Variable clinical phenotype in merosin-deficient congenital muscular dystrophy associated with differential immunolabelling of two fragments of the laminin α2 chain

PERGAMON

Neuromuscular Disorders 7 (1997) 169-175

Variable clinical phenotype in merosin-deficient congenital muscular dystrophy associated with differential immunolabelling of two fragments of the laminin or2 chain C.A. Sewry a'b'*, I. Naom a, M. D'Alessandro a, L. Sorokin c, S. Bruno a, L.A. Wilson a, V. Dubowitz a, F. M u n t o n i a aNeuromuscular Unit, Department of Paediatrics and Neonatal Medicine and bMuscle Cell Biology Group, MRC Clinical Sciences (.'entre, Royal Postgraduate Medical School Hammersmith Hospital Du Cane Road, London W12 ONN, UK Clnstitute of Experimental Medicine, Connective Tissue Research, University of Erlangen-Nuremberg, Schwabachanlage 10, D-91054 Erlangen, Germany Received 10 October 1996; received in revised form 12 December 1996; accepted 23 December 1996

Abstract Approximately half the cases of classical congenital muscular dystrophy (CMD) have a pronounced deficiency or absence of the laminin c~2 chain of laminin-2 (merosin). This is caused by mutations in the LAMA2 gene that codes for laminin c~2, and all informative cases so far studied show linkage to the appropriate region on chromosome 6q. Most CMD patients with a deficiency of laminin c~2 have a severe phenotype that involves skeletal muscle, and the central and peripheral nervous system. We have identified four cases that have minimal reduction of laminin c~2 using a commercial antibody that only recognises a C-terminal 80 kDa fragment, but show a pronounced reduction using an antibody to the 300 kDa fragment. Haplotype analysis is compatible with linkage to the LAMA2 locus in three informative families, whilst the fourth family was not informative. Two of the affected children are ambulant and have a mild phenotype. The third case is unusual in having severe muscle weakness but does not show the white matter changes on magnetic resonance imaging of the brain that is usually seen in merosin-deficient cases of CMD; the fourth case has a severe phenotype, typical of merosin-deficient patients but shows good immunolabelling of the 80 kDa fragment of laminin c¢2, corresponding to the C-terminal region. Our data show that there is a broad spectrum of phenotype and protein expression associated with a primary deficiency in laminin a2, and that a wider range of clinical cases need to be screened for a deficiency of merosin. It is also important to study the expression of laminin ~2 with more than one antibody. © 1997 Elsevier Science B.V.

Keywords: Congenital muscular dystrophy; Laminin; Laminin c~2; Merosin

I. Introduction Congenital muscular dystrophies (CMD's) are a heterogeneous group of autosomal recessive disorders characterised by muscle weakness and hypotonia at birth, or within the first few weeks of life [1]. Muscle biopsies show dystrophic features with abnormal variation in fibre size, a variable degree of connective tissue, and adipose tissue proliferation [2]. The congenital dystrophies have recently been classified according to the involvement of the brain and the eyes and it is apparent that defects in * Corresponding author. Tel.: +44 181 3833148; fax: +44 181 7462187; e-maih csewry @rpms.ac.uk

0960-8966/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PI1 S 0 9 6 0 - 8 9 6 6 ( 9 7 ) 0 0 4 2 5 - 2

different genes are responsible for each type [1,3]. A deficiency of the o~2 chain of laminin 2 (merosin) occurs in about 4 0 - 5 0 % of cases with the classical form of CMD [4,5]. Linkage data, and the identification of mutations, have demonstrated that this is a primary phenomenon, caused by defects in the gene for laminin a 2 (LAMA2) on chromosome 6q22 [6-10]. Patients with an absence of laminin oz2 have a severe phenotype. They rarely achieve independent ambulation, creatine kinase activity is elevated, magnetic resonance imaging (MRI) of the brain shows white matter changes, visual evoked responses are abnormal, nerve conduction velocities are reduced and impaired cardiac function sometimes occurs [11-14]. Some patients with this severe phe-

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notype show traces of laminin o~2 on a small proportion of fibres, in contrast to a complete absence [5], but all these cases, when informative, have been shown to link to chromosome 6q2 [10]. More recently it has become apparent that milder cases of CMD and atypical cases are also caused by primary abnormalities in the laminin or2 chain [15,16] and that partial expression of the laminin or2 chain occurs [8,15]. LAMA2 codes for a protein of 390 kDa which is synthesised as one chain but processed into two fragments which on immunoblots have molecular masses of approximately 300 and 80 kDa [17]. Many patients have been assessed for merosin status using a commercial antibody (Chemicon MAB 1922) which only detects the C-terminal, 80 kDa fragment, and may not always reveal abnormalities in the 300 kDa fragment. We report here the assessment of merosin status with two different antibodies, and the clinical features of four patients with CMD that have a pronounced reduction in the immunolabelling of the 300 kDa fragment of laminin a2 but well preserved detection of the 80 kDa fragment.

2. Patients The clinical features of four cases, two males and two females, are summarised in Table 1. All patients were from non-consanguineous families. Case AM and RS had one healthy sibling, case LW had no siblings and case ZG had an affected brother. There was no other positive family history of neuromuscular disorders in any of the families. Presentation of cases LW and RS was at 4 and 6 months of age, respectively, whilst cases AM and ZG presented later, at 8 and 22 months, respectively. All cases had delayed early motor milestones. The maximum motor achievment in cases LW and RS was sitting unsupported and standing in calipers, but they are now only able to sit. Cases AM and ZG are able to walk, although with difficulty. Contractures of the hips were severe in cases LW and RS; case AM had mild contractures of the ankles. Contractures were absent in case ZG who had joint laxity. Creatine kinase activity was elevated in all cases, with a range of 800-4025 IU/1 (upper limit of normal 180 IU/I;

Table 1). MRI of the brain showed unequivocal white matter changes in cases AM, ZG and RS, but was normal in case LW. Motor nerve conduction velocities were normal in cases AM and LW, at the lower limit of normal in case RS and reduced in case ZG. Intelligence was normal in all cases and none of them had any involvement of the eyes. The degree of clinical severity was severe in cases LW and RS, relating to the severity of weakness and contractures, but was relatively static, or with slight improvement, in cases AM and ZG.

3. Morphological and immunocytochemical studies Needle muscle biopsies were taken from the quadriceps in cases AM, ZG and RS. In case LW, laminin expression was assessed in a skin biopsy which we have previously shown can be used for the diagnosis of CMD [18]. All samples were frozen in isopentane cooled in liquid nitrogen, as previously described [5,18]. Cryostat sections were stained with a panel of histological and histochemical stains according to standard methods [19]. Unfixed cryostat sections (5 /~m) were immunolabelled for 30 min with a mouse monoclonal antibody to the 80 kDa fragment of laminin or2 (MAB 1922, Chemicon 1:4000) and with a previously characterised rat monoclonal antibody to the 300 kDa fragment (4H8-2, 1:2)[18,20]. Serial sections were also labelled with a mouse antibody to laminin oil (Chemicon MAB1924;l:4000; see discussion) and rat monoclonal antibodies to laminin /31 and 71 (Chemicon MAB 1928 and 1914;1:4000). Preservation of the plasma membrane was confirmed with an antibody to /3-spectrin (Novocastra 1:20). Sections were labelled with an appropriate biotinylated secondary antibody (Amersham 1:200) for 30 min, followed by streptavidin conjugated to Texas Red for 15 min (Amersham 1:200). All washings and dilutions were in 0.2 M phosphate buffered saline. Sections were mounted in UVinert (BDH) and viewed under epifluorescence with a Texas Red specific filter. All sections (muscle and skin) were compared with control samples labelled in parallel from patients with other neuromuscular disorders, or from patients biopsied to exclude a neuromuscular disorder. Control sections labelled

Table 1

Case

Sex

Current

AM LW

F M

5 years 5 years

ZG RS

F M

6 years 12 years

A g e at presentation

C K ~ U/1

Maximum m o t o r ability

Contractures

MRI

MNCV a

8 months 4 months

800 2200

Walks Stood supported

Abnormal Normal

Normal Normal

23 months 6 months

4025 1600

Walks Stood supported

Ankles - - mild H i p - - severe talipes - - severe None H i p - - severe

Abnormal Abnormal

Reduced L o w limit o f normal

- U p p e r limit o f n o r m a l = 180 IU/1. a M N C V = m o t o r nerve conduction velocity.

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without primary antibody, with only the secondary antibodies and Texas Red, were consistently negative, except for autofluorescence.

The two affected siblings (ZG and affected brother) shared the same genotype whilst the healthy, unaffected sibling in the family of AM did not share the same haplotype.

4. Linkage analysis

6. Discussion

Linkage analysis was performed in three families (AM, ZG, RS) with a set of 13 microsatellite markers spanrting a 13 cm interval on chromosome 6q2, where the locus for the LAMA2 gene lies [10].

Our results show that the expression of laminin c~2 in congenital muscular dystrophy is more variable than originally described [4,5] and that there is also phenotypic variability within the group of patients with merosin-deficient classical CMD. Other recent reports also illustrate this variability [ 15,16]. Our cases showed only minimal abnormalities in the immunocytochemical expression of laminin a2 when first assessed with the commercial antibody to the 80 kDa fragment, and only careful examination of the preparations and a wide experience of the normal expression of laminin and other sarcolemmal proteins led to a suspicion of an abnormality. We were able to confirm an abnormality in laminin o~2 with a second antibody that recognises the 300 kDa fragment and by linkage to the LAMA2 gene locus. We have also observed a similar finding in an unusual adult case of muscular dystrophy with a primary deficiency of the laminin a2 chain [16]. It is therefore important to consider defects in merosin in a wider group of patients with muscular dystrophy. Detection of the C-terminal 80 kDa fragment of laminin or2 but pronounced reduction of the 300 kDa fragment illustrates the need to assess merosin status with more than one antibody. This is analagous to dystrophin in Duchenne dystrophy, in which the use of antibodies that recognise different regions of the protein are essential for assessment for Duchenne dystrophy (2). The LAMA2 gene is very large, with more than 64 exons and a transcript of more than 10 kb [21,22]. The potential for mutations in different parts of the gene is therefore great. A wider panel of antibodies to different regions of the protein is required to avoid false positive or negative results. The abnormal protein expression we have observed in our cases, with good preservation of the 80 kDa fragment but reduced detection of the 300 kDa fragment, is similar to that reported in the dyZJ/dy2J mouse [23,24]. This mutant has several splicing events in the N-terminal region of the gene, one of which gives rise to an in-frame product of lower abundance and lower molecular mass on immunoblots. Immunolabelling of the C-terminal fragment, however, is normal in this mouse mutant. It is also interesting to note that the dy2J/dy2Jmutant has a milder phenotype than the allelic merosin-deficient dy/dy mouse. Another reason for the difference in labelling between the two antibodies may relate to masking of the epitope for the rat monoclonal antibody, 4H8-2. If the mutant laminin c~2 is incorporated into a laminin heterotrimer it may influence the normal configuration of the molecule and conceal some epitopes. The laminin c~2 chain is synthesised as one protein but the

5. Results

5.1. Histology and histochemistry All three muscle biopsies (cases AM, ZG, RS) showed dystrophic features with a wide variation in fibre size and an increase in endomysial connective tissue. Hypertrophied fibres, split fibres and whorled fibres were also present. Necrosis was a not feature of any of the muscle biopsies.

5.2. Immunocytochemistry Labelling of the laminin ~2 chain using the antibody to the 80 kDa fragment (Chem{con MAB 1922) was almost normal, or only slightly reduced, in the muscle of cases AM, ZG and RS (Fig. 1). The skin sample from case LW showed slightly reduced expression with this antibody (Fig. 2). In contrast, sections labelled with the antibody to the 300 kDa fragment (4H8-2) showed absence (RS) or very reduced labelling in the muscle (AM, ZG. Fig. 1). Similarly, the skin from case LW showed almost no labelling at the dermal-epidermal junction with this antibody (Fig. 2). Sections from control cases showed intense, normal labelling of all fibres and of the skin with both antibodies (Figs. 1 and 2). Expression of/31 and -¢1 chains of laminin was normal in the muscle biopsies from cases AM, ZG and RS, and was localised to the muscle fibres and blood vessels. In the skin sample of LW, laminin 131 expression was slightly reduced but the 3/1 chain showed normal expression at the dermal/ epidermal junction and blood vessels (data not shown). The muscle from cases AM, ZG and RS showed over-expression of laminin ~1 laminin in the muscle, compared to controls (data not shown). Normal skin expresses laminin c~l and no abnormalities were seen in case LW. In the skin sample of case LW, sensory nerves showed a normal intensity of label with both antibodies to laminin or2, and with antibodies to 131 and 3~1 laminin (Fig. 2).

5.3. Linkage analysis The three families in which linkage analysis was performed are small but haplotype analysis was compatible with linkage to the LAMA2 locus on chromosome 6q2.

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80kDa

300kDa

Fig. l. Immunocytochemical labelling of laminin ~x2 in muscle biopsies from a control (a,b) and cases AM (c,d), ZG (e,f) and RS (g,h) with the mouse monoclonal antibody to the 80 kDa fragment (Chemicon MAB 1922) and the rat monoclonal antibody to the 300 kDa fragment. Note the good preservation of the 80 kDa fragment and the marked reduction or absence of the 300 kDa fragment in the CMD patients. × 190.

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80kDa

173

300kDa

a

d Fig. 2. Immunocytochemicallabelling of laminin ~2 in skin biopsies from a control (a,b) and case LW (c,d) with the mouse monoclonal antibody to the 80 kDa fragment and the rat monoclonal antibody to the 300 kDa fragment. Note the slight reduction in expression of the 80 kDa fragment but the marked reduction of the 300 kDa fragment at the dermal/epidermal junction in LW. Expression in the sensory nerves of the skin appears to be normal with both antibodies (arrows). × 190. reasons for detecting two fragments are not clear. It is a consistent feature, however, that occurs with several methods of extraction [17]. Another fragment of 150 kDa has also been detected [20] and antibodies that recognise this fragment give the same results as the 4H-8 rat monoclonal reported here (personal observations). W e have been unable to perform immunoblot analysis on our CMD cases because of the limited tissue available, and the poor recognition of denatured laminin by the rat monoclonal antibody to the 300 kDa fragment. It is also important to note that, although laminin ~2 has been detected immunocytochemically, the laminin heterotrimer may not be correctly assembled and may not be fully functional. Genetic analysis in three of the families presented is compatible with linkage to the region of the ~2 gene on chromosome 6q2, suggesting that the abnormalities in protein expression are caused by mutation(s) in the L A M A 2 gene. A few mutations in this gene have recently been identified but these have been associated with an absence or partial expression of laminin c~2 [8,9]. Mutational analysis in our cases is in progress but detection of the 80 kDa fragment, in

contrast to the deficient 300 kDa fragment, suggests that the mutation is in-frame, as the globular, C terminal 80 kDa portion of the protein is present. The phenotype, in the cases presented, is variable. Two cases (AM and ZG) are considerably milder than most cases of merosin-deficient CMD reported previously, whilst the other two cases have the typical severe phenotype [ 11-14]. The two milder cases are ambulant, a feature not usually observed when laminin c~2 is absent. Their motor nerve conduction velocities, however, are slightly reduced, and in cases A M and ZG white matter changes on MRI of the brain, typical of those seen in merosin-deficient patients, are present [1 l]. Thus, muscle function in these two cases (AM an ZG) is relatively well preserved but their central and peripheral nervous system defects are similar to those in severe merosin-deficient cases. The two cases with poor muscle function (LW and RS) differ in their involvement of the nervous system. Case L W is unusual in having no detectable abnormalities on brain imaging and a normal motor nerve conduction velocity. Expression of laminin c~2 was also normal on the sensory

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nerves. This suggests that the mutation(s) in this child affect muscle, nerve and the brain to different degrees. Case RS, however, has typical white matter changes on brain MRI and is severely affected, but is unusual in having appreciable expression of the 80 kDa laminin a2 fragment. The fact that the four cases presented have appreciable expression of the C-terminal region of laminin o~2 suggests that an inframe transcript must be produced. There is no molecular explanation for the phenotypic differences at present, but differential splicing in different tissues is a possibility. Identification of the causative mutations in these cases may provide a better understanding of these observations. The precise functional role of laminin a2 is not yet clear. In muscle the globular C-terminus binds to a-dystroglycan, a component of the dystrophin-associated glycoprotein complex. This complex is thought to link the cytoskeleton with the extracellular matrix and stabilise the sarcolemmal [25]. In nerves laminin or2 also binds to c~-dystroglycan [26] and it is assumed that abnormal expression of laminin or2 in the Schwann cells, interfers with myelination. This is supported by reduced motor nerve conduction velocities in the severe merosin-deficient CMD patients [13] and the arnyelination reported in the dyldy merosin-deficient mouse [27,28]. Abnormalities in nerve function, even in severe merosin-deficient patients are only slight, suggesting that abnormalities in laminin or2 may only have a minimal effect on nerve function. In the brain all chains of laminin-2 are confined to blood vessels and there is no labelling of the neural cells [29,30]. The reasons for the MRI abnormalities in CMD are not yet known but our cases show that the brain is affected to a variable extent in different patients. Further work may determine if these differences relate to specific mutations. Severe merosin-deficient cases of CMD have an overexpression of laminin otl [4,5] and it is noteworthy that this also occurs in the cases reported here. Laminin o~l is highly expressed on fetal muscle fibres ]31] and regenerating fibres [5] but its presence in merosin-deficient CMD is not related to immaturity [5]. It is not yet known if the expression in CMD patients is due to continued expression resulting from the absence of laminin u2, or if it is a reexpression process. Irrespective of the reason, the presence of laminin c~l does not compensate functionally for the absence of laminin c~2, as it is highly expressed in severe cases. Caution in interpreting the data on laminin o~1 is needed, however, as the specificity of all antibodies that recognise laminin ~1 is currently under review [32]. The specificity of the antibody used in this study has been questioned because the restricted expression of laminin c~l mRNA does not correspond to the immunolabelling observed. In summary, we report four cases of classical CMD with differential immunolabelling of the 80 and 300 kDa fragments of laminin a2, and variable phenotype. In particular, 2 cases have milder clinical features than previously described in merosin-deficient cases and we have observed

variable involvement of the brain and peripheral nervous system. Linkage to chromosome 6q is strongly supportive of a primary defect in the gene. Our cases emphasise the broad spectrum of phenotype associated with defects in laminin c~2 and the variable expression of protein which can only be detected when more than one antibody is used.

Acknowledgements We are grateful for financial support from the Muscular Dystrophy Group of Great Britain and Northern Ireland and the Medical Research Council. We thank K. Davidson for photographic assistance.

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