ORIGINAL ARTICLE Journal of Pediatric Neurology 2004; 2(4): 213-218 www.jpneurology.org Peripheral neuropathy in merosin-negative congenital muscular dystrophy H. Jacobus Gilhuis 1, Oebele F. Brouwer 2, Aad Verrips 3, Machiel J. Zwarts 1 1 Department of Neurology and Clinical Neurophysiology, Neuromuscular Centre Nijmegen, University Medical Centre St Radboud, HB Nijmegen, The Netherlands 2 Department of Neurology, University Hospital Groningen, RB Groningen, The Netherlands 3 Department of Neurology, Canisius-Wilhelmina Hospital, GS Nijmegen The Netherlands Abstract Peripheral neuropathy in patients with merosin-negative congenital muscular dystrophy (MN-CMD) has been sporadically investigated and has been considered to be motor and demyelinating in nature on the basis of nerve conduction studies. We performed neurophysiologic studies in 12 children with MN- CMD to establish the spectrum and evolution of peripheral nervous system involvement. In our patients, nerve conduction studies for both motor and sensory nerves were near normal in the children younger than six months and abnormal in the older children. The older children had the relatively slowest nerve conduction velocities suggesting a progressive, age-related dysmyelinating neuropathy. We hypothesize that the findings are due to a myelination arrest as a result of insufficient synthesis and maintenance of the peripheral myelin sheath. (J Pediatr Neurol 2004; 2(4): 213-218). Key words: merosin-negative congenital muscular dystrophy, peripheral nervous system. Introduction The congenital muscular dystrophies (CMDs) form a heterogeneous group of autosomal recessive Correspondence: H. Jacobus Gilhuis, M.D., Neuromuscular Centre Nijmegen, Department of Neurology and Clinical Neurophysiology, University Medical Centre St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel: + 31-24-3613394, fax: + 31-24-3617018. E-mail: h.gilhuis@neuro.umcn.nl Received: March 22, 2004. Revised: June 01, 2004. Accepted: June 03, 2004. disorders clinically characterized by generalized muscle weakness with an onset in early infancy, often with congenital contractures. Muscle biopsy shows a dystrophic pattern (1). CMDs without severe mental retardation or malformations of the brain are named pure or classical forms of CMD (1). Merosin is a heterotrimeric glycoprotein consisting of a heavy chain (laminin α2) and two light chains (laminin β1 and γ1). The discovery of a deficiency in the laminin α2 chain of merosin, an extracellular matrix protein, separated classical CMD into merosin-positive and merosin-negative congenital muscular dystrophy (MN-CMD) (2). MN-CMD is caused by mutations in the LAMA2 gene located on chromosome 6q22-23, encoding for the laminin α2 chain of merosin (3). Laminin α2 is expressed in skeletal and cardiac muscles, pancreas, lungs, spleen, kidneys, adrenal glands, skin, testes, peripheral nerves and brain (4-6). In peripheral nerves, merosin is expressed in the basal lamina of Schwann cells whereas its receptors are expressed in the basement membrane of Schwann cells around myelinated axons (4). Clinical manifestations of MN-CMD occur at birth, and consist of severe muscle weakness, markedly delayed motor milestones (more severe than in merosin-positive CMD), and early contractures, often associated with joint deformities. Blood tests reveal elevated serum creatine kinase (2,3,7,8). Peripheral neuropathy has been reported in most, but not all patients with MN-CMD and has been considered to be motor and demyelinating in nature on the basis of nerve conduction studies (9,10). In this study we performed neurophysiologic studies in 12 children with MN-CMD to establish the spectrum and evolution of peripheral nervous system involvement. Materials and Methods From 1993 till 2003, 12 MN-CMD patients were identified in four participating hospitals in the
214 Table 1. Clinical features of 12 MN-CMD patients Parameters Patients 1 2 3 4 5 6 7 8 9 10 11 12 Sex M M F F F M M M F F M M Age at nerve 0.2 0.3 0.5 1 8 9 0.3 0.1 0.5 0.2 0.2 3 conduction studies 4 1 13 2 (years) 7 4 Joint contractures + - + + + + - - + - - - Consanguinity - - + - - + + - - - - - M: Male; F: Female. Netherlands. The methodology for preparation of histologic sections and immunostaining is described elsewhere (11). All children were found to be negative for merosin in quadriceps muscle on immunostaining (mouse monoclonal antibodies clone MER 3/22 B2 or clone 5H2 on muscle biopsy). All patients showed severe muscle weakness, were wheelchair bound (if older than 1 year), and had an estimated IQ of between 70 and 90. Children older than 6 months had cerebral white matter abnormalities. Nerve conduction studies were repeated once in two children and twice in two other children. Informed consent was obtained for the repeated measurements (Tables 1 and 2). During nerve conduction studies, skin temperature was maintained at a minimum of 30 ºC. Some of the data of six of the children were used in a previous study (12). Results We performed 18 neurophysiologic studies in 12 children to establish the spectrum and evolution of MN-CMD associated peripheral nervous system involvement. Sensory and motor nerve conduction velocities and amplitudes of the peroneal nerve (only motor nerve conduction), median nerve, and sural nerve were compared with normal values obtained from the literature (Table 2) (13). Nerve conduction velocities were normal in the children younger than six months, and abnormal in the older children. The gradual increase in nerve conduction velocities in our patients lagged behind the agedependent increase of conduction velocities seen in healthy children. The amplitudes of the motor studies were low for both peroneal and median nerve without a clear age-related change. The median sensory amplitudes were normal regardless of patient age. The sural sensory amplitudes were (near) normal when compared to normal confidence intervals or could not be measured (Figure 1). The oldest children had the relatively slowest nerve conduction velocities. Repeated nerve conduction studies in four individual children over the years showed a similar pattern (Table 2). There were no sensory deficits in the three oldest children, in the remaining younger children, no sensory data could be obtained because of young age and low IQ. Discussion We performed nerve conduction studies on 12 MN-CMD children and found nerve conduction velocities were near normal in the children younger than six months and abnormal in the older children. The older children had the relatively slowest nerve conduction velocities as compared to healthy children. The gap in nerve conduction velocities between patients and healthy children increased with time. Repeated nerve conduction studies in individual children over the years showed a similar pattern. The amplitudes in the motor nerve studies differed widely in patients and did not show a clear age-related decrease. This decrease in amplitudes is probably due to secondary axonal injury and loss of muscle tissue due to the myopathy. Sensory nerve studies (excluding the influence of the myopathy) of the median nerve showed conduction velocities slowing with age, but no decrease of amplitudes. This strongly suggests that the neuropathy is due to a myelination problem, and not to axonal degeneration. In case of the sural nerve studies, all amplitudes were near normal or could not be measured, probably due to secondary axonal injury. Shorer et al. (10) found mildly to significantly reduced patients, which they thought was compatible with a demyelinating motor neuropathy. In a nerve conduction study of 2 siblings with MN-CMD (aged 5 and 17 months), the amplitudes were reduced in both children with near normal conduction velocities (14). Mercuri et al. (9) reported a slowing in nerve conduction velocity in a sequential neurophysiological study in one patient, and suggested a failure of the physiological maturation process of myelination of the peripheral nervous system. Di Muzio et al. (15) found reduction of large myelinated fibers, short internodes, enlarged nodes, excessive variability of myelin thickness with no evidence of segmental demyelination in a nerve biopsy of a patient with MN-CMD and slow motor
215 Table 2. Nerve conduction studies of the 12 MN-CMD patients Patient Age Peroneal nerve Median nerve Sural nerve No (years) Motor velocity Sensory velocity Motor velocity Sensory velocity (reference values) a (reference values) a (reference values) a (reference values) a Amplitude Conduction Amplitude Conduction Amplitude Conduction Amplitude Conduction (mv) velocity (m/s) (uv) velocity (m/s) (mv) velocity (m/s) (uv) velocity (m/s) 1 0.2 1.6 (5.2 ± 2.4) 25 (35 ± 4.0) 12.2 (15.9 ± 5.2) 19 (36 ± 6.6) ND ND ND ND 4 2.5 (7.0 ± 4.8) 34 (56 ± 5.0) ND ND ND ND 17.5 (22.7 ± 5.4) 33 (53 ±3.0) 7 1.8 (8.2 ± 4.2) 40 (57 ± 4.5) 36.7 (26.7 ± 9.4) 41 (54 ± 3.3) 3.8 (12.4 ± 4.8) 41 (57 ± 3.4) 13.1 (26.8 ± 6.6) 44 (54 ± 4.2) 2 0.3 2.2 (5.2 ± 2.4) 34 (35 ± 4.0) 12.7 (15.9 ± 5.2) 38 (36 ± 6.6) 1.3 (7.4 ± 3.2) 41 (34 ± 6.6) 8.0 (11.7 ± 3.6) 33 (35 ± 5.4) 1 1.9 (5.8 ± 2.5) 38 (51 ±3.0) ND ND ND ND 0 0 4 0 0 38.0 (24.3 ± 5.5) 53 (49.5 ± 3.3) 2.0 (9.6 ± 4.4) 50 (54 ± 5.3) 0 0 3 0.5 2.7 (5.2 ± 2.4) 27 (35 ± 4.0) 8.5 (15.9 ± 5.2) 32 (36 ± 6.6) 8.5 (7.4 ± 3.2) 32 (34 ± 6.6) ND ND 4 1 3.6 (5.8 ± 2.5) 43 (51 ± 3.0) ND ND ND ND ND ND 5 8 1.8 (8.2 ± 4.2) 34 (57 ± 4.5) 43.0 (26.7 ± 9.4) 32 (54 ± 3.3) ND ND 16.6 (26.8 ± 6.6) 36 (54 ± 4.2) 1 3 1.5 (8.2 ± 4.2) 23 (57 ± 4.5) 24.6 (26.7 ± 9.4) 37 (54 ± 3.3) 1.2 (12.4 ± 4.8) 36 (57 ± 3.4) 0 0 6 9 1.8 (8.2 ± 4.2) 41 (57 ± 4.5) ND ND ND ND 7.0 (26.8 ± 6.6) 38 (54 ± 4.2) 7 0.3 ND 37 (35 ± 4.0) ND ND ND 26 (34 ± 6.6) ND ND 8 0.1 4.4 (5.2 ± 2.4) 30 (35 ± 4.0) ND ND ND ND ND ND 2 7.4 (6.1 ± 3.0) 48 (56 ± 4.5) 3.9 (24.3 ± 5.5) 31 (49.5 ± 3.3) 3.9 (9.6 ± 4.4) 44 (54 ± 5.3) 27.0 (23.3 ± 6.7) ND 9 0.5 ND ND 16.0 (15.9 ± 5.2) 26 (36 ± 6.6) 6.5 (7.4 ± 3.2) 30 (34 ± 6.6) ND ND 10 0.2 2 (5.2 ± 2.4) 38 (35 ± 4.0) ND ND 5.0 (7.4 ± 3.2) 32 (34 ± 6.6) ND ND 11 0.2 6 (5.2 ± 2.4) 31 (35 ± 4.0) 4.0 (15.9 ± 5.2) 34 (36 ± 6.6) ND 24 (34 ± 6.6) ND ND 12 3 0.8 (6.1 ± 3.0) 37 (56 ± 4.5) ND 50 (49.5 ± 3.3) 2.8 (9.6 ± 4.4) 44 (54 ± 5.3) 40.0 (23.3 ± 6.7) 38 (52.6 ± 2.3) a mean ± SD, from Parano et al. (13); ND: Not determined.
216 Figure 1. Normal range (dotted lines), average (straight line) and results of the 12 MN-CMD patients, of motor nerve conduction velocity (m/s), SNAPs (uv) and CMAPs (mv) for the median nerve, peroneal nerve and sural nerve, plotted against age (note that the X-axis is not linear). Normal values adopted from Parano et al. (13). and sensory nerve conductions. In another case of a 19-year-old girl with only mildly reduced laminin α2 chain in muscle but virtually absent in peripheral nerve, nerve conductions were impaired. Sural nerve biopsy fibers showed a globular hypermyelination which the authors attributed to alteration of the feedback control during the process of nerve fiber myelination (16). In humans, maturation of myelination of the peripheral nervous system begins during the fourth month of fetal life and it is completed at around five years of age (17). These changes are reflected in the normal increase of conduction velocities during the first five years (Figure 1). In peripheral nerves, Schwann cells cover axons. Merosin is present in the basement membrane of Schwann cells around myelinated axons, and is undetectable in unmyelinated fibers, suggesting that laminin α2 may be important for fiber myelination (18). Merosin is supposed to promote Schwann cell migration and neurite outgrowth, and upregulation of laminin α2 has been demonstrated during peripheral nerve development (19-21). In the peripheral nervous system, Schwann cells normally deposit a basal
lamina consisting of laminin among other things. The development of a basal lamina correlates with the Schwann cells ability to myelinate axons. For the synthesis and the maintenance of myelin sheath integrity, the expression of laminin α2 receptors like integrins and dystroglycan and the cellsurface bound enzyme lipoprotein lipase (LPL) are essential (17-23). Expression of active cell-surface LPL depends on sufficient extracellular matrix development in order to anchor the LPL molecules effectively (24). Merosin deficiency, resulting in an abnormal basal lamina construction and in a deficient extracellular matrix composition, may thus lead to an arrest of myelin formation of both central and peripheral nervous system. The inability of Schwann cells to myelinate axons sufficiently may be due to the loss of LPL activity, which is necessary to maintain myelin sheath integrity, and for the myelin synthesis, as LPL only works in an intact basal membrane (23,24). Animal studies done so far are not consistent with findings in humans. In the dy/dy dystrophic mouse, an animal model for congenital muscular dystrophy, laminin α2 is deficient in peripheral nerves. Their peripheral nervous system is characterized by naked axons in the nerve roots and multiple discontinuities in the basal lamina (25-28). However, another study suggested that the basal lamina is not an absolute requirement for myelination and that laminins α4 and 5 may play a critcal role in myelination instead of laminin α2 (29). A study of laminin chains in rats suggested that laminin α2 plays an important role in postnatal nerve development and axonal regeneration after injury (30). MN-CMD has also been reported in 2 cats. One of the cats which was examined in detail had decreased motor nerve conduction velocities. Biopsy of the nerves showed generalized Schwann cell abnormalities and various stages of demyelination (31). In addition, deficiencies of ligands that interact with laminin α2 such as dystroglycans and integrins also play a role in myelination of peripheral nerves (32,33). It was postulated that β1 integrin participates in the deposition of the immature basal lamina in premyelinating Schwann cells, followed by the expression of α-dystroglycan, β4 and β1 integrin during maturation of the basal lamina in myelinating Schwann cells (22). Shorer et al. (10) compared peripheral nerve function between merosin-positive CMD and MN-CMD children and found that all merosinpositive CMD had normal results, whereas 8 of 10 MN-CMD children had reduced nerve conduction velocities. Analysis of the two cases of MN-CMD with normal nerve conduction showed that they produced merosin in reduced amounts, in contrast to the others who had no traces of merosin in their biopsies (10). This confirms that neuropathy is a feature of MN-CMD and may help to differentiate between MN-CMD and other muscular dystrophies. None of our patients achieved ambulation. MN- CMD patients have a more severe muscle weakness when compared with merosin-positive patients. It is unclear if the peripheral nerve involvement contributes to the severity of the clinical condition, as the patients already suffer from a severe myopathy. There were no sensory deficits in the three eldest children, in the other children no gross sensory deficits were found. In summary, we found a pattern of relative age-dependent nerve conduction velocitiy slowing in MN-CMD. This feature, combined with recent findings in biopsies, suggests a disruption in the physiological maturation process of the myelination of both motor and sensory nerves. Acknowledgements We thank Dr. R. Ten Houten and Dr K.P.J. Braun for their cooperation, and Dr. J. Pomp and Dr. H.J. ter Laak for their comments. References 1. Dubowitz V. 22nd ENMC sponsored workshop on congenital muscular dystrophy held in Baarn, The Netherlands, 14-16 May 1993. Neuromuscul Disord 1994; 4: 75-81. 2. Tome FM, Evangelista T, Leclerc A, et al. Congenital muscular dystrophy with merosin deficiency. C R Acad Sci III 1994; 317: 351-357. 3. Hillaire D, Leclerc A, Faure S, et al. Localization of merosin-negative congenital muscular dystrophy to chromosome 6q2 by homozygosity mapping. Hum Mol Genet 1994; 3: 1657-1661. 4. Leivo I, Engvall E. Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development. Proc Natl Acad Sci U S A 1988; 85: 1544-1548. 5. Villanova M, Malandrini A, Toti P, et al. Localization of merosin in the normal human brain: implications for congenital muscular dystrophy with merosin deficiency. J Submicrosc Cytol Pathol 1996; 28: 1-4. 6. Vuolteenaho R, Nissinen M, Sainio K, et al. Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues. J Cell Biol 1994; 124: 381-394. 7. Fardeau M, Tome FM, Helbling-Leclerc A, et al. Congenital muscular dystrophy with merosin deficiency: clinical, histopathological, immunocytochemical and genetic analysis. Rev Neurol 1996; 152: 11-19 (in French). 8. Philpot J, Sewry C, Pennock J, Dubowitz V. Clinical phenotype in congenital muscular dystrophy: correlation with expression of merosin in skeletal muscle. Neuromuscul Disord 1995; 5: 301-305. 9. Mercuri E, Pennock J, Goodwin F, et al. Sequential study of central and peripheral nervous system involvement in an infant with merosin-deficient 217
218 congenital muscular dystrophy. Neuromuscul Disord 1996; 6: 425-429. 10. Shorer Z, Philpot J, Muntoni F, Sewry C, Dubowitz V. Demyelinating peripheral neuropathy in merosindeficient congenital muscular dystrophy. J Child Neurol 1995; 10: 472-475. 11. ter Laak HJ, Leyten QH, Gabreels FJ, Kuppen H, Renier WO, Sengers RC. Laminin-alpha2 (merosin), beta-dystroglycan, alpha-sarcoglycan (adhalin), and dystrophin expression in congenital muscular dystrophies: an immunohistochemical study. Clin Neurol Neurosurg 1998; 100: 5-10. 12. Gilhuis HJ, ten Donkelaar HJ, Tanke RB, et al. Nonmuscular involvement in merosin-negative congenital muscular dystrophy. Pediatr Neurol 2002; 26: 30-36. 13. Parano E, Uncini A, De Vivo DC, Lovelace RE. Electrophysiologic correlates of peripheral nervous system maturation in infancy and childhood. J Child Neurol 1993; 8: 336-338. 14. Brett FM, Costigan D, Farrell MA, Heaphy P, Thornton J, King MD. Merosin-deficient congenital muscular dystrophy and cortical dysplasia. Eur J Paediatr Neurol 1998; 2: 77-82. 15. Di Muzio A, De Angelis MV, Di Fulvio P, et al. Dysmyelinating sensory-motor neuropathy in merosin-deficient congenital muscular dystrophy. Muscle Nerve 2003; 27: 500-506. 16. Deodato F, Sabatelli M, Ricci E, et al. Hypermyelinating neuropathy, mental retardation and epilepsy in a case of merosin deficiency. Neuromuscul Disord 2002; 12: 392-398. 17. Engvall E, Earwicker D, Day A, Muir D, Manthorpe M, Paulsson M. Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells. Exp Cell Res 1992; 198: 115-123. 18. Villanova M, Malandrini A, Sabatelli P, et al. Localization of laminin alpha 2 chain in normal human central nervous system: an immunofluorescence and ultrastructural study. Acta Neuropathol 1997; 94: 567-571. 19. Anton ES, Sandrock AW Jr, Matthew WD. Merosin promotes neurite growth and Schwann cell migration in vitro and nerve regeneration in vivo: evidence using an antibody to merosin, ARM-1. Dev Biol 1994; 164: 133-146. 20. Engvall E, Earwicker D, Day A, Muir D, Manthorpe M, Paulsson M. Merosin promotes cell attachment and neurite outgrowth and is a component of the neurite-promoting factor of RN22 schwannoma cells. Exp Cell Res 1992; 198: 115-123. 21. Jaakkola S, Savunen O, Halme T, Uitto J, Peltonen J. Basement membranes during development of human nerve: Schwann cells and perineurial cells display marked changes in their expression profiles for laminin subunits and beta 1 and beta 4 integrins. J Neurocytol 1993; 22: 215-230. 22. Previtali SC, Nodari A, Taveggia C, et al. Expression of laminin receptors in schwann cell differentiation: evidence for distinct roles. J Neurosci 2003; 23: 5520-5530. 23. Huey PU, Marcell T, Owens GC, Etienne J, Eckel RH. Lipoprotein lipase is expressed in cultured Schwann cells and functions in lipid synthesis and utilization. J Lipid Res 1998; 39: 2135-2142. 24. Saxena U, Klein MG, Goldberg IJ. Metabolism of endothelial cell-bound lipoprotein lipase. Evidence for heparan sulfate proteoglycan-mediated internalization and recycling. J Biol Chem 1990; 265: 12880-12886. 25. Arahata K, Hayashi YK, Koga R, et al. Laminin in animal models for muscular dystrophy. Proc Japan Acad 1993; 69: 259-264. 26. Bradley WG, Jenkison M. Neural abnormalities in the dystrophic mouse. J Neurol Sci 1975; 25: 249-255. 27. Sunada Y, Bernier SM, Kozak CA, Yamada Y, Campbell KP. Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin M chain gene to dy locus. J Biol Chem 1994; 269: 13729-13732. 28. Xu H, Christmas P, Wu XR, Wewer UM, Engvall E. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc Natl Acad Sci U S A 1994; 91: 5572-5576. 29. Nakagawa M, Miyagoe-Suzuki Y, Ikezoe K, et al. Schwann cell myelination occurred without basal lamina formation in laminin alpha2 chain-null mutant (dy3k/dy3k) mice. Glia 2001; 35: 101-110. 30. Wallquist W, Patarroyo M, Thams S, et al. Laminin chains in rat and human peripheral nerve: distribution and regulation during development and after axonal injury. J Comp Neurol 2002; 454: 284-293. 31. O Brien DP, Johnson GC, Liu LA, et al. Laminin alpha 2 (merosin)-deficient muscular dystrophy and demyelinating neuropathy in two cats. J Neurol Sci 2001; 189: 37-43. 32. Feltri ML, Graus Porta D, Previtali SC, et al. Conditional disruption of beta 1 integrin in Schwann cells impedes interactions with axons. J Cell Biol 2002; 156: 199-209. 33. Masaki T, Matsumura K, Saito F, et al. Expression of dystroglycan and laminin-2 in peripheral nerve under axonal degeneration and regeneration. Acta Neuropathol 2000; 99: 289-295.