Clinical Correlations in 16 Patients With Total or Partial Laminin α2 Deficiency Characterized Using Antibodies Against 2 Fragments of the Protein

Background  Many patients with classic congenital muscular dystrophy have been found to have partial or total deficiency of the α2 chain of laminin 2 (merosin). This deficiency has mostly been studied using only 1 antibody against a fragment of the protein.

Objectives  To characterize the expression of laminin α2 in the skeletal muscle of patients with laminin α2 deficiency using antibodies against 2 different portions of the protein and to correlate the immunochemical findings with clinical phenotype.

Methods  We studied 4 patients with total lack of laminin α2 and 12 with partial laminin α2 deficiency with immunohistochemical techniques and Western blot analysis. We used antibodies recognizing an 80-kd fragment toward the C-terminus and a 300-kd fragment toward the amino-terminal. Patient characteristics examined were functional compromise, magnetic resonance imaging or computed tomography of the brain, electromyography, evoked potentials, and creatine kinase levels.

Results  In 4 patients, immunohistochemical analysis revealed no reactivity to either antibody; in 2 patients, the 300-kd fragment alone was partially expressed; in 2 patients, the 80-kd fragment alone was partially expressed; and in 8 patients, both fragments were partially expressed. Immunoblot analysis revealed bands of reduced intensity and normal molecular weight generally corresponding to the immunohistochemical findings. Absence of both fragments or of one with reduction of the other always produced a severe clinical phenotype, while a milder clinical phenotype was observed when both fragments were partially expressed.

Conclusions  Extent of laminin α2 deficiency in most cases correlates with clinical phenotype but not with peripheral and central white matter abnormalities. Skin biopsy specimens may reveal laminin α2 deficiency in patients who have normal laminin α2 levels in muscle biopsy specimens.

ALTHOUGH congenital muscular dystrophy (CMD) is clinically and genetically heterogeneous, all forms are characterized by early onset and recessive inheritance, the main findings in muscle being connective tissue proliferation, variable fiber size, central nuclei, and occasional necrotic fibers. A subgroup of CMD (the so-called muscle-eye-brain diseases, including Fukuyama, Walker-Warburg, and Santavuori dystrophies) presents, in association with muscle involvement, developmental defects of the central nervous system and eye abnormalities. Patients with classic CMD typically manifest only skeletal muscle involvement.¹ In 1994, Tomé et al² discovered a deficiency of merosin in some patients with classic CMD; the corresponding genetic defect was subsequently localized to the LAMA2 locus of chromosome 6q2.^3,4 Complete lack of the protein is usually found in these patients, most of whom show severe muscle weakness; however, milder muscle involvement has been recently reported in association with partial merosin deficiency.^5-8 A clinically asymptomatic abnormality of the central white matter signal is present on magnetic resonance images (MRIs) in nearly all laminin α2–deficient patients with CMD.⁹

Laminins are heterotrimeric proteins, consisting of a heavy α chain and light β and γ chains that assemble into cross-shaped molecules. The several laminins are expressed differently in the basal lamina of different tissues.^10,11 Merosin is the heavy α2 chain (previously called the M chain) of laminin 2, the muscle isoform that is also expressed in Schwann cells, trophoblast, and skin.^12,13 Laminin α2 is now the preferred name for merosin.¹⁴

The functional role of laminin 2 is not completely understood, but it is thought to help stabilize the cell membrane.¹⁵ It is known that laminin 2 binds to extracellular α-dystroglycan (through the large G globular domain of the α2 chain¹⁶) and that the latter is part of the large transmembrane protein complex that tightly binds subsarcolemmal dystrophin and links the extracellular matrix to the intracellular cytoskeleton.¹⁷ Laminin 2 also links muscle fibers through α7β1 integrins, recently shown to be laminin 2 receptors in skeletal muscle and abnormally expressed in laminin α2–deficient human patients and mice.¹⁸

We present the results of our investigation of laminin α2 expression by immunohistochemical and immunoblot analysis using several antibodies. We studied 16 patients: 11 with classic CMD and 5 with dystrophic histopathologic findings and clinical myopathy.

Eleven patients had classic CMD, according to the clinical criteria for diagnosis summarized by Kobayashi et al.¹⁹ Five others (patients 8, 11, 12, 13, and 16) were included after a partial laminin α2 deficiency was detected in muscle biopsy specimens, obtained routinely as part of the diagnostic workup of patients with dystrophy ( Table 1).

In 12 of 16 patients, central white matter was investigated on MRI scans (using a 0.5- or 1.5-T scanning unit). Sagittal spin echo T₁-weighted images, axial and coronal intermediate images, and coronal spin echo T₁- and T₂-weighted images were obtained. In patients 7 and 13, a second MRI scan was performed 18 and 12 months later, respectively. In some patients, computed tomography was performed. Electromyography, evoked potentials, and creatine kinase levels were always investigated.

Immunohistochemical analysis was performed on 6-mm-thick cryosections of muscle biopsy specimens. Skin biopsy specimens were also obtained from patients 6 and 12. Muscle and skin biopsy specimens were snap frozen in isopentane and liquid nitrogen and maintained in liquid nitrogen.

Immunohistochemical expression of laminin α2 was determined using a commercial monoclonal antibody (Chemicon, Temecula, Calif) and the monoclonal antibody 4H8-2, which was produced and characterized by Schuler and Sorokin.²⁰ The former recognizes an 80-kd fragment toward the C-terminus and the latter a 300-kd fragment toward the amino-terminal. Dilutions were 1:800 and 1:10, respectively, in phosphate-buffered saline plus appropriate serum. Immunostaining was detected by immunofluorescence or immunoperoxidase using biotin-avidin amplification.

Laminin α2 expression by immunoblot was evaluated using the anti–80-kd monoclonal antibody in all cases and using an affinity purified polyclonal antibody raised against a recombinant human M-chain fragment of 300-kd in most cases. This polyclonal antibody was previously characterized by Sunada et al.²¹ Muscle cryosections were solubilized in edetic acid extraction buffer (10-mmol/L edetic acid, 50-mmol/L TRIS–hydrochloric acid [pH, 7.5], 150-mmol/L sodium chloride, 1-mmol/L phenylmethylsulfonyl fluoride, 0.75-mmol/L benzamidine, 1-mg/mL aprotinin, 1-mg/mL leupeptin, and 1-mg/mL pepstatin A). After centrifugation, the samples were separated on 3% to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose. The nitrocellulose transfers were stained with the primary anti–α2 chain antibodies. The dilutions were 1:200 and 1:25 for the 80-kd monoclonal and 300-kd polyclonal antibodies, respectively.

The electromygraphic findings were myopathic; the findings of histopathologic examination were dystrophic; and evoked potentials were normal, unless otherwise stated. The clinical features are summarized in Table 1.

Laminin α2 immunostaining was completely absent from muscle fiber surfaces with both antibodies in 4 cases (patients 3, 4, 5, and 6) (Figure 1), and immunoblot analysis with both antibodies failed to reveal a specific laminin α2 band (Figure 2). These patients had the typical clinical features of merosin-negative CMD, with muscle hypotonia at birth, weakness, and severe delay in motor development. They all could sit unsupported but were unable to walk. Patient 5, a severely mentally retarded boy, could also crawl and stand at 5 years. Magnetic resonance imaging was not performed in patients 4 and 5 but in patients 3 and 6 disclosed (T2) white matter signal hyperintensity. A skin biopsy specimen was obtained from patient 6 and also showed a complete absence of laminin α2 (Figure 3).

In patients 1 and 2, there was no immunostaining on muscle fibers with the anti–80-kd antibody, while the anti–300-kd antibody elicited a weak nonhomogeneous signal on most fibers (Figure 4). Immunoblot analysis showed absence of the laminin α2 band with both antibodies ( Figure 2). These patients were as severely affected as the merosin-negative patients. Patient 1 died of recurrent respiratory infection at 3 years of age. The plasma creatine kinase level was particularly high, and the MRI showed hyperintensity of brain white matter involving periventricular and posterior hemispheric regions.

In patients 7 to 14, laminin α2 was partially expressed with both antibodies. Positivity on muscle fiber surfaces was patchy, and intensity varied from fiber to fiber and fascicle to fascicle ( Figure 4). Immunoblot analysis with the 80-kd antibody showed 2 or 3 bands of variably reduced intensity at about 80 kd ( Figure 2). Immunoblot analysis with the 300-kd polyclonal antibody (performed in 3 patients) showed a normal band in patient 9, no band in patient 13, and a reduced intensity band in patient 14 ( Figure 2).

Patients 7 through 14 all acquired autonomous walking. When present, proximal limb muscle weakness was very slowly progressive. Only patient 8, the daughter of first cousins, with a Duchenne-like phenotype plus severe mental retardation, lost her walking ability at 15 years of age. Mild mental retardation was also observed in patient 9, in whom brain MRI detected only a slight white matter hyperintensity. Neurophysiological investigation revealed moderate peripheral motor neuropathy and abnormal evoked potentials in patient 7 and mild peripheral demyelinating neuropathy and abnormalities on visual evoked potentials and somatosensory evoked potentials in patient 11. Patient 11, a 39-year-old man, demonstrated moderate limb girdle weakness, elbow and knee retractions, and neck and forearm muscle contractures. His symptoms began when he was about 15 years old and progressed slowly. His MRI (T₂) showed diffuse and symmetrical hyperintensity involving subcortical and lobar white matter but sparing the periventricular areas. There was family consanguinity. A 36-year-old sister (patient 12) with bilateral hip dislocation had a similar but less severe phenotype. Her MRI (T₂) showed only a few small areas of signal hyperintensity in the posterior paraventricular white matter. A muscle biopsy specimen revealed marked dystrophic features, consisting of a few surviving fibers with conspicuous fibrotic and fatty connective tissue proliferation. In the surviving muscle fibers, laminin α2 labeling was normal with both antibodies ( Figure 3). However, her skin biopsy specimen revealed reduced positivity to laminin α2 immunostaining ( Figure 3), similar to that observed in her brother's muscle biopsy specimen.

Perinatal symptoms, with hypotonia and bilateral talipe equinovarus, were observed in patient 13, a 1-year-old son of consanguineous parents. On admission, he was hypotonic, with hypotrophic muscles in the proximal aspect of his upper limb and mild weakness of his neck muscles. He was unable to crawl or stand without support. His MRI scan showed marked cerebral and cerebellar white matter signal abnormalities, sparing the brainstem, corpus callosum, and basal ganglia. One year later, when he was reexamined, he was no longer able to walk and fell frequently (autonomous walking achieved at 20 months). Findings of subsequent MRI confirmed those of the previous scan.

Patients 10 and 14, investigated for high plasma creatine kinase levels determined at the ages of 30 and 11 years, respectively, had normal neurologic examination results and muscle strength; in both, muscle biopsy specimens revealed myopathic features, with only mildly increased connective tissue. In patient 14, laminin α2 immunostaining was slightly reduced and relatively uniformly distributed on cell surfaces; the findings of MRI and evoked potentials were normal. When he was reexamined at the age of 13 years, he was still clinically normal. In patient 10, MRI and neurophysiological investigations were not performed.

Laminin α2 immunostaining in patients 15 and 16 showed reduced intensity with the 80-kd antibody and negative results with the 300-kd antibody ( Figure 4). Immunoblot analysis showed that patient 15 had an 80-kd band that was reduced in intensity and no 300-kd band, while patient 16 had an 80-kd band that was reduced in intensity and a 300-kd band of normal molecular weight but slightly reduced intensity ( Figure 2). Patient 15, a 16-month-old girl, had normal motor development up to 9 months of age, followed by an inability to crawl and get up and reduced limb movement. Her MRI scan showed a slight diffuse hyperintensity of subcortical and periventricular white matter (compatible with incomplete myelination). A second examination at 36 months showed improvement: she had acquired the ability to stand with support and to walk slowly with callipers.

Patient 16, a 1-year-old girl, was severely hypotonic and weak from birth. When examined, she could not move her head or limbs against gravity. Hypoplasia of the vermis and dilation of the cisterna magna were observed on computed tomographic scan; MRI was not performed.

To our knowledge, this is the first report on a consistent series of patients with CMD evaluated with 3 antibodies against 2 different fragments of laminin α2. The most severe phenotype, characterized by hypotonia and weakness at birth and an inability to achieve the upright position, was observed in patients with absence of both fragments, as expected, and in the 2 patients (Nos. 1 and 2) with absence of the 80-kd band and reduction of the 300-kd fragment. The abnormal protein present in the latter 2 patients is presumably unable to ameliorate the clinical phenotype. An abnormal protein that is unable to ameliorate the clinical phenotype may also be the cause of the severe phenotype in patients 15 and 16, in whom the 80-kd fragment was reduced and the 300-kd fragment was absent on immunohistochemical analysis, while immunoblot analysis showed that the 300-kd fragment was present in patient 16; these patients were as severely compromised as those with total absence of laminin α2. The different expression of the 300-kd fragment by immunohistochemical and immunoblot analysis in patient 16 is probably because the 2 antibodies against this part of laminin α2 recognize different epitopes (the monoclonal antibody was raised against mouse heart laminin α2, and the polyclonal antibody was raised against a recombinant human M-chain fragment.^20,21 Note that the former works in immunohistochemical procedures and the second in immunoblot only).

Clinical compromise was rather variable in patients with partial expression of both fragments. However, the onset of symptoms was late; all patients acquired the ability to walk; and most patients were only moderately affected. These patients probably express a protein that is more functional than the one that is expressed in the patients discussed above.

The findings of immunoblot analysis of laminin α2 confirmed the immunohistochemical results in most patients and showed that, when present, the 2 α2-chain fragments were reduced in quantity but of normal molecular weight. The presence of multiple 80-kd bands is probably due to degradation of this fragment, which seems to occur more readily in patients than in controls ( Figure 2). In patient 13, however, the 300-kd band was absent by immunoblot, while immunohistochemical staining of this part of the molecule showed reduced positivity on most fibers. Again, this is probably because different 300-kd antibodies were used for immunoblot and immunohistochemical analysis.

Sewry et al²² recently evaluated 4 patients by immunohistochemical analysis with the same anti–80-kd and anti–300-kd monoclonal antibodies and found that the 300-kd epitope was very reduced or absent in all 4 patients and that the 80-kd epitope was preserved; however, 2 patients were severely affected (as were our patients 15 and 16) and 2 had a milder phenotype. Allamand et al⁸ described 2 siblings with a relatively mild phenotype and a mutation leading to internally deleted laminin α2. In muscles, they found near normal laminin α2 expression with 2 antibodies against the C-terminus and a more evident reduction of the protein with the antibody against the 300-kd fragment at the amino-terminal.

A mild phenotype is observed in the dy^2jdy^2j mouse, one of the animal models of laminin α2 deficiency. In this animal, laminin α2 immunolabeling with C-terminus antibody is normal, while the amino-terminal immunolabeling is reduced.²³

In view of these findings, it seems that in some cases partial function of the protein is maintained (with consequent mild clinical phenotype) when the C-terminus is preserved. The situation is reminiscient of that in dystrophinopathies in which out-of-frame deletions lead to the Duchenne phenotype and in-frame deletions, which preserve C-terminus expression, give rise to the Becker phenotype. Note that little is known of the laminin α2 gene and its product, however. Like the dystrophin gene, the laminin α2 gene is very large (64 exons); the transcript is 9.5 kilobases encoding a polypeptide of 3110 residues.²⁴ Therefore, as in dystrophinopathies, merosin expression should ideally be investigated using a range of antibodies that "sample" most of the protein. The results of such studies would suggest which part of the gene to investigate for mutations, eliminate or reduce false-positive or false-negative diagnoses, and allow more detailed correlation between genotype and clinical phenotype. Furthermore, in view of the large spectrum of clinical manifestations in laminin α2–deficient patients, we propose that the term CMD be abandoned for a more systematic nomenclature.

Laminin 2 is also expressed in the skin.¹³ We performed a skin biopsy in patient 12, sister of patient 11, because unlike her brother, she expressed laminin α2 normally in muscle, although her muscle specimen showed more severe dystrophic features. We suppose that the laminin α2 expression was normal because we were observing only surviving fibers that had more protein. Our finding of reduced laminin α2 expression in the skin of patient 12 suggests that in suspected and familial cases the diagnosis may be confirmed by the findings of skin biopsy.

The role of laminin 2 in the central and peripheral nervous systems is not clear; it has been suggested that the protein is involved in myelination.²⁵ Peripheral nervous system alterations may be due to defective expression of laminin 2 in Schwann cells. Although laminin 2 messenger RNA has been detected in various regions of the fetal human brain,²⁶ the protein itself has been detected only at the blood-brain barrier.²⁷

Data on histopathologic features of the brain in laminin α2–deficient patients are very limited. Among 3 unconfirmed but probable cases in which there were extensive cerebral white matter abnormalities and autopsies were performed, microscopic examination revealed spongy myelin and moderate astrocytosis in 1 case and no sign of myelin breakdown in the other 2 cases.^28-30

White matter abnormalities in our patients did not correlate with clinical severity, with age at onset, or with complete vs partial merosin deficiency. This was true for both central and peripheral white matter. Thus, MRI white matter abnormalities were most extensive in an adult (patient 11) who had a slowly progressive, late-onset myopathy with partial laminin α2 deficiency (but his affected sister, with reduced expression of laminin α2 in skin but not in skeletal muscle, had only punctate hyperintensity). Furthermore, cerebral MRI abnormalities do not seem to progress, since a second scan obtained 18 and 12 months later in patients 7 and 13, respectively, revealed the same features observed previously. With regard to peripheral white matter abnormalities, a mild peripheral demyelinating motor and sensory neuropathy was present in patients with severe clinical involvement and total lack of laminin α2 expression, as well as in patients with mild clinical involvement and partial expression of both laminin α2 fragments. Further investigation of the role and expression of laminin 2 in the central and peripheral nervous system is required.

Accepted for publication April 14, 1998.

The polyclonal antibody used in this study was kindly donated by Kevin P. Campbell, PhD, Howard Hughes Medical Institute and University of Iowa College of Medicine, Iowa City.

We thank Flavia Blasevich and Sergio Daniel for technical assistance and Don Ward for help with the English.

Reprints: Lucia Morandi, MD, Department of Neuromuscular Diseases, Istituto Nazionale Neurologico "C. Besta", via Celoria 11, 20133 Milano, Italy.