Abstract
Ullrich disease is a form of congenital muscular dystrophy characterized clinically by generalized muscle weakness, contractures of the proximal joints, and hyperflexibility of the distal joints from birth or early infancy. Recently, mutations of the collagen VI gene have been associated with Ullrich disease. The authors report on a boy with Ullrich disease who has complete deficiency of collagen VI and harbors compound heterozygous mutations in the collagen VI alpha 2 gene. Absence of microfibrils on EM, together with normal collagen fibrils and basal lamina, suggests that loss of a link between interstitium and basal lamina may be a new molecular pathomechanism of muscular dystrophy.
In 1930, Ullrich described two boys with an unusual form of congenital muscular dystrophy.1 The clinical findings included generalized muscle weakness, contractures of the proximal joints, and hyperflexibility of the distal joints from birth or early infancy. He labeled this disorder ‘Die kongenitale atonisch-sklerotische Muskeldystrophie.’ High-arched palate, protuberant calcanei, and normal intelligence are other characteristics of the disease. Muscle biopsies revealed dystrophic changes.2 Recently, two groups found complete deficiency of collagen VI in patients with Ullrich disease.3,4⇓ All patients had frameshift type mutations in the gene encoding collagen VI alpha 2 (COL6A2). So far, four different mutations were reported; a single nucleotide insertion at exon 13, a 26-bp deletion in exon 14, an aberrant splicing at the border between intron 17 and exon 18, and exon 24 skipping.
We report a new patient with Ullrich disease and complete deficiency of collagen VI with morphologic abnormalities at the EM level.
Materials and methods.
Patient.
The male patient was the first child of healthy parents who denied consanguinity. At birth, he was noted to have generalized muscle weakness, hypotonia, and contractures of proximal joints. His motor developmental milestones were delayed. He was able to sit unassisted at age 2 years 6 months.
The patient was diagnosed as having Ullrich disease based on clinical and muscle histology findings at age 3. We examined the patient at the age 4. He could sit but could not walk without assistance (figure 1A). He had generalized muscle weakness and atrophy that was most prominent in the neck. Although facial muscle weakness was equivocal, his palate was clearly high arched. He had severe contractures of the proximal joints, including neck, shoulder, elbow, hip, and knee joints, and had marked kyphoscoliosis. In contrast, distal joints such as hand, ankle, toe, and finger joints were hyperextensible (see figure 1B,C). Other findings included enlarged calcanei (see figure 1C) and diminished or absent tendon jerks. His intelligence was normal. Laboratory examination revealed no specific diagnostic abnormalities. The serum creatine kinase level was slightly elevated (220 to 480 IU/L; normal, 51 to 197 IU/L). Results of a brain CT were normal.
Figure 1. Clinical features of Ullrich disease. (A) The patient is unable to extend his arm due to the contractures of elbow and shoulder joints. Note slender stature with generalized muscle wasting. (B) Hyperextension of the wrist joint. (C) Hyperlaxity of toe joints and protuberant calcanei.
Histochemical and immunohistochemical analyses.
Transverse serial frozen muscle sections of 6 μm or 8 μm thickness were stained with hematoxylin and eosin, modified Gomori trichrome, and a battery of histochemical techniques. We also immunostained skeletal muscles with the monoclonal antibody against collagen VI (1:500) (ICN Biomedicals, Aurora, OH) and the polyclonal antibody against collagen IV (1:2000) (Advance, Tokyo, Japan).
Sequence analysis of collagen VI alpha 2 gene (COL6A2).
Total RNA was extracted from frozen muscle using Totally RNA Kit (Nippon Gene, Tokyo, Japan) and was reverse transcribed into cDNA with oligo (dT)20 primer using the ThermoScript RT-PCR System (Life Technologies, Carlsbad, CA). We amplified two overlapped fragments, encompassing nt 1 to 1330 and nt 1313 to 3147 (nucleotide number is based on AY029208), which cover the entire open reading frame. We directly sequenced the amplified fragments with the PCR primers5 and relevant internal primers using BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster, CA), and then electrophoresed the samples using an ABI PRISM 377 DNA sequencer (PE Biosystems). RT-PCR using a forward primer in exon 13 (AGG GCC ACA GGT GCT GCC AA) and a reverse primer in exon 15 (GGG CCC GCT TAG CAC CAT GGA) gave at least four bands. Each band was excised and sequenced. To identify the intronic mutations, we amplified the genomic fragments encompassing intron 14 through exon 15 and intron 23 through exon 24. We sequenced the amplified fragments with the PCR primers.3
Electron microscopy.
Skeletal muscle from the patient was fixed with 2% glutaraldehyde and postfixed in osmium tetroxide, dehydrated in graded alcohol series, and then embedded in Epon (Taab Laboratories Equipment, Ltd., Aldermaston, UK). Ultrathin sections, stained with uranyl acetate and lead citrate, were examined.
Results.
Histochemical and immunohistochemical analysis.
On histochemical examination, scattered necrotic and regenerating fibers were identified. Moderate endomysial fibrosis was present (figure 2A). There was type 2 fiber atrophy, and marked type 1 fiber predominance was seen. Fiber type distribution differed from fascicle to fascicle (see figure 2B). On immunohistochemistry, collagen VI was completely absent in the muscle from the patient (see figure 2D), whereas collagen VI was present in sarcolemma and interstitial tissue in normal muscle (see figure 2C) and in other disease control muscle, including Duchenne muscular dystrophy and Fukuyama type muscular dystrophy (data not shown). In contrast, collagen IV, which is a major component of basal lamina, was intact in the patient’s muscle (see figure 2F).
Figure 2. Muscle pathology findings (biceps brachii). (A) Hematoxylin and eosin staining showing necrosis and regeneration with endomysial fibrosis. (B) Histochemical staining for myosin ATPase with preincubation at pH 4.6. Type 1 fiber predominance and scattered type 2C fibers are seen. Fiber-type distribution differs from fascicle to fascicle. (C and D) Immunostaining for collagen VI: normal (C) and the patient’s result (D). Collagen VI was completely absent in the patient’s muscle. (E and F) Immunostaining for collagen IV: normal (E) and the patient (F). Collagen IV is normally present in sarcolemma in the patient. (G, H, I ) Electron micrographs: (G) The basal lamina (arrowheads) is intact even in degenerating muscle fibers. (H) Fukuyama type muscular dystrophy for control. Both collagen fibrils (asterisks), with typical periodic pattern of about 65 nm and about 50 nm in diameter, and microfibrils (arrowheads), with much smaller diameter, are present in the interstitium. (I) In the patient, collagen fibrils (asterisks) are present but microfibrils are totally absent. (Bar =100 μm in A and B; 50 μm in C–F; 1 μm in G; and 0.5 μm in H and I.)
Sequence analysis of collagen VI alpha 2 gene (COL-6A2).
We found compound heterozygous mutations in the COL6A2 gene. One allele had a G-to-C substitution at position −1 in intron 14 (see part B of the supplementary figure, which can be found on the Neurology Web site. Go to www.neurology.org and scroll down the Table of Contents for the title link to this article.). Direct sequence of RT-PCR products showed overlapping peaks after exon 14 when read with a forward primer (see part A of supplementary figure on the Web site) and before exon 15 when read with a reverse primer. The sequencing of the shortest band showed that exon 14 was connected to the last 53 bp of intron 14 and exon 15. Likewise, the second shortest band showed that exon 14 was connected to the last 247 bp of intron 14 and exon 15. We were unable to isolate and purify the other bands because they were too close to each other in the gel. The second COL6A2 allele had a C-to-G substitution at position −3 in intron 23 (see part D of supplementary figure on the Web site.). The direct sequence of the RT-PCR products showed skipping of the entire exon 24. Both mutations were absent in 100 normal chromosomes.
Electron microscopy.
The basal lamina was intact even in degenerating muscle fibers (see figure 2G). Collagen fibrils in the interstitium appeared normal, with a periodic pattern of about a 65-nm interval. In contrast, microfibrils, which are usually seen in the interstitium associated with collagen fibrils, were totally absent (see figure 2I).
Discussion.
We have identified complete collagen VI deficiency in a patient with Ullrich disease and found compound heterozygous mutations of COL6A2 in the patient. In one allele, a C at −3 position in intron 23 was changed to G. The nucleotide at this position is conserved in only 65% of introns;6 however, we did not identify any other polymorphisms in this region. This mutation caused the skipping of the entire exon 24 and is predicted to cause frameshift of the codon, resulting in the elimination of most of the C-terminal globular domain. The same exon 24 skipping was reported in an Italian patient, although the patient had a nucleotide substitution at −1 position in intron 23. The second allele had a G-to-C transition at position −1 in intron 14, causing the insertion of intron 14 of variable size. The inserted fragments that we identified were 53 bp and 247 bp in length; thus, both are predicted to cause a frameshift of the codon, resulting in the elimination of the triple-helical domain and the C-terminal globular domain. RT-PCR analyses revealed abnormal splicing of exons contiguous to the polymorphism, indicating that both are pathogenic mutations.
Collagen VI is thought to be a major protein in microfibrils. The complete absence of microfibrils in our patient with complete collagen VI deficiency suggests this idea. Collagen VI has been shown to anchor the basement membrane in skeletal muscle by interacting with collagen IV, a major component of basal lamina.7 Intact basal lamina and collagen fibrils on electron microscopy, together with intact collagen IV expression on immunohistochemical study, suggests that loss of anchoring between the basal lamina and the interstitium may be a new molecular mechanism of congenital muscular dystrophy.
Acknowledgments
Acknowledgment
The authors thank the patient and his family for their cooperation and vigorous support of this research work; Dr. Michio Hirano for reviewing the manuscript; and Ms. Fumie Uematsu and Ms. Kumiko Murayama for their technical assistance.
Footnotes
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Additional material related to this article can be found on the Neurology Web site. Go www.neurology.org and scroll down the Table of Contents for the September 24 issue to find the link for this article.
- Received March 4, 2002.
- Accepted May 29, 2002.
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