Am. J. Hum. Genet. 66:1398±1402, 2000 Report Classical Ehlers-Danlos Syndrome Caused by a Mutation in Type I Collagen Lieve Nuytinck,1 Margarida Freund,2 Lieven Lagae,3 Gerald E. Pierard,4 Trinh Hermanns-Le,4 and Anne De Paepe1 1Center for Medical Genetics, University Hospital Gent, Gent, Belgium; 2Centre de GeÂneÂtique Humaine, Universite Catholique de Louvain, Brussels; 3Department of Pediatric Neurology, University Hospital Leuven, Leuven, Belgium; and 4Laboratory of Experimental Dermatology and Dermatopathology, University of LieÁge, LieÁge, Belgium Classical Ehlers-Danlos syndrome (EDS) is characterized by skin hyperelasticity, joint hypermobility, increased tendency to bruise, and abnormal scarring. Mutations in type V collagen, a regulator of type I collagen ®brillogenesis, have been shown to underlie this type of EDS. However, to date, mutations have been found in only a limited number of patients, which suggests genetic heterogeneity. In this article, we report two unrelated patients with typical features of classical EDS, including excessive skin fragility, in whom we found an identical argininercysteine substitution in type I collagen, localized at position 134 of the a1(I) collagen chain. The arginine residue is highly conserved and localized in the X position of the Gly-X-Y triplet. As a consequence, intermolecular disul®de bridges are formed, resulting in type I collagen aggregates, which are retained in the cells. Whereas substitutions of glycine residues in type I collagen invariably result in osteogenesis imperfecta, substitutions of nonglycine residues in type I collagen have not yet been associated with a human disease. In contrast, argininercysteine substitutions in type II collagen have been identi®ed in a variety of chondrodysplasias. Our ®ndings show that mutations in other ®brillar collagens can be causally involved in classical EDS and point to genetic heterogeneity of this disorder. The Ehlers-Danlos syndrome (EDS) comprises a hetero- Insights into the molecular basis of the common, clas- geneous group of disorders affecting skin, ligaments and sical types of EDS (EDS I and II [MIM 130000 and MIM joints, and blood vessels (Steinmann et al. 1993). The 130010]) have only recently become available. These most recent classi®cation recognizes six subtypes, most EDS subtypes are characterized mainly by hyperelasticity of which have been linked with genetic defects affecting of the skin, hyperlaxity of the joints, increased tendency one of the ®brillar collagens (Beighton et al. 1998). Mu- to bruise, and abnormal scarring. Transgenic mouse ex- tations in type III collagen have been identi®ed in the periments (Andrikopoulos et al. 1995) provided the ®rst vascular type of EDS (EDS IV [MIM 130050]) (Kui- evidence for the causal involvement of type V collagen vaniemi et al. 1997). Structural mutations affecting the in this disorder. So far, however, <10 mutations in the procollagen I N-proteinase cleavage site were found in COL5A1 and COL5A2 genes have been reported in pa- the rare arthrochalasis variants of EDS (EDS VII A and tients with classical EDS (Nicholls et al. 1996; Wenstrup B [MIM 130060]) (Byers et al. 1997), whereas defects et al. 1996; De Paepe et al. 1997; Burrows et al. 1998; of the enzymes lysylhydroxylase and procollagen I N- Michalickova et al. 1998; Richards et al. 1998). This proteinase are implicated in the recessive occuloscoliotic raises the question of genetic heterogeneity in this EDS (EDS VI [MIM 225400]) (Kuivaniemi et al. 1997) and variant. In this study, we found an identical argininer dermatosparaxis (EDS VII C [MIM 225410]) (Colige et cysteine substitution in the a1(I) collagen chain in two al. 1999) variants, respectively. unrelated patients with classical EDS. These ®ndings provide the ®rst evidence for the causal involvement of Received September 20, 1999; accepted for publication January 31, another ®brillar collagen in this disorder. 2000; electronically published March 17, 2000. Patient 1, a 5-year-old girl and the only child of a Address for correspondence and reprints: Dr. Anne De Paepe, Center healthy nonconsanguineous couple, was born at 137 wk for Medical Genetics, 0K5, 185 De Pintelaan, B-9000 Gent, Belgium. of gestation, after premature rupture of membranes. She E-mail: [email protected] q 2000 by The American Society of Human Genetics. All rights reserved. had a history of easy bruising and scarring after minimal 0002-9297/2000/6604-0021$02.00 trauma and presented a soft, velvety, and hyperexten- 1398 Reports 1399 Figure 1 Ultrastructural ®ndings in the skin of patient 2. A, On the transverse section (28,400#), the collagen ®brils show a distinct variability in diameter; some ®brils are larger and irregular in outline (arrowhead). B, On the longitudinal section (28,400#), the deposition of granulo®lamentous material along the collagen ®brils is visible (arrowhead). In some foci, collagen ®brils have an unraveled and disorganized aspect (arrow). sible skin. In addition, she had atrophic paper scars on nation indicated that she had no signs of osteopo- the face, elbows, knees, and shins; ecchymoses on the rosis. lower legs; and generalized joint hyperlaxity. Her facial Patient 2 was a 7-year-old boy, the only affected child appearance, which included redundant skin folds on the in a sibship of four children from healthy, nonconsan- eyelids and very soft earlobes, was reminiscent of clas- guineous parents. He was born at 137 wk of gestation sical EDS. Her sclerae were white, and X-ray exami- and showed hypotonia in the ®rst months of life. An 1400 Am. J. Hum. Genet. 66:1398±1402, 2000 type I and type III collagen compared with control cell lines. In patient 1, analysis of the cell-layer collagens showed the presence of an additional band correspond- ing to dimeric collagen a-chains (®g. 2), which migrated higher up in the gel. In patient 2, a similar collagen pro®le was seen, although the additional band repre- senting the collagen dimers was of lesser intensity. The dimers disappeared after reduction with b-mercapto- ethanol, which suggests the presence of disul®de- bounded collagen a-chains. Two-dimensional cyanogen bromide peptide mapping showed that this additional band was composed of a1(I) collagen chains (data not shown). The thermal stability of the collagen dimers was decreased by 17C compared with the normal a1(I) col- lagen chains. Mutation analysis of the COL1A1 cDNA sequence was performed, and in half the clones a CrT transition (nucleotide 934rexon 14) was identi®ed, which resulted in the substitution of arginine (CGC) for cysteine (TGC) in position 134 of the procollagen a1(I) chain (®g. 3). Analysis of the sequencing of the genomic DNA con- ®rmed the mutation in both patients and showed it to Figure 2 SDS-PAGE of radiolabeled, pepsin-digested collagens be absent in the parents. Because the a1(I)-R134C mu- from the medium (left) and the cell layer (right), produced by the tation was not detectable by restriction digestion, ge- ®broblasts of patient 1. The pattern of the medium collagens is com- nomic DNA from 50 control individuals was investi- parable to that of controls. In the cell layer, a population of dimeric collagen a-chains is seen (arrow) that disappears after reduction. P = gated by SSCP analysis for the COL1A1±exon 14 area patient 1; N = control samples. and surrounding sequences, but no abnormal migration shift was seen. The mutation was not found in 30 un- related patients with classical EDS in whom a COL5A1 operation was performed for strabismus. When clinically and COL5A2 mutation screening was negative. Muta- examined at age 5 years, he had typical features of clas- tion analysis of the COL3A1, COL5A1, and COL5A2 sical EDS, including a soft and doughy skin texture, cDNA was normal in both patients. moderate skin hyperextensibility, and joint hyperlaxity. The mutation identi®ed in both EDS patients was a In addition, he had a pronounced tendency for splitting substitution of a basic arginine in the X position of the of the skin, easy bruising, and impaired wound healing. Gly-X-Y triplet of the type I collagen a1 chain by an He also presented an unusual tenderness of the skin and uncharged polar cysteine, an amino acid residue, which soft tissues, evident when he was touched. He had a is not normally present in the triple helical domain of pectus excavatum and ¯at feet. His sclerae were white, type I collagen. The most common type of mutation in and radiographic examination showed no signs of osteo- type I collagen is the substitution of a glycine residue by porosis. a bulkier amino acid, which has invariably been asso- Ultrastructural studies performed on a skin biopsy ciated with osteogenesis imperfecta (OI) (Kuivaniemi et from patient 2 showed, on transverse section, a distinct al. 1997). The presence of a glycine residue in every third variability in the size of the collagen ®brils, with their position of the collagen a-chain is essential for correct diameter in the range of 70±250 nm (®g. 1A). Some of folding of the helical domain of ®brillar collagen mol- the ®brils were thicker and showed an irregular outline. ecules. Glycinercysteine substitutions in the a1(I) col- Longitudinal sections showed the presence of granulo- lagen chain are easily detected on biochemical analysis ®lamentous material along the ®brils (®g. 1B). In some by the presence of an additional band migrating in the foci, collagen ®brils had an unraveled aspect and ran in upper part of the gel, which consists of disul®de- a disorganized pattern. Mild dilatation of the rough en- bounded a1(I) collagen chains. doplasmic reticulum was seen. In this study, however, the cysteine substitution in- A dermal ®broblast culture was established from both volves an arginine residue in the X position of the Gly- patients, and collagen and procollagen were studied by X-Y triplet.