Muscular dystrophy due to dysferlin deficiency in Libyan Jews

Brain (2000), 123, 1229–1237

Muscular dystrophy due to dysferlin deficiency in Libyan Jews Clinical and genetic features Zohar Argov,1 Menachem Sadeh,4 Kineret Mazor,1 Dov Soffer,2 Esther Kahana,5 Iris Eisenberg,3 Stella Mitrani-Rosenbaum,3 Isabelle Richard,6 Jacques Beckmann,6 Sharon Keers,7 Rumaisa Bashir,7 Kate Bushby7 and Hanna Rosenmann1 Departments of 1Neurology, 2Pathology and 3Development of Molecular Biology and Genetic Engineering, Hadassah University Hospital and the Hebrew University, Hadassah Medical School, Jerusalem, 4Department of Neurology, Wulfson Hospital, Holon, 5Department of Neurology Barzilai Hospital, Ashkelon, Israel, 6URA 1922, Genethon, Evry, France and 7Department of Biochemistry and Genetics, University of Newcastle upon Tyne, UK

Correspondence to: Zohar Argov, MD, Department of Neurology, Hadassah University Hospital, Jerusalem 91120, Israel

Summary The cluster in Jews of Libyan origin of limb-girdle muscular dystrophy type 2B due to a dysferlin 1624delG mutation is described. The carrier frequency of this mutation is calculated to be ~10% in this population, in which the disease prevalence is at least 1 per 1300 adults. Twenty-nine patients from 12 families were all homozygous for the same mutation. However, clinical features were heterogeneous even within the same family: in half of the patients onset was in the distal muscles of the legs, which is similar to Miyoshi myopathy, while in others onset was in the proximal

musculature, which is similar to other forms of limbgirdle dystrophies. Age at onset varied from 12 to 28 years (mean 20.3 ⍨ 5.5 years). One patient was presymptomatic at age 28 years. Progression was slow regardless of age of onset, patients remaining ambulatory until at least 33 years. Five patients described subacute, painful enlarged calves as an early, unusual feature. The variable features in this ethnic cluster contribute to the definition of the clinical spectrum of dysferlinopathies in general. The cause of the observed heterogeneity remains unclear.

Keywords: muscular dystrophy; distal myopathy; dysferlin; muscle hypertrophy Abbreviations: CK ⫽ creatine kinase; LGMD ⫽ limb girdle muscular dystrophy; PCR ⫽ polymerase chain reaction

Introduction The muscular dystrophies are a group of genetically determined, primary degenerative myopathies. To date at least eight forms of recessively inherited muscular dystrophies, affecting mainly the limbs and the limb-girdle musculature, have been defined genetically (Bushby, 1999). They share the features of progressive muscular weakness of the limbs and limb girdles, very high levels of serum creatine kinase (CK), and histological features of necrotizing myopathy with regeneration and connective tissue infiltration without specific ultrastructural abnormalities (Bushby, 1995, 1999). Although these disorders are still grouped under the general term of limb-girdle muscular dystrophy type 2 (LGMD2) (Bushby and Beckmann, 1995), various genetically defined subtypes can now be identified within this group (subtypes LGMD2A–H). © Oxford University Press 2000

The distal myopathies are also a very heterogeneous group of myopathic disorders, and are characterized by the predominant involvement of the distal muscles of the limbs (Griggs and Markesbery, 1994). While most of these disorders are recognized as myopathies with specific structural abnormalities and mild elevation of serum CK, one of the forms (Miyoshi myopathy) shows histological features compatible with muscular dystrophy (Miyoshi et al., 1986). In this myopathy, the gastrocnemius is the most frequently and the most severely affected muscle, especially in the initial phases. LGMD2B was first defined as an entity in 1994 by linking the muscular dystrophy in two families of different ethnic origin to a site on chromosome 2p13 (Bashir et al., 1994). The clinical features of LGMD2B, as reported for five families

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(Mahjneh et al., 1996), varied in severity and onset was in the late teens. Weakness at onset was usually found in the proximal musculature, though CT examination of the gastrocnemius showed subclinical involvement in some patients. Later, the distal Miyoshi myopathy was linked to the same site on chromosome 2p13 (Bejaoui et al., 1995). Studies in two large kindreds showed the co-existence of LGMD2B and of Miyoshi myopathy phenotypes in the same family (Illarioshkin et al., 1996; Weiler et al., 1996). The gene itself (dysferlin) was identified recently and found to be mutated both in families with LGMD2B (Bashir et al., 1998) and in families with Miyoshi myopathy (Liu et al., 1998). Ethnic clusters of autosomal recessive LGMD have been reported previously. Calpainopathy (LGMD2A), for example, is seen at a high frequency among the Amish and Basque populations and on the island of Re´ union (Fardeau et al., 1996; Bushby, 1999). We found recently a cluster of LGMD2B among Libyan Jews originating in the area of Tripoli (Argov et al., 1998). The identification of this cluster was important for the characterization of the dysferlin gene, as one of the first mutations identified in this gene was in this population (Bashir et al., 1998). The clinical features, including those which seem to be unique to the ‘dysferlinopathies’, and the high carrier frequency in Libyan Jews, with a clear founder effect, are reported in this paper. We show that, even among patients of the same small ethnic community who are homozygous for an identical, recessively inherited point mutation, the clinical features can be very variable.

Methods Patient group Twelve Libyan Jewish families with one or more members affected by muscular dystrophy are included in this report. Twenty-nine patients of these families agreed to be tested for this study. In four of these families, 17 additional subjects with a similar muscle disease are known (five are dead) but none of the 12 living members gave consent to be tested. In three pairs of families, various degrees of distant relationship were established. In 10 families, at least one subject (not necessarily one of those who were tested in the current study) had a muscle biopsy that confirmed the diagnosis of muscular dystrophy or myopathy. Most patients had serum CK measurements available and many had had an EMG at some stage of the disease. Information about these tests was obtained by reviewing their medical records. A presymptomatic patient is also included in this report (see below). All patients were re-examined during the period 1997–98 and the detailed history of the onset and progression of the disease was taken again. The clinical examination performed for this report (for many patients it was done at home) included detailed testing of muscle power [five grades, according to the Medical Research Council (MRC) grading method] and evaluation of functional status according to the scale of Walton and Gardner-Medwin (Walton and Garder-

Medwin, 1981). At the age of onset of symptoms, the patient is defined as being at functional grade I (Fardeau et al., 1996). For some patients the definitions in the above system placed them between grades. To obtain a measure of the rate of disease progression, the change in grade over time in years from onset to the time of examination for this study was assessed. This rate of progression, expressed in grades per year, was calculated for each patient individually. Averages for each group were taken from these values. It should be remembered that the functional scale is not linear, so the calculated figure is only a general estimate. All participants in the study gave informed consent. The study was approved by the Helsinki Committee of Hadassah University Hospital.

Mutation detection DNA was prepared from leucocytes using conventional methods and amplified by the PCR (polymerase chain reaction) using primers described previously (Bashir et al., 1998). The PCR was performed under the following conditions: 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min and 72°C for 10 min, to yield a fragment of 178 base pairs. Samples of amplified DNA were analysed by single-strand conformation polymorphism on a non-denaturing MDETE gel (mutation detection enhancement; FMC Bioproducts, Rockland, Me., USA) under conditions described previously (Bashir et al., 1998). In samples showing mobility shifts, the presence of the mutation was determined by restriction enzyme analysis using BmyI to cut the wild-type allele and either AvaII or Sau96I to cut the mutant allele (1624delG). In addition to the previously reported nine families who carried a similar dysferlin mutation (Bashir et al., 1998), we tested five newly identified Libyan Jewish patients with muscular dystrophy from three families and the presymptomatic subject for the presence of the mutation by restriction enzyme analysis. To assess the mutation carrier frequency in this community, DNA samples from 82 unrelated Libyan Jews (stored in our DNA bank for genetic screening of other diseases in this population) were also tested for the presence of the 1624delG mutation. The 95% confidence interval was calculated for the carrier frequency.

Results Representative case reports Family LGL-2 (family II) In this consanguineous Libyan Jewish family, nine patients had a myopathy but only six were studied (Fig. 1). This family was the first of this ethnic cluster to be genotyped as LGMD2B, and all patients carry the same point mutation. The following two patients of this family demonstrate the different modes of clinical presentation of LGMD2B in our cluster.

Dysferlinopathy in Libyan Jews

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Fig. 1 Family tree of the first genotyped LGMD2B Libyan Jewish kindred (LGL-2 or family II). Note that the exact relationship between subjects I-3 and I-5 cannot be established today.

Patient III-10 (D.A.). A 38-year-old man had noticed difficulty climbing stairs since the age of 26 years. When examined at that age, weakness in the proximal musculature of the lower limbs had been found (affecting the quadriceps, iliopsoas and hamstrings to similar degrees). Serum CK was ~3000 international units (IU)/l in repeated tests (normal value for males is ⬍200 IU/l). His first muscle biopsy was not conclusive, but two subsequent biopsies showed evidence of a necrotizing myopathy with endomysial fibrosis without inflammation, confirming the diagnosis of muscular dystrophy. His weakness progressed slowly and upper limb involvement was detected by detailed testing at the age of 36 years, although the patient himself did not report it. At that age, there was clear biceps weakness (–4/5 on the MRC scale) and mild scapular muscle wasting without weakness in any of the other muscles of the upper limbs. In the lower limbs the iliopsoas, quadriceps, gluteus and hamstrings were 3/5, plantar flexion was 2/5 and dorsiflexion was mildly weak (–5/5). The patient is still ambulant and operates motorized agricultural equipment. His functional score on the Walton and Garder-Medwin (1981) scale was 3. This patient represents the typical proximal type of presentation in our cluster. Patient III-2 (G.M.). This 30-year-old woman was first evaluated by us at the age of 16 years. At age 15 years she had noted a painful swelling of her left calf. She described a dragging of her left leg during walking, could not stand on tiptoes and had an episode of subluxation of the left ankle during one of her frequent stumbles and falls. When she was evaluated at that age, only weakness of the left gastrocnemius was reported. As pain and weakness slowly progressed and a serum CK level of 4800 IU/l (normal value for females is ⬍170 IU/l) was found, she was referred for muscle biopsy. She was not aware then of any other affected members in the family, and a diagnosis of inflammatory myopathy was suggested. At age 16 years, weakness was limited to the

lower limbs: plantar flexion was 2/5 (MRC scale) on the left and 4/5 on the right, and dorsiflexion was 5/5 on the right and –5/5 on the left. There was also very mild weakness (–5/5) of the proximal musculature of the left leg and some pain in the calf muscles, which was greater on the right. EMG showed numerous polyphasic units of high amplitude and reduced recruitment in the gastrocnemii. Right gastrocnemius biopsy showed an active necrotizing myopathy without inflammation (Fig. 2). A short course of steroids (prednisolone 40 mg/day for 3 months) failed to improve her condition. The disease has progressed slowly, and at age 30 years the distribution of weakness is very similar to that of patient III-10: biceps only in the upper limbs (4/5) and weakness of all muscles of the lower limbs (2–3/5). Her functional score is also similar to that of her distant cousin, D.A. Serum CK at age 29 years was 2300 IU/l. This patient exemplifies the distal type of onset observed in our patients, with its unusual subacute, painful weakness and swelling of the calves.

Family XII Only one subject was tested in this family. Y.M. is a 28year-old woman who was referred recently for evaluation of persistent high serum CK (3900–5300 IU/l). At age 26 years she gave birth to her first child. For a few months after delivery she experienced general fatigue without myalgia or noticeable weakness, which gradually disappeared. She denies any current neuromuscular symptoms and describes herself as completely healthy. She was found to have cystinuria, a common disorder in Libyan Jews (Bonne-Tamir and Adam, 1992). At age 28 years she has no signs of weakness or muscle wasting on detailed testing. CT of the leg muscles showed areas of atrophy, which was pronounced mainly in the calves and less so in the vastus lateralis. Because of her Libyan Jewish origin she was tested for the 1624delG

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Fig. 2 Gastrocnemius muscle biopsy of patient III-2 of family LGL-2, taken in the initial phase of painful calf hypertrophy. Note myofibre necrosis and phagocytosis, mild endomysial fibrosis but no inflammation. Haematoxylin and eosin, ⫻200.

dysferlin mutation and found to be homozygous. This patient is our only known presymptomatic subject.

Summary of clinical features (Table 1) Mean age at onset was 20.3 ⫾ 5.5 years (range 12–28 years). There was no significant difference between those with distal onset (mean 19.7 years) and those with proximal onset (mean 20.3 years). Age at onset can vary by about a decade in the same family (e.g. families I and II). It should be noted that, even in retrospect, patients reported good physical condition before the onset of disease. Those with onset in the early teens had completely normal milestones and were able to participate normally in physical activity at school. Those with onset in the third decade completed mandatory Israeli military service (2.5–3 years) without noticeable limitations. In all patients, weakness was noted initially in the legs. In half of the patients, initial symptoms were related to distal weakness: inability to stand on tiptoes (13 patients), instability of the ankles with episodes of subluxation (three patients), difficulty walking downstairs only (seven patients) and drop feet (one patient). An unusual feature was transient painful calf swelling, similar to that described by patient G.M., which was reported by five patients (four females) from two families. This was symmetrical in three and asymmetrical in two patients, lasted for a few months to ~1 year, and was followed by weakness and wasting in the same muscles. Two patients had a calf muscle biopsy during this phase which showed florid non-inflammatory necrotizing myopathy, as shown in Fig. 2. Three of these patients received a short course of high-dose steroids without any change in symptoms. These patients constitute the subgroup with distal onset. In the other patients, early symptoms were related to proximal leg musculature (mainly difficulty climbing stairs, rising from chairs and laboured gait) and many of them showed no

clinical evidence of distal muscle weakness initially. This subgroup represents the proximal type of onset in our cluster. In six families the two different modes of onset were noted, even within the same sibship (Table 1). Information about upper limb involvement comes from 22 patients since, in the remaining patients, weakness is still limited to the legs. The first symptoms of upper-limb involvement are usually related to biceps weakness, and the few patients tested at this intermediate stage did have marked weakness of this muscle with minimal or no weakness in any other upper limb or shoulder-girdle muscle, as in patient D.A. The upper limb involvement was noted by the patients between 1 and 16 years after the onset of gait difficulty, at a mean age of 28.5 years. Although the mean age at which patients reported upper-limb involvement was later in the distal onset group than in the proximal onset group (31 versus 26 years), the difference was not statistically significant. Patients underwent detailed muscle testing for this study between 1 and 29 years after the onset of the disease, and at different stages of their disease. Thus, only a few general comments about the relative muscle involvement at different stages can be made. (i) In the patients with distal onset, the gastrocnemius was always weaker than the tibialis anterior, which is similar to observations made in Miyoshi myopathy. However, this difference gradually disappeared as the disease progressed and at advanced stages all distal leg muscles were similarly very weak. (ii) Among the proximal muscles involved at onset, the quadriceps was always the weakest muscle, but the iliopsoas and hamstrings were also involved at an early stage. Again, these differences gradually disappeared as the patients became more disabled. (iii) In the advanced stages, there was no differential weakness in the proximal muscles of the upper limbs (deltoid, biceps, triceps) despite a previously transient phase of isolated biceps weakness that might have lasted for a few years. The shoulder


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Table 1 Clinical features of 29 Libyan Jewish patients with dysferlinopathy Family, patient

Age, gender

Onset at onset (years)

Onset mode

Age at upper limb onset (years)

Functional grade

Family I I-1 I-2 I-3 I-4 I-5

58, 57, 46, 42, 41,

20 18 17 26 28

Distal* Proximal Distal* Distal* Distal*

42 30 ? 32 39

VII VII V–VI V IV

F F F F F

Family II II-1 II-2 II-3 II-4 II-5 II-6

44, 42, 36, 31, 29, 27,

F M M M F M

25 23 26 25 16 16

Distal Proximal Proximal Proximal Distal* Proximal

36 35 33 NP 25 20

VI II–III III II–III III IV

Family III III-1 III-2 III-3

38, F 35, M 27, M

12 16 16

Proximal Distal Distal

21 25 25

VIII–IX IV–V III–IV

Family IV

Comments

One additional affected WCB at 36 WCB at 40 Biopsy: dystrophy Biopsy: dystrophy

Three additional patients, one dead Biopsy: dystrophy Biopsy: dysferlin deficiency

Six more patients, three dead Biopsy: dystrophy WCB at 37 years

Two patients died Related to family III Biopsy: myopathy

IV-1

19, F

12

Distal

NP

III

Family V V-1

54, F

18

Distal

28

VIII

V-2

47, M

17

Proximal

22

VIII

Family VI VI-1 VI-2

39, M 38, F

20 21

Proximal Proximal

30 35

VI VI

Biopsy: dystrophy

Family VII VII-1

32, F

16

Distal

26

VII

WCB at 33 years

Family VIII VIII-1 VIII-2 VIII-3

35, F 32, F 30, F

15 18 18

Distal Proximal Proximal

25 25 19

VI V V

Biopsy: dystrophy

Family IX IX-1 IX-2 IX-3

44, F 37, F 23, F

26 25 22

Distal Distal Proximal

42 NP NP

III II II

Family X X-1

26, M

18

Proximal

21

III

Related to family XI Biopsy: dystrophy

Family XI XI-1

29, F

18

Proximal

28

IV

Two additional affected sibs Biopsy: dysferlin deficiency

Family XII XII-1

28, F

WCB at 33 years. Biopsy: dystrophy WCB at 36 years Related to family IX

Distal* ⫽ transient calf hypertrophy; WCB ⫽ wheelchair-bound; NP ⫽ not present.

Three additional patients Biopsy: dystrophy

High CK only

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Fig. 3 Change in functional status (Walton and Gardner-Medwin scale) between onset and examination in the present study. Onset is defined as grade I. (A) Fourteen patients with distal onset. (B) Fourteen patients with proximal onset. There was no apparent difference in disease onset and progression rate between patients with distal onset and those with proximal onset.

girdle musculature was always less involved and scapular winging was not a feature in this cluster. The distal muscles of the upper limbs were usually spared until a very advanced stage of the disease. (iv) Once the disease had progressed from one muscle group to another (i.e. from distal onset to proximal involvement or vice versa) it was impossible to find a difference in the pattern of weakness. The progression of the disease was slow (Fig. 3). There was no apparent difference between the subgroups with distal (Fig. 3A) and proximal (Fig. 3B) onset with respect to the rate of progression. The means of the progression rates were similar in the distal and proximal subgroups (0.21 and 0.24 grades per year, respectively). Six patients were wheelchairbound; three of them had distal and three had proximal onset (Table 1). The age at which the subjects lost the ability to walk was 艌33 years (in at least one of those who became wheelchair-bound at 33 years, it was related to a traumatic cause). In all patients, serum CK levels were 10–30 times the upper limit of normal on repeated tests throughout the early years of the disease. CK levels were not tested regularly afterwards, but they remained very high even after 15 years of disease. On the other hand, results obtained in patient XII (our only presymptomatic subject) suggest that serum CK can be high for a few years before the onset of weakness. EMG in all tested patients showed no evidence of spontaneous activity, even in the distal muscles involved at an early stage. Motor units were usually of small amplitude and short duration, which is compatible with a myopathic pattern. Quantitative EMG measurements were not made.

Molecular genetics An identical mutation in the dysferlin gene, a single G base deletion at codon 1624 (1624delG), was found in all 12 families. This mutation is predicted to cause a premature stop codon 9 base pairs downstream (Bashir et al., 1998).

Fig. 4 Single-strand conformation polymorphism analysis of Libyan Jewish subjects. Homozygous patients with the 1624delG mutation in the dysferlin gene (lanes 2 and 4) show a pattern different from that of healthy unrelated subjects (lanes 5 and 6); heterozygous carriers present a combined pattern (lanes 1 and 3).

All patients were homozygous for this mutation, including the presymptomatic subject Y.M. Figure 4 shows a single-strand conformation polymorphism profile of homozygous patients, heterozygous carriers and normal individuals of Libyan Jewish ancestry. Determination of the genotype by restriction enzyme analysis is shown in Fig. 5. When this modified screening test was applied to samples from 82 unrelated Libyan Jews, eight out of 164 alleles had the common 1624delG dysferlin mutation. This suggests a carrier frequency of 9.75% in this population (95% confidence interval 4.6–18.8%).

Discussion A high frequency of muscular dystrophy in Libyan Jews was reported briefly in 1991 (Fried, 1991). This was, however, before the introduction of the modern genetic classification of LGMD. It is possible that, at that stage, this inbred community harboured either multiple forms of recessive LGMD (similar to the situation in the Amish community, which has a cluster of both LGMD2A and LGMD2E) or a common type with high prevalence. The identification of this Libyan Jewish cluster contributed to the identification of dysferlin as the gene for LGMD2B (Bashir et al., 1998). The detection of the 1624delG mutation in this ethnic cluster


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Fig. 5 Mutation analysis of 1624delG. (A) Digestion of the PCR product by BmyI digestion of wild-type DNA yielded fragments of 98 and 80 base pairs (bp) (lanes 2 and 6–8), whereas the mutant DNA remained intact as a 177 bp fragment (lane 3). All three fragments were detected in heterozygous individuals (lanes 1, 4 and 5). (B) Digestion of PCR product by AvaII or Sau96I. Digestion of the mutant DNA yielded fragments of 99 and 78 bp (lane 1), whereas the wild-type DNA remained intact as a 178 bp fragment (lanes 3–6). All three fragments were detected in heterozygous individuals (lanes 2 and 7–8).

allowed us to evaluate further the features of this type of muscular dystrophy, giving us the opportunity to examine a cohort of genetically identical patients and to evaluate intraallelic variability. The observations in this ethnic group enlarge the known clinical (phenotypic) spectrum of the dysferlinopathies. The Libyan Jewish community has been clustered around the city of Tripoli since at least the third century AD, probably after the destruction of the second Jewish Temple. Jews expelled from Spain in the 14th and 15th centuries are a second, large source of this community (Meiner et al., 1997). It is difficult to differentiate clearly between the two sources, and we do not have any information about a common ancestral origin for the 12 families described here. In Israel today there are ~55 000 adults (over the age of 15 years) with both parents of Libyan origin (based on the last population survey of Israel, in 1996). This community, which practised endogamy until very recent times, is affected by several genetic diseases. The best known is Creutzfeldt– Jakob disease, linked to codon 200 in the prion protein gene (Meiner et al., 1997), but high frequencies of cystinuria and familial Mediterranean fever have also been recorded (BonneTamir and Adam, 1992). So far we are aware of at least 41 living patients with dysferlinopathy in this community (our 29 definite patients plus 12 additional close relatives with chronic myopathy who declined testing). In addition, a few more families with a similar disorder have been evaluated by us in the past but they were not available for this study. Thus, the prevalence of muscular dystrophy due to a dysferlin mutation in the Libyan Jewish community of Israel is estimated to be very high: at least 1 in 1300 adults (41/ 55 000) and probably even higher. Our data show a carrier frequency for the common 1624delG mutation close to 10% in this community. This very high carrier rate suggests that determination of the carrier status for the dysferlin mutation 1624delG should be an essential part of genetic counselling in the Libyan Jewish community. This is the first ethnic cluster of dysferlin-deficient muscular dystrophy that has been identified, and there is probably a strong founder effect. Only a few reports of the clinical features of LGMD2B are available. Mahjneh and colleagues described five families of different ethnic background (Mahjneh et al., 1996). There was no weakness in the distal muscles initially, although the

gastrocnemii were abnormal by CT scan in the early stages. Little intrafamilial variability was found, and the observed interfamilial variability could have been the result of different mutations in the (at that stage unknown) gene. Weiler and colleagues described a Canadian aboriginal kindred, with nine patients, linked to chromosome 2p13 (Weiler et al., 1996). Seven patients had onset of weakness in the proximal muscles of the lower limbs in their early to late teens, and all also had distal weakness when examined later. All but one were wheelchair-bound by age 25–42 years. Two patients manifested primarily distal weakness in their mid-teens and were still walking at age 24–25 years. The authors suggest that patients with distal onset have a slower progression, although this was not proved conclusively because of the short follow-up period. The large Avar family from the province of Caucasus, reported by Illarioshkin and colleagues is even more heterogeneous (Illarioshkin et al., 1996). There are 12 affected individuals in this family and two are presumed to have Duchenne muscular dystrophy. Of the remaining 10, seven had proximal onset at age 15–30 years and became wheelchair-bound at age 38–45 years, and three patients had distal onset at 15–21 years and were ambulatory at age 23–24 years. Again, a different rate of progression cannot be ascertained for the two subgroups, since patients with distal onset were much younger at the age of evaluation than the patients with proximal onset. Both reports suggest that the same gene defect can be associated with variable presentation, and in the Canadian kindred this has now been confirmed (Weiler et al., 1999). One of the families reported by Liu and colleagues also had a variable phenotype with an identical dysferlin mutation (Liu et al., 1998). The finding of an ethnic cluster of dysferlinopathy, as described here, provides an opportunity to better define the clinical features of LGMD2B in a genetically homogeneous population. With such a genetic background, one would expect a priori the resulting disease to be clinically uniform. However, our patients showed marked inter- and intrafamilial heterogeneity. Distal onset (resembling Miyoshi myopathy) was observed in half the patients, while in the others onset was in the proximal lower-limb musculature. It should be emphasized that the distal and proximal phenotypes are based on clinical symptoms and signs and it is possible that subclinical involvement of other muscles could be detected

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at onset by sensitive methods such as CT. Our report describes additional families in which both distal and proximal onset was found in sibs with an identical mutation. Age at onset also varied from 12 to 28 years. The rate of progression was relatively slow and we could not detect any statistically significant difference in progression rate between patients with distal and proximal onset. Patients retained walking ability until at least the middle of the fourth decade of life, when some became wheelchair-bound. The gastrocnemius was always involved as the disease progressed (it also showed changes on CT in a presymptomatic patient), but in the advanced stages all muscles of the lower limbs became severely atrophic and weak. A hitherto unreported feature of dysferlinopathy is presentation with transient painful calf hypertrophy, which was seen in five of 14 patients with distal onset, most of them women. Such a phenomenon in a single patient raises the diagnostic possibility of an inflammatory myopathy, even if the patient has other family members with known muscular dystrophy. Another possibility is a transient inflammatory phase, similar to that reported for facioscapulohumeral dystrophy (Munsat, 1994). Although there are other diagnostic possibilities, this one carries important therapeutic implications. The histological features, as seen in gastrocnemius biopsies performed during this phase, and the lack of response to short-term steroid therapy given to three such patients do not support the possibility that inflammation is of major pathogenic importance. It should be noted, however, that in muscle biopsies of the patients with distal onset in the study of Illarioshkin and colleagues inflammatory cell infiltrates were seen (Illarioshkin et al., 1996), and they were also seen in a large Jewish Yemenite family with a new dysferlin mutation (E. McNally and Z. Argov, unpublished results). The reason for the clinical heterogeneity that we describe in subjects homozygous for the same point mutation is not clear. One possibility is the presence of modifying factors, which may be genetically determined, related to the function of dysferlin in muscle fibres (Weiler et al., 1999). However, it is difficult to hypothesize how such modifier genes could operate differently in the same family belonging to an inbred community. Other modifying factors might be related to behavioural differences, such as the level of physical activity and even environmental factors. We could not statistically test the relationship between physical activity during the presymptomatic period and disease onset, as the information available was very subjective. There were, however, no clear behavioural differences in these patients (before or after disease onset) and most of the patients of a single family reside in the same village, which also excludes any clear environmental factors. Interestingly, four of the five patients with transient calf hypertrophy were women, but whether gender is a contributing factor cannot be established statistically at this point. The diagnosis of dysferlin-related muscular dystrophy should be considered in any patient with a muscular dystrophy syndrome (progressive weakness, very high serum CK and

necrotizing myopathy on muscle biopsy). Clues to the diagnosis of dysferlinopathy in general are: (i) late teenage or adult onset (physical activity before onset is normal, even in retrospect); (ii) weakness starting in the lower limbs only; (iii) mixed proximal and distal muscle weakness at onset; (iv) distal (gastrocnemius) weakness with transient painful calves; (v) lower limb weakness with isolated biceps weakness; (vi) slow progression without loss of walking ability before age 30 years; and (vii) variable clinical features in the same family. If a recessively inherited trait appears in a large kindred suitable for linkage analysis, the diagnosis of this type of muscular dystrophy can be sought by this method. However, in a single patient or in a small family this is not readily feasible, as the scope of mutations has not yet been determined and the gene is relatively large for a practical approach to mutation searching. Immunohistochemistry for dysferlin may be a very useful diagnostic method in such patients (Anderson et al., 1999). The clinical features of dysferlinopathy are different from the common features of other frequent forms of the socalled limb-girdle muscular dystrophies. The age of onset of calpainopathies (LGMD2A) is usually slightly earlier (end of the first decade to early in the second), but the range of age at onset that has been recorded is wide and could overlap with that for the dysferlinopathies. However, in the calpainopathies (LGMD2A), involvement of the upper limb and shoulder girdle is usually present at onset and distal involvement is rare at this stage. Also, the pattern of upper limb involvement in the calpainopathies is markedly different from that in the dysferlinopathies, as in the latter the shoulder girdle musculature is much less affected and no winging of the scapulae is seen. Most cases of sarcoglycanopathy (LGMD2C–F) have their onset in the first decade of life, a feature not seen in any of our families, although again adult onset has been recorded. Interestingly, patients with these disorders describe some early difficulty in keeping up with their friends during physical activity, unlike in the dysferlinopathies, when even in retrospect patients report good physical performance before the onset of symptoms. Lower-limb involvement in these disorders is usually manifested in the proximal musculature, distal weakness at the early stage is rare, and calf hypertrophy is common. In such patients, when upper-limb involvement is present, scapular winging is common. However, it should be recognized that, with the increased use of molecular genetics and immunohistochemical stains in muscular dystrophy diagnosis, the clinical spectrum of each genetically defined type of muscular dystrophy may yet broaden and phenotypic characteristics may become less distinct. While we want to avoid discussion of terminology, it seems that the current grouping of ‘limb-girdle’ dystrophies is evolving progressively to a classification based on the defective protein.

Acknowledgements We thank the patients and their families for their cooperation. This study was supported by the Hilda Katz Blaustein

Dysferlinopathy in Libyan Jews Fund, the Lena P. Harvey Endowment Fund for neurological research in the Department of Neurology, Hadassah University Hospital, Jerusalem, Israel, a special donation to Z.A. for research into hereditary myopathies in Jews in memory of Nataly Hollo-Bencze, and the Muscular Dystrophy Group and the Medical Research Council of Great Britain (Department of Biochemistry and Genetics, University of Newcastle upon Tyne).

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