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. 2001 Jun 19;98(13):7516-21.
doi: 10.1073/pnas.121027598. Epub 2001 May 29.

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI

O Camacho Vanegas  1 , E Bertini, R Z Zhang, S Petrini, C Minosse, P Sabatelli, B Giusti, M L Chu, G Pepe
Affiliations

Ullrich scleroatonic muscular dystrophy is caused by recessive mutations in collagen type VI

O Camacho Vanegas et al. Proc Natl Acad Sci U S A. .

Abstract

Ullrich syndrome is a recessive congenital muscular dystrophy affecting connective tissue and muscle. The molecular basis is unknown. Reverse transcription-PCR amplification performed on RNA extracted from fibroblasts or muscle of three Ullrich patients followed by heteroduplex analysis displayed heteroduplexes in one of the three genes coding for collagen type VI (COL6). In patient A, we detected a homozygous insertion of a C leading to a premature termination codon in the triple-helical domain of COL6A2 mRNA. Both healthy consanguineous parents were carriers. In patient B, we found a deletion of 28 nucleotides because of an A --> G substitution at nucleotide -2 of intron 17 causing the activation of a cryptic acceptor site inside exon 18. The second mutation was an exon skipping because of a G --> A substitution at nucleotide -1 of intron 23. Both mutations are present in an affected brother. The first mutation is also present in the healthy mother, whereas the second mutation is carried by their healthy father. In patient C, we found only one mutation so far-the same deletion of 28 nucleotides found in patient B. In this case, it was a de novo mutation, as it is absent in her parents. mRNA and protein analysis of patient B showed very low amounts of COL6A2 mRNA and of COL6. A near total absence of COL6 was demonstrated by immunofluorescence in fibroblasts and muscle. Our results demonstrate that Ullrich syndrome is caused by recessive mutations leading to a severe reduction of COL6.

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Figures

Figure 1
Figure 1
Clinical aspect during the neonatal period of the patient in family A. (A) A remarkable distal hyperlaxity of wrist and fingers. (B) A peculiar congenital kyphotic contracture of the spine in the baby.
Figure 2
Figure 2
Pedigrees of UCMD families. Family A shows consanguinity between the parents. Family B displays the presence of two affected sons and two healthy parents formally demonstrating the presence of recessive mutations. In family C, the proband carries a de novo mutation identical to one of family B. The second mutation has not been found. We do not know which one of the two parents carries a mutation.
Figure 3
Figure 3
Diagrammatic representation of mutations in families A, B, and C. (A) The proband in family A has a homozygous insertion of a C in a stretch of Cs between nucleotides 1147 and 1151 in exon 13 encoding the triple helix of COL6A2. Both consanguineous parents are heterozygous for this mutation, which causes a frame shift and the formation of a premature termination codon at nucleotides 1347–1349 inside exon 16. (B) One of the COL6A2 alleles in both affected sons of family B has an A → G substitution at the conserved splice acceptor site (ag) at position −2 in intron 17, causing the activation of a cryptic acceptor site within exon 18. The aberrant splicing causes the deletion of 28 bp (nucleotides 1459–1486 in exon 18 in the cDNA), the slippage of the reading frame, and the formation of a premature termination codon at nucleotides 1631–1633. Their apparently healthy mother and also the proband in family C are heterozygous for the same mutation. (C) The second COL6A2 allele of both affected sons in family B has a G → A substitution at position −1 of intron 23, causing the skipping of the entire exon 24 of 46 bp (from nucleotide 1671 to nucleotide 1716) and the changing of the reading frame thereafter. Their apparently healthy father is heterozygous for this mutation. The nucleotide positions are numbered according to the complete coding sequence of the COL6A2 cDNA (GenBank accession no. AY029208) with nucleotide +1 corresponding to the translation start site.
Figure 4
Figure 4
Immunofluorescence of cultured fibroblasts in proposita of family A and B. (A) Immunofluorescence with anti-COL6 (see Materials and Methods) of normal fibroblasts. COL6 is secreted in the extracellular matrix and seems organized in a dense three-dimensional network that is entangled with the cells. (B) In fibroblasts of propositus of family A, COL6 is not detectable in the extracellular matrix, whereas a moderate labeling is present inside the cytoplasm of the cells. (C) In the propositus of family B, COL6 is not detectable inside the cytoplasm of the cells whereas a residual labeling is present in the extracellular matrix as small multiple dots. (Magnification = ×20).
Figure 5
Figure 5
Analysis of COL6 mRNA and protein in fibroblasts from the proband of family B. (A) Northern blot analysis showing little α2(VI) mRNA in the patient's fibroblasts. Fifteen mg of total RNA from fibroblasts of the proband in family B (lane 1) and a control (lane 2) were run on a 1% denaturing agarose gel, transferred to a nylon membrane, and hybridized with 32P-labeled cDNA probes for the α1(VI), α2(VI), and α3(VI) collagen chains. The sizes of the α1(VI), α2(VI), and α3(VI) collagen mRNAs are 4.2, 3.5, and 8–10 kb, respectively. Multiple α3(VI) mRNAs result from alternative splicing of exons encoding the N-globular domain (23). The amount of each splice variant varies in different control fibroblast strains according to the growth state of cells. Therefore, the difference in the intensity of the α3(VI) mRNA splice variants between the control and the patient probably is not a direct consequence of the mutation. (B) Immunoprecipitation showing a marked reduction in COL6 secretion. Fibroblasts from a control (lane 1), the proband of family B (lane 2), and a patient with BM (lane 3) were metabolically labeled overnight. The culture media were immunoprecipitated with antibodies specific for the α3(VI) collagen chain (25). The immunoprecipitated samples were reduced with 5% 2-mercaptoethanol and run on a 5% SDS/polyacrylamide gel. The α1(VI) and α2(VI) collagen chains comigrated at 140 kDa, whereas the α3(VI) collagen chains migrated at 260–300 kDa.
Figure 6
Figure 6
Immunofluorescence of muscle in the proposita of family B (A–D) and C (E–H). Immunolocalization of COL6–140 kDa (2C6 antibody; A, B, E, and F) and merosin (C, D, G, and H) in skeletal muscle from controls (A, C, E, and G) and patients (B, D, F, and H). The expression of COL6 was almost absent in muscle fibers of the patient of family B (B), and drastically reduced in the patient of family C (F), whereas merosin-staining seemed to be normal (D and H). (Bar = 80 μm.) These results show that this recessive disorder can be screened by immunofluorescence using monoclonal antibodies directed against one of the three COL6 subunits.
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