Deficiency of syntrophin, dystroglycan, and merosin in a female infant with a congenital muscular dystrophy phenotype lacking cysteine-rich and C-terminal domains of dystrophin

Dystrophin, a cytoskeletal protein with a molecular mass of 427 kDa, is absent in muscle in patients with Duchenne muscular dystrophy(DMD).¹ Dystrophin is associated, through its cysteine-rich and C-terminal domains, with the dystrophin-associated glycoproteins (DAGs) in the sarcolemma.² The DAGs are composed of three subcomplexes:^3-6 the dystroglycan, the sarcoglycan, and the syntrophin complexes. The dystroglycan complex contains 156 kDa α-dystroglycan that links to laminin 2 (merosin) and 43 kDaβ-dystroglycan that binds to α-dystroglycan extracellularly and to the cysteine-rich/C-terminal domains of dystrophin intracellularly. The sarcoglycan complex contains 50 kDa α-sarcoglycan, 43 kDaβ-sarcoglycan, and 35 kDa γ-sarcoglycan. Thus, merosin is linked to the subsarcolemmal cytoskeleton via the DAGs. The syntrophin complex located intracellularly includes α₁-, α₂-, andβ₂-syntrophins. The absence of dystrophin causes a severe reduction of all DAGs in DMD muscle and disrupts the linkage between the extracellular matrix and the cytoskeleton.^7,8 This presumably and eventually leads to muscle cell death. In particular, deletion of the cysteine-rich and C-terminal domains of dystrophin is always associated with a severe phenotype,⁹ indicating that these domains play an important role in sarcolemmal stabilization. Primary deficiencies of the components of the sarcoglycan complex cause three forms of severe autosomal recessive muscular dystrophies-LGMD¹⁰ 2C,¹¹ D,¹² and E^13,14-while the primary deficiency of merosin causes the classic form of CMD^15,16 associated with extensive brain white matter abnormalities.¹⁷

Although secondary deficiency of merosin is present in patients with Fukuyama-type congenital muscular dystrophy (FCMD), merosin is expressed normally in patients with dystrophinopathy or other forms of neuromuscular disorders.¹⁸ We report a female infant who showed a phenotype of CMD but had a deletion of the cysteine-rich and C-terminal domains of dystrophin. We found a severe reduction of syntrophin, dystroglycan, and merosin in the biopsied skeletal muscle. These findings indicate that secondary deficiency of merosin can occur as a result of dystrophin abnormality in dystrophinopathy patients.

Methods. Patient report. An 18-month-old girl was admitted to our hospital for evaluation of delayed motor development and elevated serum CK level. There was no history of neuromuscular disorders in her family. She was born after a normal pregnancy and delivery. The neonatal period was uneventful. Her motor milestones were delayed; she had head control at 5 months. At the age of 18 months she could not roll over and crawl. On physical examination she had generalized hypotonia with facial muscle weakness and contracture of hip and knee joints. All tendon reflexes were absent. Biochemical examinations revealed serum CK of 9,640 IU per liter(normal, 30 to 210), aldolase 119 IU per liter (normal, 1.7 to 5.7), and LDH 1,443 IU per liter (normal, 190 to 440). Chromosomal anomalies were not found in the patient or either parent. T2-weighted brain MRI showed high-density signals in the white matter (figure 1).

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Figure 1. T2-weighted brain MRI showing abnormal high-density signals in the periventricular white matter.

Immunocytochemistry. Skeletal muscle biopsy was performed on her femoral muscle. Indirect immunofluorescence analysis of biopsied muscle was performed as previously described^7,8,10 using the following antibodies: monoclonal antibodies against dystrophin N-terminal domains (A1C, DYS3), rod domain (DYS1), C-terminal domains (V1A4z, DYS2), merosin (5H2), β-dystroglycan (8D5), α-sarcoglycan (IVD31), and polyclonal antibodies against α-dystroglycan, γ-sarcoglycan, and the syntrophins.

RT-PCR analysis of dystrophin messenger RNA (mRNA). Total RNA was isolated from peripheral blood of the patient and both parents, and the biopsied muscle of the patient. The complementary DNA (cDNA) was prepared as described previously.¹⁹ Nested 10 sets of primer encompassing all exons of dystrophin transcript were used for reverse transcriptase polymerase chain reaction analysis of the patient's dystrophin mRNA, as previously described.²⁰ An amplified fragment encompassing all exons of dystrophin cDNA was sequenced as previously described.²⁰

Results. Conventional histochemical analysis. The muscle biopsy showed an increased variability in the size of fibers, a number of degenerating and regenerating fibers, and massive infiltration of interstitial connective tissue (figure 2).

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Figure 2. Transverse section of biopsied muscle showed an increased variability in fiber size, a number of degenerating and regenerating fibers, and massive infiltration of interstitial connective tissue (H&E, original magnification ×120 before 27% reduction).

Immunocytochemical analysis. The skeletal muscle from the patient showed normal sarcolemmal staining with antibodies against N-terminal and rod domains of dystrophin. But, antibodies against the C-terminal domains of dystrophin did not stain the sarcolemma of muscle fibers(figure 3). Immunostaining for α-, β- (not shown), and γ-sarcoglycan was well preserved in the sarcolemma. However, immunostaining for syntrophins, α- and β-dystroglycan, and merosin was greatly reduced (figure 4).

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Figure 3. Immunocytochemical analysis of dystrophin in the skeletal muscle from a normal control (Normal), the present patient (Patient), and a typical Duchenne muscular dystrophy (DMD) patient. The patient's muscle showed normal sarcolemmal staining with antibodies against N-terminal (N′-Ter-1 and N′-Ter-2) and rod (Rod) domains of dystrophin. Antibodies against C-terminal (C′-Ter-1 and C′-Ter-2) domains showed no sarcolemmal staining. Bar, 50µm.

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Figure 4. Immunocytochemical analysis of dystrophin-associated proteins and merosin in the skeletal muscle from a normal control (Normal), the present patient (Patient), and a typical Duchenne muscular dystrophy (DMD) patient. Expression of merosin (Lamα2), α- and β-dystroglycan (α-DG and β-DG), and syntrophin (SYN) was greatly reduced in the patient's muscle. γ- andα-sarcoglycans (γ-SG and α-SG) were normally expressed. Bar, 50 µm.

Multiplex PCR screening on lymphocytes did not reveal any deletions of the dystrophin gene in the patient or either parent.

RT-PCR analysis of dystrophin mRNA. PCR amplification of dystrophin cDNA encompassing hotspot regions revealed no gross rearrangement. The size of the amplified product encompassing exons 67 to 79 showed two fragments of normal 642 base pair (bp) and mutated 330 bp on lymphocytes cDNA from the patient. On muscle cDNA from the patient (figure 5), only the mutant gene (330 bp) was expressed. Sequence analysis of amplified product disclosed that exon 70 was directly joined to exon 75 and that exons 71 to 74 were completely absent on muscle cDNA from the patient(figure 6).

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Figure 5. Reverse transcriptase polymerase chain reaction analysis of the dystrophin messenger RNA in RNA from muscle (a) and peripheral blood (b) by using primers encompassing exons 71 to 74. (a) The size of the amplified product is 642 bp in normal control muscle. The size of the amplified product in the patient's muscle is reduced to 330 bp showing a lack of exons 71 to 74. (b) The size of the amplified product from the control's blood is 642 bp. The size of the amplified product from the patient's blood is 642 bp and 330 bp. The size marker is a size ladder HaeIII-digested Phix 174 phage DNA.

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Figure 6. Sequence of the amplified product from patient and control muscle cDNA. The sequence of the region joining exons 70 to 75 are presented. In the patient's muscle cDNA, the sequence of the 3′ end of exon 70 (5′-GAAAC-3′) is joined directly to the 5′ end of exon 75 (5′-GAATCT-3′).

Discussion. Our patient was phenotypically similar to CMD. Moreover, she had white matter abnormalities on brain MRI and reduced expression of merosin in the skeletal muscle. This suggested that she might have merosin-deficient CMD. Surprisingly, however, dystrophin analysis showed a lack of the C-terminal domains of dystrophin, and nested RT-PCR analysis of mRNA in the patient's muscle showed a complete lack of exons 71 to 74 (a region from cysteine-rich to C-terminal domains) of the dystrophin gene. The patient's muscle expressed only the mutant gene. Thus the patient was a symptomatic DMD carrier with skewed X-inactivation.²¹ This is important since our patient illustrates, for the first time, that a dystrophin abnormality can cause a secondary reduction of merosin in dystrophinopathy.

DMD patients who lack the C-terminal domains of dystrophin have a severe phenotype,⁹ and all DAGs are severely reduced in their muscles.²² In our patient, on the other hand, α- and β-dystroglycan and syntrophin were greatly reduced, but the components of the sarcoglycan complex were well preserved. Theβ-dystroglycan binding site is localized to the second half of hinge 4 and the cysteine-rich domains of dystrophin.^23,24 The syntrophin directly interacts with dystrophin through more than one binding site in exons 73 and 74.²⁵ The binding site forα₁-syntrophin, which is predominantly expressed in skeletal muscle, coincides with the region with a deletion that causes a severe phenotype.²⁶ Since our patient lacked exons 71 to 74 of the dystrophin gene, syntrophin and dystroglycan were probably reduced due to the loss or disturbance of their binding to the dystrophin cytoskeleton. The mechanism by which the sarcoglycan complex was preserved in our patient is unclear. The reduction of α-dystroglycan might have led to the reduction of merosin in our patient, because merosin is bound toα-dystroglycan.

In our patient, the reduction of merosin in muscle may correlate with the phenotype of CMD, rather than that of typical symptomatic DMD carriers. In DMD and Becker muscular dystrophy muscles, merosin is normally expressed around the muscle fibers, while a moderate reduction in merosin occurs in FCMD.¹⁸

Merosin promotes neurite outgrowth and Schwann cell migration, and may have a role in the normal development of central²⁷ and peripheral²⁸ nervous systems. Merosin-deficient CMD is characteristically associated with extensive white matter abnormalities indicative of dysmyelination on brain CT and MRI.¹⁷ FCMD patients also have central dysmyelination.²⁹ Our patient also had extensive white matter abnormalities. These findings suggest that the deficiency of merosin, primary or secondary, may correlate with the brain white matter abnormalities, and that merosin may have a role in myelinogenesis of the CNS.

Acknowledgments

We thank Drs. Kevin P. Campbell (University of Iowa) and Louise V.B. Anderson (Newcastle General Hospital) for the generous supply of antibodies.