Abstract
The authors identified eight patients with Ullrich disease in whom collagen VI was present in the interstitium but was absent from the sarcolemma. By electron microscopy, collagen VI in the interstitium was never linked to the basal lamina. These findings suggest that in these patients it is not the total absence of collagen VI from the muscle but the failure of collagen VI to anchor the basal lamina to the interstitium that is the cause of Ullrich disease. Only one of the patients had a mutation in the collagen VI gene, suggesting that the primary abnormality in most of the patients involved some other molecules.
Ullrich disease is an autosomal recessive disorder characterized clinically from birth or early infancy by congenital muscular dystrophy, with contractures of the proximal joints and hyperextensibility of the distal joints, high-arched palate, and protuberant calcanei with normal intelligence.1 Recently, complete loss or reduction of collagen VI due to collagen VI gene mutations has been associated with Ullrich disease.2–6⇓⇓⇓⇓ Collagen VI is thought to play a role in connecting the basal lamina to the interstitium and a defect in this function is implicated in Ullrich disease.5,7⇓ We report eight patients with Ullrich disease in whom collagen VI was present in the interstitium but was specifically absent in the sarcolemma, bolstering the hypothesis that Ullrich disease is due to the loss of mechanical anchoring of the basal lamina to the interstitium.
Materials and methods.
Patients.
We studied eight Japanese patients with the diagnosis of Ullrich disease based on typical clinical features, i.e., delayed motor milestones, hyperextensibility of distal joints, and contractures of proximal joints. All were sporadic cases. Serum creatine kinase (CK) was mildly elevated in two-thirds of the patients (table). Biceps brachii muscle was biopsied in all patients.
Table Clinical summary of the patients
Histochemical and immunohistochemical analysis.
Muscle biopsy samples were frozen in liquid nitrogen-cooled isopentane for histochemistry and immunohistochemistry. Eight-micrometer-thick transverse serial sections were stained with hematoxylin and eosin, modified Gomori trichrome, and a battery of histochemical techniques. We also immunostained biopsy sections with monoclonal antibodies against collagen VI (1:500) (ICN Biomedicals, Aurora, OH), fibronectin (1:200) (CHEMICON, Temecula, CA), integrin α7,8 and α-dystroglycan (1:100) (Upstate, Lake Placid, NY), and the polyclonal antibody against collagen IV (1:2000) (Advance, Tokyo, Japan). We visualized the monoclonal antibodies by avidin-biotin-peroxidase complex method (Vector Laboratories, Burlingame, CA) using biotinylated goat anti-mouse IgG (Vector) and 3,3′-diaminobenzidine except for double immunostaining for collagen IV and VI, for which we used two secondary antibodies—FITC-labeled anti-mouse immunoglobulin G (IgG) (Leinco Technology, St. Louis, MO) and rhodamine-labeled anti-rabbit IgG (Leinco)—and the sections were examined by fluorescence microscopy.
Sequence analyses.
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).
In COL6A1 and COL6A2, we amplified two overlapping fragments, encompassing nt 35 through 1299 and nt 1280 through 3133 (NM001848) (all nucleotide numbers are based on the open reading frame [ORF] in the cDNA sequence indicated by each accession number), and nt 21 through 1390 and nt 1292 through 3136 (NM001849), respectively, which cover the entire ORF. In COL6A3, we amplified three overlapping fragments, encompassing nt 64 through 3406, nt 3323 through 6347, and nt 6257 through 9610 (NM400369). We directly sequenced the amplified fragments with the PCR primers9 and relevant internal primers using BigDye Terminator Cycle Sequencing Kit (PE Biosystems, Foster, CA), and electrophoresed the samples using ABI PRISM 377 and 3100 DNA sequencer (PE Biosystems). We also sequenced the amplified COL6A2 genomic fragments in lymphocyte DNA from Patient 5 encompassing intron 22 through 23 and intron 26 through exon 27.
With biglycan and decorin, we amplified each exon and flanking sequences by PCR in DNA from the patients and directly sequenced the amplified fragments (primer information available upon request).
Electron microscopy.
For electron microscopy, a portion of the muscle biopsy was fixed in 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 were stained with uranyl acetate and lead citrate.
Results.
Histochemical and immunohistochemical analyses.
All the biopsies showed variation in muscle fiber size, increased endomysial connective tissue, and regenerating fibers. There were necrotic fibers in six biopsies (figure).
Figure. Pathologic features of the disease. (A–C) Immunostaining for collagen VI (A: normal, B: Fukuyama-type CMD [FCMD], and C: Ullrich disease). Collagen VI is present in the interstitium but is markedly reduced or absent in the sarcolemma in an Ulrich disease patient. (D–F) Immunostaining for collagen IV (D: normal, E: FCMD, and F: Ullrich disease). Collagen IV is present in the sarcolemma. (G–I) Superimposed images (G: nonFCMD, H: FCMD, and I: Ullrich disease). Both collagen VI and collagen IV are present in sarcolemma in other congenital muscular dystrophies, as indicated by yellow (G and H). In contrast, only red is seen in the sarcolemma in Ullrich disease although interstitium is stained green (I). (J and K) Electron micrographs (J: FCMD and K: Ullrich disease). Microfibrils usually link to the basal lamina, as exemplified in FCMD muscle (arrowheads) (J). In contrast, in Ullrich disease, the basal lamina is intact and microfibrils are present in the interstitium (encircled), but is never associated with the basal lamina (arrowheads) (K). (Bar = 50 μm in A-I; 1 μm in J and K.)
By immunohistochemistry, collagen VI was present in the interstitium and sarcolemma in normal controls and in muscle samples from patients with other forms of congenital muscular dystrophy (CMD) (see the figure, A and B). In our patients, however, collagen VI was markedly reduced or absent in the sarcolemma while it was present in the interstitium (see the figure, C). We confirmed the specific absence of collagen VI from the sarcolemma by double immunostaining for collagen VI (see the figure, A through C) and collagen IV (see the figure, D through F), a major component of basal lamina. In Fukuyama-type CMD (FCMD) and non-Fukuyama-type CMD (nonFCMD) muscles, collagen VI and collagen IV were colocalized in the sarcolemma (see the figure, G and H). By contrast, in patients with Ullrich disease, only collagen IV, but not collagen VI, was present in the sarcolemma although collagen VI was present in the interstitium (see the figure, I).
On immunostaining for proteins interacting with collagen VI, integrin α7, α-dystroglycan, and fibronectin, all were present in the sarcolemma, as in controls (data not shown).
Sequence analyses.
None of our patients had mutations in genes encoding collagen VI subunits, biglycan, and decorin, except that Patient 5 had a compound heterozygous mutation in COL6A2 gene. On direct sequencing of the RT-PCR products from Patient 5, there were skipping of entire exon 23 and a six-bp deletion in exon 26, both in a heterozygous manner. In the genomic DNA, we found a heterozygous G-to-A substitution at position +5 in intron 23 and the corresponding heterozygous six-bp deletion in exon 26. The latter mutation deleted one of the two tandem repeats of the sequence CATCGG in nt 2268–2273 and 2274–2279 in COL6A2 ORF, which is predicted to delete isoleucine and glycine, at residues 759 and 760 (or 757 and 758) (data not shown). We also sequenced DNA from the parents of Patient 5 and found the former mutation in the mother and the latter in the father, both in heterozygous mode. Both mutations were absent in 100 normal chromosomes.
Electron microscopy.
The basal lamina was intact even in degenerating muscle fibers (see the figure, J). Collagen fibrils in the interstitium appeared normal with a periodic pattern of about 65 nm intervals. Microfibrils, which are known to be collagen VI, were present in the interstitium, but they were never linked to the basal lamina by electron microscopy (see the figure, K).
Discussion.
Three types of collagen VI immunostaining pattern have been reported in Ullrich disease: normal, complete absence, and generalized reduction (partial deficiency).3–6⇓⇓⇓ The eight patients in this study had a new mode of collagen VI involvement, its almost complete absence specifically from the sarcolemma, but not from the interstitium.
Among our eight patients, only one had a compound heterozygous mutation in the COL6A2 gene, but even these mutations may not be pathogenic because they are in-frame, although they were absent in 100 normal chromosomes in our study. Alternatively, mutations may exist in the noncoding regions, which we did not sequence. Collagen VI is thought to anchor the basement membrane in skeletal muscle by interacting with collagen IV, a major component of the basal lamina.7 However, by electron microscopy, there was no connection between collagen VI microfibrils in the interstitium with the basal lamina even though both the basal lamina and collagen fibrils were morphologically intact. These findings suggest that not only the absence of collagen VI from skeletal muscle but also the absence of collagen VI from the sarcolemma alone, both of which result in the loss of anchoring between the basal lamina and the interstitium, can cause Ullrich disease. Thus, our findings indicate genetic heterogeneity in Ullrich disease with collagen VI abnormality.
Proteins interacting with collagen VI are natural candidates to be the molecule primarily responsible in our patients. Indeed, mice deficient in the sarcolemmal protein, biglycan, which is also thought to bind to collagen VI, were reported to show a reduction in collagen VI, especially in the sarcolemma.10 We, therefore, investigated the proteins that potentially bind to collagen VI, including biglycan, decorin, integrin α7, α-dystroglycan, and fibronectin. However, integrin α7, α-dystroglycan, and fibronectin were all present by immunohistochemistry. Furthermore, no mutations were found in biglycan and decorin genes, suggesting that these proteins are unlikely to be involved. Nevertheless, there still remains the possibility that other proteins that interact in a similar fashion with collagen VI could be responsible for the disease in our patients and further studies are necessary to identify the primary cause.
Acknowledgments
The authors thank the patients and their families for their cooperation and support of this research and F. Uematsu, K. Tatezawa, and K. Murayama for technical assistance.
Footnotes
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See also page 529
- Received May 19, 2003.
- Accepted November 3, 2003.
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