at sports or to the muscular dystrophy (LGMD) 2B, and Miyoshi ability to cope well with physically demanding jobs myopathy (MM).1 2 While LGMD2B is charac- although this is suggested by mainly anecdotal terised by proximal muscle involvement, patients evidence.19 20 This observation contradicts the affected by MM present with distal muscle weak- clinical course of other muscular dystrophies (MDs) ness at onset.12Subsequently, distal anterior where the majority of patients describe some sort compartment myopathy and a proximodistal mode of impaired muscle function before onset of overt of onset have been described as further phenotypes/ weakness. This aspect is difficult to address, has not e modes of presentation in dysferlin deficiency,3 5 yet been formally assessed but may be a distinct fi fi This paper is freely available and some patients have been reported with hyper- feature in dysferlin de ciency. In this rst study on 5 online under the BMJ Journals CKaemia. During the course of the disease, the the clinical spectrum of dysferlinopathy in the UK, unlocked scheme, see http:// phenotypes show a considerable amount of overlap we present clinical, genetic, pathology and jnnp.bmj.com/site/about/ so that weakness may extend from proximal to biochemical data on patients with dysferlinopathy, unlocked.xhtml KlingeCopyright L, Aboumousa Article A, Eagle M,authoret al. J (or Neurol their Neurosurg employer) Psychiatry (2010). 2010. doi:10.1136/jnnp.2009.178038 Produced by BMJ Publishing Group Ltd under licence. 1 of 8 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.2009.178038 on 14 June 2009. Downloaded from Research paper and specifically address the aspect of pre-symptomatic fitness abduction, flexion, horizontal adduction, elbow flexion including levels. brachioradialis and biceps, elbow extension, wrist flexion and extension, finger flexion and extension. In the lower limb, hip fl fl MATERIALS AND METHODS exion, extension, abduction and adduction, knee exion and fl fl Thirty-six patients with dysferlin deficiency from 32 families extension and in the ankle plantar exion, dorsi exion, inversion were assessed retrospectively. The diagnosis of dysferlinopathy and eversion. Each muscle group was assessed in several different was made based on reduction or absence of dysferlin in the positions to determine antigravity activity as well as the ability muscle biopsy and mutation analysis. Patients were referred to to resist a manually applied force. Strength was graded using an the Newcastle Muscle Centre as part of the National Commis- 11 point scale from 5 (normal strength) to 0 (no activity). The sioning Group designated specialised diagnostic service for same order of assessment was followed in each case. Each LGMDs in the UK for examination and further evaluation. patient was also asked to demonstrate their ability to stand on fi During the clinic visits a complete history and family history tiptoes as manual resistance cannot suf ciently demonstrate fl were taken, and patients were specifically asked about their normal strength plantar exion. muscle strength and athletic performance before onset of Upper and lower limb score symptoms, such as their performance at school sports. A detailed muscle assessment was undertaken by the same Muscles of the upper or lower limb were scored on an 11 point e examiner in 21 patients, and muscle strength scored.21 numerical scale (0 10) according to the strength grade (also 11 point). The score of the patient was calculated as the per cent of Immunoanalysis of muscle biopsies the maximum score achievable. Muscle biopsies were analysed as part of the National Statistical analysis Commissioning Group designated specialised diagnostic service ’ ’ for LGMD. Optimised immunohistochemical and multiplex Fisher s exact test for count and the Student s t test were fi < western blot protocols were used for the demonstration and applied. Statistical signi cance was established when p 0.05. resolution of muscular dystrophy associated proteins, as previ- ously described.22 23 Primary antibodies used for immunohisto- RESULTS chemistry were directed against: b-spectrin (RBC2/3D5), Muscle immunoanalysis findings b-dystroglycan (43DAG/8D5), C terminus dystrophin (Dy8/ Immunohistochemistry 6C5) and N terminus dystrophin (Dy10/12B2), a-sarcoglycan Dysferlin was either severely reduced or absent on tissue (Ad1/20A6), b-sarcoglycan (1/5B1), g-sarcoglycan (35DAG/ sections with no apparent correlation to the clinical presentation 21B5), d-sarcoglycan (3/12C1) (all from Novocastra, Newcastle, or underlying mutation. b-dystroglycan was reduced in 17% of UK) and caveolin-3 (Transduction Laboratories, Oxford, UK). samples, and caveolin-3 showed reduced sarcolemmal labelling Antibodies used for western