30th and 31st ENMC international workshops, Naarden, The Netherlands, Held 6–8 January 1995

Neuromusc. Disord., Vol. 5. No. 4, pp. 337-343, 1995

Pergamon

Copyright © ElsevierScience Ltd Printed in Great Britain. All rights reserved 0960-8966/95 $9.50 + 0.00

0960-8966(95)00005.-4 WORKSHOP REPORT THE LIMB-GIRDLE

MUSCULAR

DYSTROPHIES

-- PROPOSAL

FOR

A NEW NOMENCLATURE 30TH AND

31 S T E N M C

INTERNATIONAL

THE NETHERLANDS,

HELD

INTRODUCTION

Clinicians and other scientists from Europe, Tunisia, Brazil and the U.S.A. gathered in Naarden on 6-8 January 1995 for the second meeting of the ENMC-sponsored consortium for the study of the limb-girdle muscular dystrophies (LGMD). The participants were able to discuss a number of exciting developments since the last meeting of the consortium in Soest in January 1992. Four genes for the autosomal recessive forms of L G M D have now been localized [1-4] and there is evidence for at least one other gene causing ARLGMD. A deficiency of the 50-kDa dystrophin associated glycoprotein, adhalin, has been demonstrated in patients with autosomal recessive muscular dystrophy, both as a primary feature (due to mutations in the adhalin gene [4]) and as a secondary event [5]. The main focus of discussion of the meeting was to discuss experience in these areas and to try to make sense of the heterogeneity of this group of diseases, through the collaboration of clinicians, pathologists, protein chemists and molecular biologists. C H R O M O S O M E 15-LINKED M U S C U L A R D Y S T R O P H Y (LGMD2A)

Jacqui Beckmann (Paris, France) reported the positional cloning of a muscle specific gene from the LGMD2A region on chromosome 15q. cDNA capture was used to identify muscule genes from a region chosen as most likely to contain the mutation by the use of linkage Address for correspondece: Dr K. M. D. Bushby, Dept of H u m a n Genetics, University of Newcastle upon Tyne, UK.

WORKSHOPS, 6-8 JANUARY

NAARDEN,

1995

disequilibrium. A range of mutations was identified in different families and some correlation was noted between the different mutations and disease course, with clusters of similarly affected patients sharing similar mutations. Several different mutations have been identified unexpectedly in the families from Reunion Island, inconsistent with there being a single founder effect. This raises the possibility that LGMD2A might not be a s~mple monogenic disorder, but rather a digenic disorder with the production of symptoms being determined by the presence of a second "modifier" gene, unlinked to chromosome 15. In order to address this issue. Dr Beckmann proposed the establishment of a consortium to look for mutations in all chromosome 15-1inked families. Gene Jackson (Detroit, U.S.A.) reported the finding of genetic heterogeneity in the L G M D patients from the Amish population of North America, with linkage to chromosome 15 shown amongst the affected families in the Northern Indiana and excluded in the Southern Indiana pedigrees. Some phenotypic differences have emerged between the linked and unlinked Amish families, with slightly more variability in the age at onset, age at confinement to a wheelchair and age at death in those unlinked to chromosome 15 and the presence of calf hypertrophy and macroglossia in this group. The disease in the Amish chromosome 15-1inked families was consistent with that reported in the Reunion families, who also show chromosome 15-linkage. In two biopsies from Amish families not linked to chromosome 15 adhalin staining of muscle biopsies was abnormal. No real consensus was reached on the clinical phenotype of chromosome 15-1inked families, with data from Mayana Zatz (Sao Paulo, 337

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Brazil), Jim Gilchrist (Providence, U.S.A.) and Fernando Tom6 (Paris, France) all confirming the variability of the phenotype in many respects, both within and between families, though this variability may be better understood once all the mutations are identified. Age at onset in the families presented ranged from 8 to 23 years, with age at loss of mobility also variable, but seen in some patients in the late teens or early twenties. Other parameters, such as the presence of calf hypertrophy, could also vary both within and between families. The urgent need for the publication of good clinical studies of all of the different clinical groups was reiterated. CHROMOSOME 2-LINKED MUSCULAR DYSTROPHY (LGMD2B)

Rumaisa Bashir (Newcastle, U.K.) reported on the progress made on genetic and physical mapping of LGMD2B. The locus has been mapped to chromosome 2p13.3, based on FISH hybridization with YAC clones encompassing the recombinant flanking markers, DS291-5cM-D2S286. An insertional mutant on mouse chr6 (mnd2) [6, 7], is within a region showing conserved synteny with human chromosome 2, and maps to within the contig. A human EST corresponding to a mouse single copy sequence within the non-recombinant area in mouse is under investigation, but the nonrecombinant area in LGMD2B has not yet been fully refined by genetic mapping. Further microsatellites in the area are currently being developed to accomplish this task. An overview of the phenotypic features of families showing definite linkage to chromosome 2p was given by Kate Bushby (Newcastle, U.K.), and more details of individual families were presented by Ibrahim Mahjneh (Florence, Italy), Mayan Zatz and Jim Gilchrist. Age at onset in the chromosome 2p-linked families was usually after 15 yr, and progression in most cases was slow, though some showed more rapid deterioration. On all parameters, overlap with the families linked to chromosome 15 meant that clinical distinction between the two groups would currently be impossible in an individual case. Jim Gilchrist presented preliminary findings from applying discriminant analysis to the different family groups studied, but further data need to be collected before any definite conclusions can be drawn. Ibrahim

Mahjneh described detailed data on patterns of muscle involvement in chromosome 2p-linked families, with CT scanning a valuable tool to distinguish muscle involvement. Using CT scanning, early involvement of the soleus, abductors and the muscles of the posterior compartment of the thigh could be identified. The linkage of Miyoshi myopathy to the same region of chromosome 2p as LGMD2B was noted [8]. Professor Ben Hamida (Tunis, Tunisia) commented that the phenotype of Miyoshi myopathy is truly distinct from LGMD, with onset of muscle weakness in the gastrocnemius and soleus muscles and the first symptoms reported as impairment of standing on tip-toe. All of the reported LGMD2B patients so far have had onset in the pelvic girdle musculature with the first symptoms being difficulty with stairs or impairment of running. Similar to the Myoshi phenotype, however, is the onset most often in the late teens and massive elevation of creatine kinase in LGMD2B. CHROMOSOME 13-LINKED MUSCULAR DYSTROPHY (PREVIOUSLY SCARMDI)

Mongi Ben Hamida pointed out that the Tunisian A R D L M D reported in 1977-1980 and well documented in 1983 is localized on the pericentromeric region of chromosome 13q. There was a marked inter and intrafamilial variability in the age of onset and in the course. The course of the disease was severe in 25% of the patients, mild in 55% and slow in 20%. Muscle biopsies showed dystrophic features similar to those observed in D M D with normal dystrophin. Jeff Vance (Duke, U.S.A.) reported that good progress was being made towards the mapping of the gene on chromosome 13q, with a rare allele showing strong evidence of linkage disequilibrium with the disease. Mayana Zatz presented families with a severe phenotype and one family with milder disease in association with linkage to chromosome 13q and patchy staining for adhalin, demonstrating phenotypic heterogeneity in this group as well. CHROMOSOME 17-LINKED LGMD (PRIMARY ADHALIN DEFICIENCY, PREVIOUSLY SCARMD2)

Jean-Claude Kaplan (Paris, France) showed that chromosome 17q-linked muscular dystro-

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phy has now been identified in patients from all over Europe and North Africa. A variety of different types of mutations in the adhalin gene have now been found, with null mutations on the whole associated with less adhalin staining in immunocytochemistry of muscle samples, lower abundance of adhalin on western blotting and more severe clinical course. Adhalin mutations may, therefore, be associated with a range of adhalin staining patterns in muscle biopsies. Adhalin mutations have been described in families with a broad range of clinical presentations and intrafamilial variability of the phenotype may also be seen. The term SCARMD, including a reference to the severity of the condition is therefore not always accurate as a description of the disease associated with adhalin deficiency or adhalin mutations. This point was reinforced by the data of Rita Passos-Bueno (Sao Paulo, Brazil) who had also observed extreme variability of the phenotype in the chromosome 17-1inked families. Professor Kaplan concluded that it was currently impossible to distinguish the phenotypes, clinical presentation or pathological features of primary and secondary adhalinopathies, except for those cases that show complete absence of adhalin, suggestive of a null mutation in the adhalin gene itself. In familial cases, where linkage analysis is feasible, the type of muscular dystrophy can be distinguished using the adhalin intragenic microsatellite or the markers most closely linked to the chromosome 13 gene. When such analyses are not applicable (small, incomplete families, or sporadic cases) one has to assess adhalin in muscle and search for mutations in the gene if it is abnormal. Further heterogeneity in this group is, however, suggested by the observation in Brazil of families with absent adhalin but linkage to neither chromosome 13 nor 17. Steve Roberds (Iowa, U.S.A.) described the characterisation of adhalin, a transmembrane protein, with two N-linked glycosylation sites. Most of the protein is extracellular. Adhalin m R N A is expressed mostly in skeletal muscle, but is present in heart, and also in lung though this may reflect the presence of smooth muscle. The adhalin gene maps to chromosome 17q21 and has at least 10 exons. It is not yet known whether the chromosome 13 locus, linkage to which is also associated with adhalin deficiency, is a member of the dystrophin-glycoprotein complex that has not yet been cloned, or another

protein that binds to adhalin. Preliminary screening of the adhalin cDNA in the cardiomyopathic hamster (which shows adhalin deficiency in muscle [9], has shown no mutations. THE RELATIVE PROPORTIONS OF THE VARIOUS DEFINED LOCALIZATIONS

The presence of families unlinked to any of the loci yet defined suggests that there is at least one other gene responsible for a recessive LGMD phenotype. The relative proportions of the families showing linkage so far is bound to be biased, as only large families can currently be studied with any certainty. The only population in which any kind of proportions can be defined therefore is amongst the Brazilian population, where a large number of big families with L G M D have been identified. Amongst 15 families studied there with relatively mild disease, approximately 40% mapped to chromosome 2p, 33% to 15q, 13% to 17q and 7% to 13q. One family remained unlinked. These data must, however, be treated as provisional, until small families can be studied in a variety of populations and regional variations in the different types of L G M D determined. Preliminary data suggest that mutations in all of the genes appear to be present in a wide range of populations. EXPERIENCE WITH ADHALIN PROTEIN ANALYSIS

Fernando Tom6 described experience with abnormal adhalin in 66 muscle biopsies (including the two Amish patients from the group unlinked to chromosome 15), 30 from Europe, one from La R6union, 32 from North-Africa and the Middle-East, and one from a mixed European and North African origin. The 35 DAG was also decreased in these patients. The clinical course of these patients was variable. Conversely, Ieke Ginjaar (Leiden, The Netherlands) described her experience of adhalin analysis in 14 patients with a clinical diagnosis of SCARMD (that is severe disease with onset in childhood and rapid progression), and showed that only 30% had abnormal adhalin, suggesting that further genetic causes for this phenotype remain to be defined. In one patient with adhalin deficiency some fibres also showed reduction in dystrophin. Thomas Void (Dusseldorf, Germany) presented a group of

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children with normal adhalin, severe muscle weakness and a number of other features including macroglossia, facial weakness, extreme hyperlordosis, soft and hyperextensible skin and cardiac involvement, who may represent a distinct phenotypic group. Thomas Voit also presented patients of variable clinical severity who did show adhalin deficiency, including a pair of sibs with almost complete adhalin deficiency, a severe clinical course and deafness. This is of particular interest given the localisation of a gene for recessive deafness on chromosome 13q, and highlights the importance of looking for additional features which may not be clinically obvious. Another pair of sibs had partial adhalin deficiency, severe weakness and dilated cardiomyopathy. Preliminary sequencing of the adhalin gene in this family by Jean-Claude Kaplan has so far failed to detect a mutation. Yoshihide Sunada (Iowa, U.S.A.) described a clinically similar patient. The relationship between adhalin deficiency and cardiomyopathy remains to be fully determined, and more intensive investigation of these patients is probably indicated. Caroline Sewry (London, U.K.) presented cases illustrating the difficulty of classification of children with autosomal recessive muscular dystrophy with and without adhalin deficiency. The study of additional pathological features may sometimes help to determine differences between patients, such as fetal myosin levels or utrophin staining, which were found at higher levels in some of the more mildly affected patients. Louise Anderson and Kate Bushby (Newcastle, U.K.) presented data on 50 nonXp21-1inked patients with abnormal adhalin labelling in muscle. The only two with a total adhalin deficiency were from Qatar and Sudan and had a severe phenotype. One also showed patchy dystrophin labelling on sections but dystrophin of normal size and abundance on blots. Amongst the patients with partial adhalin deficiency the phenotype ranged from childhood onset and confinement to a wheelchair before their twenties, to adult onset and slow progression. In addition, a reduction in adhalin labelling was seen in some patients with congenital muscular dystrophy, particularly in those with complete merosin deficiency. This kind of pattern has not been seen by Fernando Tom6 or Caroline Sewry amongst the patients they had investigated with congenital muscular dystrophy. However, the finding of variable patterns of staining of the different components

of the dystrophin glycoprotein complex should lead to caution in interpretation until the whole group of proteins can be fully investigated, and mechanisms of primary and secondary deficiency fully understood. Jay Tischfield (Indianapolis, U.S.A.) suggested that cell fusion studies between 17-1inked and 13-1inked families might help to elucidate such mechanisms. BETHLEM MYOPATHY

Pieter Bolhuis (Amsterdam, The Netherlands) reported linkage of Bethlem myopathy to the telomeric region of chromosome 21q. Marianne de Visser (Amsterdam, The Netherlands) described the distinct phenotype associated with Bethlem myopathy and stressed that it should probably not be included as a true limb-girdle muscular dystrophy. CHROMOSOME 5-LINKED MUSCULAR DYSTROPHY (LGMD1)

Jim Gilchrist reported that the large dominant L G M D family in whom the disease maps to chromosome 5 has been extended, and the localisation refined. There is some evidence for anticipation of age at onset in this family. GENERAL CLINICAL STUDIES OF LGMD

Several careful population-based clinical studies of L G M D were presented by Marianne de Visser, Irena Hausmanowa-Petrusewicz (Warsaw, Poland), Hannu Somer (Helsinki, Finland) and Ibrahim Mahjneh (Florence, Italy), who also presented data illustrating the usefulness of muscle CT scanning in determining the exact pattern of muscle involvement in different types of LGMD. In all of the studies the issue of correct diagnosis and exclusion of all possible alternative diagnoses was highlighted. Berk Bakker (Leiden, The Netherlands) presented the possible usefulness of X-inactivation studies as relatively simple investigations to provide evidence of manifesting carrier status in women with presumed LGMD. CONCLUSIONS

1

Nomenclature

All of the presentations relating to clinical phenotypes highlighted the extreme variability

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Workshop Report Table 1. Geneticcauses of a limb-girdlemuscular dystrophy Gene nomenclature

Chromosomal localization

Protein involvement

Muscles involved

Progression

?

Reported

Usually mild ? anticipation

AD: LGMD1 Other gene(s) for dominant LGMD

5q Unknown

AR:

X-linked:

LGMD2A LGMD2B LGMD2C (previously SCARMD1)

15q 2p 13q

LGMD2D (previously SCARMD2) Other genes for AR LGMD

17q

DMD

? 9

Primary involvement unknown, may have secondary adhalin deficiency Primary adhalin deficiency

May be specific or not to particular localisations

Spectrum of disease severity from severe to mild

unknown Xp21

Dystrophin

Selective,we-Ii--1 documented

The table describes the various genetic causes now established for a muscular dystrophy of limb-girdle distribution. Xp21 linked muscular dystrophy is included for completeness. Information so far collected suggests that the clinical spectrum seen in association with all of the various genes for recessive LGMD will reflect that seen in the Xp21 dystrophins i.e. from early onset with fast progression to late onset, mild disease. in severity which could be associated with any of the identified genetic localizations. This led to particular difficulty with the term S C A R M D (severe childhood autosomal recessive muscular dystrophy) as a clinical diagnosis where severity need not be a part of the disease. Similarly, the gene designations SCARMD1 and S C A R M D 2 for the chromosome 13 and 17 genes become untenable when the diseases linked to these loci may be extremely mild and of adult onset. An extension of the current L G M D nomenclature was therefore suggested, with L G M D 2 C replacing SCARMD1 and L G M D 2 D replacing SCARMD2. These changes and the complete nomenclature for all the genetically determined muscular dystrophies of limb-girdle distribution are presented in Table 1. This amendment was agreed by all the participants at the workshop and will be presented to the G D B nomenclature committee by Jeff Vance. The group agreed that the main reason for implementing change at this stage was to avoid confusion, given the increasingly obvious imprecision of the term SCARMD. The new nomenclature was necessary in the face of the wider than predicted spectrum of disease severity associated with all of the genetic localisations so far identified.

2 Clinical delineation of the various phenotypes While a spectrum of clinical severity undoubtedly exists for each of the L G M D genes, there is still an urgent need for collection of clinical data relating to the families with proven linkage to one of the defined loci. It was agreed that where detailed clinical data were available on the families, these should be published separately as a high priority. In addition, a simple questionnaire will be designed and made available to all clinicians with genetically defined families, so that a set of defining clinical features can be collected about all of these families and stored on database. This questionnaire will be co-ordinated by Kate Bushby.

3 Collection of epiderniological data Estimates currently available for the prevalence of L G M D pre-date any of the molecular characterizations of the group. Clinicians working in areas where full ascertainment is possible will embark on the collection of revised epidemiological statistics. The collection of such data should also go some way to resolving the issue of the relative proportions of

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recessive and dominant disease in the L G M D group, an issue which is crucial for genetic counselling. The experience of the clinicians present at the workshop suggested that dominant L G M D occurs too frequently to be ignored when counselling a sporadic case as to the risk of recurrence. While creatine kinase does tend to be lower and disease progression slower in dominant disease, no clear distinction can be made in an individual case. An empiric risk of 5% to offspring of a sporadic case (assuming that a m a x i m u m of 10% of cases are dominant) would appear to be reasonable, until better figures can be obtained. 4

Consortia f o r genetic investigation

Continued collaboration between the various groups working on the different localizations was emphasised, in particular with the sharing of information to allow mutation analysis as the genes become identified. Co-ordinators for each group were identified. Jacqui Beckmann for L G M D 2 A (Fax No: 010 331 40180155), K a t e Bushby for L G M D 2 B (Fax No: 0191 2227143), Jean-Claude Kaplan for L G M D 2 D (Fax No: 010 331 44411522), Jeff Vance for L G M D 2 C (Fax No: 010 1 919 6846514) and Pieter Bolhuis for Bethlem M y o p a t h y (Fax No: 010 31 20 6971438). Each group will be responsible for the establishment of a database for their own mutation data, but this will also be available to other consortium members. G r o u n d rules for publication by members o f the consortium were laid down. 5

P r o t e i n studies

The different laboratories working on adhalin analysis were encouraged to share antibodies and try to standardize techniques so that results were as comparable as possible. There was general agreement that the collaborations established through the L G M D consortium have been fruitful and should continue, with a further meeting to discuss progress in due course. List of participants

L Anderson (England), B Bakker (The Netherlands), R Bashir (England), J S Beckmann (France), M Ben H a m i d a and C Ben H a m i d a (Tunisia), P Bolhuis (The Netherlands), K Bushby (England), A Emery (ENMC), M Fardeau (France), J Gilchrist

(U.S.A), I Ginjarr (The Netherlands), I Hausmanowa-Petrusewicz (Poland), C Jackson (U.S.A.), M Jeanpierre, J-C Kaplan and F Letureq (France), I Mahjneh and G P Marconi (Italy), M Passos-Bueno (Brazil), S Roberds (U.S.A.), N R R o m e r o (France), C Sewry (England), H Somer (Finland), Y Sunada and J Tischfield (U.S.A.), F Tom6 (France), J A Urtizberea (France, AFM), J Vance (U.S.A.), G - J Van O m m e n and M de Visser (The Netherlands), T Voit (Germany), S Yates (England M D G ) , M Zatz (Brazil). workshop was sponsored by ENMC, with the financial support of: Association Franqaise contre les Myopathies, Italian Telethon Committee, Muscular Dystrophy Group of Great Britain and Northern Ireland, Unione Italiana Lotta Distrofia Musculare, Vereniging Spierziekten Nederland, and other Associate Member Associations and Contributors.

Acknowledgements--This

D R K. M. D. B U S H B Y Dept of H u m a n Genetics University of Newcastle upon Tyne U.K. D R J. S. B E C K M A N N Genethon Evry Paris, France REFERENCES

1. Beckmann J S, Richard I, Hillaire D et al. A gene for limb-girdle muscular dystrophy maps to chromosome 15 by linkage. C R Acad Sci Paris 1991; 312: 141-148. 2, Ben Othmane K, Ben Hamida M, Pericak-Vance M A et al. Linkage of Tunisian autosomal recessive Duchenne-like muscular dystrophy to the pericentromeric region of chromosome 13q. Nature Genet 1992; 2: 315-317. 3. Bashir R, Strachan T, Keers, Set al. A gene for autosomal recessive limb-girdle muscular dystrophy maps to chromosome 2p. Human Molec Genet 1994; 3: 455-457. 4. Roberds S, Leturcq F, Allamand V e t al. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 1994; 78: 625-633. 5. AzibiK, Bachner L, Beckmann J Set al. Severe childhood autosomal recessive muscular dystrophy with the deficiency of the 50kDA dystrophin-associated glycoprotein maps to chromosome 13q. Human Molec Genet 1993; 2: 1423-1428. 6. Jones J M, Albin R L, Feldman E Let al. mnd 2: a new mouse model of inherited motor neuron disease. Genomics 1993; 16: 669-677. 7. Barrow L L, Simin K, Jones J M, Lee D C, and Meisler M H. Conserved linkage of early growth reponse 4, annexin 4, and transforming growth factor alpha on mouse chromosome 6. Genomics 1994; 19: 388-390. 8. Bejaoui K, Hirabayashi K, Hentati F et al. Linkage of Miyoshi Myopathy (distal autosomal recessive

Workshop Report muscular dystrophy) locus to chromosome 2p 12-14, Neurology (in press). 9. Yamanouchi Y, Mizuno Y, Yamamoto H et al. Selective defects in dystrophin-associated glycoproteins

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50DAG (A2) and 35 DAG (A4) in the dystrophic hamster: an animal model for severe childhood autosomal recessive muscular dystrophy (SCARMD). Neuromusc Disord 1994; 4: 49-54.