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Neuromuscular Disorders 22 (2012) 522–527 www..com/locate/nmd

Relative frequency of congenital subtypes: Analysis of the UK diagnostic service 2001–2008

E.M. Clement a, L. Feng a, R. Mein b, C.A. Sewry a, S.A. Robb a, A.Y. Manzur a, E. Mercuri a,c, C. Godfrey a, T. Cullup b, S. Abbs b, F. Muntoni a,⇑

a Dubowitz Neuromuscular Centre, Institute of Child Health and Great Ormond Street Hospital, London WC1N 1EH, United Kingdom b DNA Laboratory, GSTS Pathology, Genetics Centre, Guy’s Hospital, London, United Kingdom c Department of Child Neurology, Catholic University, Rome, Italy

Received 29 November 2011; accepted 26 January 2012

Abstract

The Dubowitz Neuromuscular Centre is the UK National Commissioning Group referral centre for congenital muscular dystrophy (CMD). This retrospective review reports the diagnostic outcome of 214 UK patients referred to the centre for assessment of ‘possible CMD’ between 2001 and 2008 with a view to commenting on the variety of disorders seen and the relative frequency of CMD subtypes in this patient population. A genetic diagnosis was reached in 53 of 116 patients fulfilling a strict criteria for the diagnosis of CMD. Within this group the most common diagnoses were VI related disorders (19%), dystroglycanopathy (12%) and merosin deficient congenital muscular dystrophy (10%). Among the patients referred as ‘possible CMD’ that did not meet our inclusion criteria, congenital and congenital myasthenic syndromes were the most common diagnoses. In this large study on CMD the diagnostic outcomes compared favourably with other CMD population studies, indicating the importance of an integrated clinical and pathological assessment of this group of patients. Ó 2012 Elsevier B.V. All rights reserved.

Keywords: Congenital muscular dystrophy; Epidemiology; Differential diagnosis

1. Introduction CMDs are a heterogeneous group of disorders. They most commonly present with muscle weakness and hypoto- The Dubowitz Neuromuscular Centre was commis- nia in the first months of life; are common and sioned in 2001 by the UK Department of Health National serum elevated in several but not all sub- Commissioning Group (NCG) to provide a national com- types. Other clinical features include muscle wasting or prehensive service for the assessment, investigation and hypertrophy and abnormalities of eye, skin and brain. management of patients affected by congenital muscular Respiratory and cardiac involvement is also common. dystrophy (CMD). In 2005, the service expanded to include There are at least 13 genetically distinct forms of CMD the congenital myopathies. The NCG service can be reported, for summary of known variants see Table 1 accessed by the referring clinical team at various levels, supplementary information. In addition there is a further including clinical review of patient, assessment of muscle group of as yet undefined patients in whom all known biopsy or molecular genetic analysis and is freely available forms can be excluded. A proportion of these cases is to UK patients. affected by CMD subtypes that are clinically and patholog- ically indistinguishable from genetically defined forms, while others, despite their clinical and/or pathological ⇑ Corresponding author. Tel.: +44 20 7905 2111; fax: +44 20 7905 2832. similarities to CMD, clearly belong to a different group E-mail address: [email protected] (F. Muntoni). of conditions. Typical examples of the latter are myotonic

0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2012.01.010 E.M. Clement et al. / Neuromuscular Disorders 22 (2012) 522–527 523 dystrophy, some congenital variants and con- Dreifuss muscular dystrophy (EDMD2) [14]. Rigid spine genital myasthenic syndromes [1–3]. muscular dystrophy (RSMD1) is a CMD caused by muta- biopsy is an important part of the diag- tions in SEPN1. Mutations in the same gene also cause nostic workup of children with suspected CMD. Muscle other pathological phenotypes including congenital fibre biopsy findings in affected individuals range from mild myo- type disproportion, multiminicore disease and desmin- pathic changes to overtly dystrophic, depending on the related myopathy with mallory body-like inclusions. The muscle biopsied and the age at biopsy. The extent of dystro- phenotypes and pathology in this group of conditions show phic changes necessary to qualify a muscle biopsy to be con- considerable overlap, however the fact that pathological sidered “dystrophic” is also subject to debate. For the changes in axial muscle are invariably dystrophic supports purpose of this report, dystrophic change is defined as mus- this condition being a dystrophic rather than myopathic cle with fibrosis and necrosis. If necrosis is not observed variant [15]. then muscle fibre regeneration must be present [4]. Few studies have looked at the incidence of CMD but Merosin (laminin2) deficient congenital muscular dys- from the reports available it has been estimated at between trophy type 1A (MDC1A) was the first CMD pathologi- 4.7 Â 10À5 live births in the North of Italy with a point cally defined due to a characteristic deficiency of the prevalence of 2.5 Â 10À5 in Western Sweden [16,17]. The a2 chain of merosin on skeletal muscle biopsy; this prevalence of different subtypes of CMD varies in different also became the first CMD variant for which the genetic populations, often due to founder mutations. MDC1A has defect, mutations in laminin a2(LAMA2), was found. historically been thought of as the most common form of The availability of a straightforward pathological diagnosis CMD with estimates suggesting that it accounts for and genetic testing, led to the rapid proliferation of manu- between 30% and 50% of cases [1,18]. In Japan however, scripts reporting the clinical features and relative frequency Fukuyama CMD is the most common form of CMD of this condition in several populations [5–7]. The collagen reported but has rarely been seen outside this population VI related disorders, Ullrich congenital muscular dystro- [19,20]. phy (UCMD) and its milder allelic variant Bethlem myop- In this study we review the diagnostic outcome of 214 UK athy (BM), represent another significant proportion of patients clinically assessed in our department between 2001 CMD cases. Although many cases of BM present after and 2008 with a view to describing the frequency of the var- the first 2 years of life, with some remaining asymptomatic ious disorders encountered in our patient population. in adulthood, early or congenital onset cases are well doc- umented and for this reason have been included here. The 2. Materials and methods biochemical and molecular diagnosis of collagen VI related disorders is more complicated due to the subtle reduction The UK referrals to the NCG Dubowitz Neuromuscular of protein expression in many UCMD patients and the lack Centre between April 2001 and January 2008 as ‘possible of clear pathological markers on muscle biopsy for most CMD’ were retrospectively reviewed. Ethical approval BM cases. However improved detection techniques such was obtained from the Hammersmith Hospital and Queen as double immunofluorescence of collagen VI and proteins Charlotte Ethics Committee. Only patients who had been such as perlecan and collagen IV on muscle biopsy, or clinically assessed by us at the centre and from whom a immunohistochemical studies on cultured fibroblasts fol- muscle biopsy was available for the diagnostic studies were lowed by the genetic screening of the three collagen 6A included in this report. Patients where biopsy sample was chains, have led to the appreciation that collagen VI defects unavailable or insufficient for analysis, or cases who had are one of the most common CMD subtypes [8–10]. Muscle not been assessed in person at the centre, but in whom a imaging also plays an important role in the diagnosis of the diagnostic muscle or DNA sample had been forwarded collagen VI related conditions [11]. A third subgroup of for diagnosis have been excluded. Where more than one patients are grouped together in the so called ‘dystrogly- member of a family was seen, only one member of the fam- canopathy’ variant , identified by defective glycosylation ily has been included for the purpose of analysis (the of alpha dystroglycan in skeletal muscle, and appears to patient with the most information available). be a relatively frequent CMD subtype [12]. The spectrum Clinical notes, genetic tests and muscle biopsy reports of clinical severity observed in the dystroglycanopathies is were reviewed for all patients fulfilling the criteria. Diag- extremely variable, ranging from early lethal conditions nostic and biopsy information was gathered firstly for all with associated structural brain and eye malformations referrals, which however consisted of a considerable num- such as walker warburg syndrome (WWS), to much milder ber of patients not affected by CMD and then subsequently variants only affecting muscle and overlapping in clinical for patients who fulfilled the tighter inclusion criteria for severity with limb girdle muscular dystrophies (for a review CMD. This included: (1) Dystrophic, myopathic or mini- see [12]). Intermediate variants exist, and include muscle mal change muscle biopsy, with evidence of regeneration. eye brain disease (MEB), Fukuyama CMD (FCMD), Patients were excluded from this subgroup analysis if MDC1D and MDC1C. Separate conditions are the defi- biopsy showed additional structural change (e.g. rods) sug- ciency of a7 integrin [13] and CMD caused by mutations gesting an alternative diagnosis. Patients with cores were in LMNA, the gene more commonly involved in Emery included, as long as RYR1 mutations had been excluded, 524 E.M. Clement et al. / Neuromuscular Disorders 22 (2012) 522–527 as cores have been reported in patients with RSMD1 and Table 1 also more rarely also EDMD2 and UCMD (Sewry and Distribution of the genetic and pathological diagnoses in the ‘CMD Muntoni, personal observation). (2) Presentation before subgroup’ (n = 116). 2 years of age with , weakness, contractures, Diagnosis CMD subgroup Number Gene delayed motor milestones, raised CK or characteristic eye Genetic (n = 53) Collagen VI related 22 Collagen VI =22 or brain abnormality. (3) Non-CMD diagnoses (or patients (BM = 5) in whom alternative diagnosis is strongly suspected) (UCMD = 17) Dystroglycanopathy 14 POMT1 =2 excluded. POMT2 =3 Diagnostic standards for diagnosis were as for Dubo- POMGNT1 =4 witz and Sewry (CMD and ) [4], God- FKRP =4 frey et al. (dystroglycanopathy) [12], Gerenmayeh et al. LARGE =1 (MDC1A) [21], Nadeau et al. (collagen VI) [22] and Kinali EDMD2 4 LMNA =4 MDC1A 12 LAMA2 =12 et al. (congenital myasthenia) [23]. All biopsy samples from RSMD1 1 SEPN1 =1 patients referred with suspected CMD underwent a panel Pathological (n = 5) Collagen VI related 2 – of routine investigation including histological, histochemi- Dystroglycanopathy 3 – cal and immunohistochemical analysis except for those Unknown (n = 58) – 58 – where sample size limited testing. For further information Total 116 on standard departmental testing procedure see www.ich. BM, Bethlem myopathy; UCMD, Ullrich congenital muscular dystrophy; ucl.ac.uk/gosh/clinicalservices/neuromuscular_services. EDMD2, autosomal dominant Emery–Dreifuss muscular dystrophy; MDC1A, merosin deficient CMD; RSMD1, rigid spine muscular Genetic testing was directed by biopsy and clinical find- dystrophy. ings and often supported by muscle magnetic resonance imaging (MRI) [24]. Molecular genetic analysis of CMD and congenital myopathy genes was provided by Guys and St Thomas’ Trust, London (part of CMD NCG ser- vice), the NCG Referral Centre for LGMD at the Institute of Human Genetics at Newcastle University and National Commissioning Group funded Oxford Congenital Myas- thenic Syndrome Service. Key references for the listed dis- eases and their diagnostic workup can be found at: www.musclegenetable.org/.

3. Results

3.1. Part 1: total cohort analysis ‘all referrals’

Fig. 1. Graph showing distribution of diagnoses (n = 58) in the ‘CMD Muscle biopsy samples were received from 415 patients subgroup’. MDC1A, merosin deficient CMD; EDMD2, autosomal referred with ‘possible CMD’ during the study period. Of dominant Emery–Dreifuss muscular dystrophy; RSMD1, rigid spine these, 218 patients were clinically assessed at our centre. muscular dystrophy. Four siblings were excluded, leaving 214 patients included in subsequent analysis, with an average age at biopsy assessment of 6.2 years (range 1 day–37.2 years). cally assigned to a pathological subgroup (4%), see Table 1. The referrals represent a very mixed group in terms of The most common CMD diagnosis made was collagen VI ethnicity reflecting the London and wider UK population. related disorders (21%; genetically confirmed in 22/116 and Patients were referred from throughout the UK with 74/ pathologically confirmed in a further two patients) fol- 214 referred from the Greater London area. lowed by dystroglycanopathy (15%; genetically confirmed All 214 patients were assessed to see if they conformed in 14/116 and pathologically confirmed in a further three to stricter CMD inclusion criteria including; (1) Dystro- patients). The relative frequency of the different CMDs is phic, myopathic or minimal change muscle biopsy, with shown in Fig. 1. evidence of regeneration. (2) Presentation before 2 years Of 57/116 patients in the CMD subgroup with clearly of age with hypotonia, weakness, contractures, delayed dystrophic biopsies, a molecular diagnosis was made in motor milestones, raised CK or characteristic eye or brain 43 (75%) and a further three patients could be unequivo- abnormality. (3) Non-CMD diagnoses excluded. cally assigned to a pathological subgroup (data not One hundred and sixteen-patients fulfilled all three crite- shown). ria. Average age at biopsy assessment was 6.4 years (range Ninety-eight of the 214 patients referred for assessment 8 days–28.4 years). of ‘possible CMD’ did not meet our strict CMD inclusion A molecular diagnosis was reached in 53 of 116 (46%) criteria. In this group a genetic diagnosis was made in 30/ patients and another five patients (4%) could be unequivo- 98 and a pathological diagnosis was made in a further 17 E.M. Clement et al. / Neuromuscular Disorders 22 (2012) 522–527 525

Table 2a Distribution of genetic diagnoses (n = 30) in the patients referred that did not fulfil stricter diagnostic criteria for CMD (n = 98). Diagnosis Number Phenotype Subtype number Gene Congenital muscular dystrophya 2 RSMD1a 2 SEPN1 =2 Congenital myopathy 11 CCD 4 RYR1 =4 Myotubular myopathy 3 MTM1 =3 3 ACTA1 =3 CFTD 1 TPM3 =1 Limb girdle muscular dystrophy 6 Dystroglycanopathy 6 FKRP =5 POMGNT1 =1 Congenital myasthenia 8 Congenital myasthenia 8 CHRNA1 =2 COLQ =1 DOK7 =3 RAPSYN =1 CHRNG =1 Other 3 EDS 1 Collagen 3a1 =1 RETT syndrome 1 MECP2 =1 Chromosomal 1 Chromosomal = 1 Total 30 RSMD1, rigid spine muscular dystrophy; CCD, ; CFTD, congenital fibre type disproportion; EDS, Ehlers–Danlos syndrome. a The two patients with RSMD1 both presented after 2 years of age but are included in the CMD group as convention states that RSMD1 is a CMD.

Table 2b glycanopathies with 17/116 (15%). The most common Distribution of pathological ‘diagnoses’ (n = 17) in referred patients not dystroglycanopathy genes found to be mutated were POM- fulfilling diagnostic criteria for CMD (n = 98). GNT1 and FKRP, with four affected cases each. A diagno- Diagnostic group Number Phenotype Subtype sis of MDC1A was made in 12/116 (10%), EDMD2 in 4/ number 116 (3%) and RSMD1 in 1/116 (1%). Two further geneti- Congenital muscular 2 Collagen VI related 2 cally proven cases of RSMD1 were excluded from this dystropy (UCMD) ‘CMD’ subgroup analysis as they presented after 2 years Congenital myopathy 13 Core myopathy 3 of age. We found no cases of integrin a7 deficiency in Centronuclear 4 our population. A final diagnosis could not be determined myopathy Myofibrillar 1 in 58 cases (50%). It should be noted that we documented a myopathy further five cases of collagen VI related disorders confirmed Nemaline myopathy 5 from analysis of skin biopsy alone in patients not assessed Other 2 Mitochondrial 1 at our centre and therefore not included in this report, rein- Pompe disease 1 forcing this as the most prevalent CMD subtype in our Total 17 referral population. UCMD, Ullrich congenital muscular dystrophy. Our results contrast those of a recent Australian study by Peat et al. where a specific histopathological diagnosis patients, see Tables 2a and 2b. Congenital myopathy (24/ could be allocated in 49% of 101 CMD cases and a defini- 98 either genetically or pathologically confirmed), congen- tive genetic diagnosis could be made in 24% [25]. It should ital myasthenia (8/98) and limb girdle muscular dystrophy be noted that patients with biopsies containing cores were (6/98) were the most common diagnoses. excluded in their study therefore direct comparison with There were a further 23/214 patients in whom a clinical our data should be done with care. Even taking this into diagnosis is strongly suspected but a definitive genetic or account, our definitive (genetic) diagnosis rate is almost pathological diagnosis could not be reached. twice as high (46% v 24%) when comparing our data to the Australian CMD cohort. The reasons for the differ- ences in the diagnostic pick up rate are most likely a reflec- 4. Discussion tion of the fact that all of the patients in our study were clinically assessed in the specialised diagnostic service. We present here the diagnostic outcome in a large The pathology department has a longstanding tradition cohort of UK patients referred to the CMD NCG centre of interpretation of protein abnormalities in muscular for clinical review and muscle biopsy analysis between dystrophies and routinely uses a very extensive panel of 2001 and 2008. antibodies to characterise not only the primary protein Of those patients fulfilling the diagnostic criteria for deficiency but also secondary protein changes which can CMD, a diagnosis of CMD was made in 50% (58/116) be very helpful. Furthermore many patients also had addi- and was genetically confirmed in 53 of 116 (46%). Within tional supportive investigations including analysis of colla- this group, collagen VI related myopathy was the most gen VI in fibroblasts and muscle MRI imaging to help common diagnosis with 24/116 (21%), followed by dystro- direct genetic investigations. We have previously reported 526 E.M. Clement et al. / Neuromuscular Disorders 22 (2012) 522–527 that muscle MRI is a particularly useful diagnostic adjunct the diagnosis cannot be confirmed, approximately 20% in investigating muscular dystrophies with a high specificity have a collagen VI-like phenotype. This suggests either that and sensitivity for different subtypes of CMD [24]. We have current collagen VI diagnostic methods are not sensitive found MRI to be especially useful in the case of collagen VI enough or that similar disorders exist but have yet to be related disorders where we have found it to be a more reli- genetically and pathologically refined. A further two able indicator of collagen VI pathology than fibroblast patients probably have a ‘syndromic’ cause of their prob- analysis. Indeed abnormal production of collagen VI in lems with others having suspected Marinesco-Sjo¨gren fibroblast cultures is a sensitive indicator but not specific syndrome and motor neuronopathy. for collagen VI abnormality (Muntoni, unpublished data). The remainder of these cases are likely to comprise a In the Australian study, dystroglycanopathy was found number who have an as yet undefined form of CMD or to be the most common group representing 25% of cases, congenital myopathy caused by mutations in a novel gene with collagen VI abnormalities found in 12% [25]. Studies and also some patients with a defined form of CMD in from Japan reveal FCMD to be the most prevalent form whom we are unable to confirm this pathologically or of CMD in the Japanese population (49.2% of CMD cases genetically. Although the sensitivity of genetic testing has in one series) but this is due to a FKTN founder mutation. steadily improved, large heterozygous deletions or duplica- The second most common form of CMD encountered in tions are likely to be missed using present techniques. In the Japanese population is collagen VI deficiency with an some patients variants of unknown significance will have estimated frequency of 7.2% in their CMD cohort. Interest- been detected that cannot be proven to be pathogenic using ingly, a 2009 paper from the North of England [26] found current methods. Some of the undiagnosed patients will MDC1A to be the most prevalent CMD disorder (0.6/ eventually ‘reveal’ their diagnosis as their symptomatology 100,000) and much more common than dystroglycanopa- and histopathology evolves. For example, recent studies thies (0.03/100,000), UCMD (0.13/100,000) and RSMD1 highlight the mild initial pathological features in UCMD (0.13/100,000). However, mutations in FKRP are more fre- [27], and the selectivity of muscle involvement which char- quently seen in that LGMD cohort (0.43/100,000). In addi- acterises many of these conditions [24]. In others, a diagno- tion, BM has an estimated prevalence of 0.77/100,000 in sis may be made as a result of new technology, in particular their series but as there is no information about the age the emergence of the hybridisation arrays for assessing of presentation of these patients, comparison between the copy number variations in DNA of affected individuals. collagen VI related disorders in that study and ours is of With regard to the clinical presentation of the CMDs; limited value. although MDC1A is usually seen as a classical presentation Analysis of our data suggests that MDC1A, in various of severe early weakness, hypotonia and later characteristic series of patients reported to be the most common form features on brain MRI, the collagen VI related disorders of CMD, is not the most frequent diagnosis in our popula- and dystroglycanopathies are frequently more challenging tion, as both collagen VI related disorders and dystrogly- to diagnose due to their often milder phenotypes at presen- canopathy are more prevalent. This may be a reflection tation. In both groups and also in EDMD2 and RSMD1, of improved diagnostic assays and greater awareness of the classification lines between CMD, myopathy and the clinical and pathological presentation of other CMD LGMD are frequently blurred. forms; we cannot exclude regional variation in the preva- The geographically diverse nature of our referral popu- lence of CMD subtypes. lation unfortunately precludes any formal estimation of It is also of interest to note the large number of non prevalence or incidence for the different CMD subtypes, CMD diagnoses (47/214, including two patients with none the less, the information gathered here we feel is an RSMD1 presenting after 2 years of age) made in the accurate reflection of clinical activity in the CMD NCG patients referred with ‘possible CMD’, reflecting the impor- service. It is of interest to note that over 50% of our tance of an integrated approach to the diagnosis of these patients were referred from outside greater London high- rare and heterogeneous conditions, which includes clinical lighting the nationwide uptake of services. examination, pathological diagnosis and muscle imaging In summary, the data presented in this study represents directing the genetic testing. the largest series of UK CMD referral data and diagnostic These results also confirm our previous observations of outcome reported. It reveals collagen VI related disorders conditions resembling CMD, which include several con- to be the most common form of CMD in our patient genital myopathies and congenital myasthenias which group, accounting for 21% of referrals that fulfil the may show significant clinical and some pathological over- CMD inclusion criteria. Patients with dystroglycanopathy lap with CMD [3]. Indeed, this is the reason why the follow closely behind accounting for 15% with MDC1A NCG service in London has expanded to include assess- seen in 10%. There remain a large proportion of cases ment and diagnostic testing for congenital myopathy cases. (50%) that fulfil the clinical and pathological criteria for Despite our integrated approach, we were unable to CMD, in whom no diagnosis can be made. This suggests reach a diagnosis in half (58/116) of the CMD subgroup. that further CMD subtypes and genes are yet to be These undiagnosed patients are a varied group. Whilst discovered. E.M. Clement et al. / Neuromuscular Disorders 22 (2012) 522–527 527

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