84686Med Genet 1998;35:846-848

A single base mutation in COL5A2 causes J Med Genet: first published as 10.1136/jmg.35.10.846 on 1 October 1998. Downloaded from Ehlers-Danlos syndrome type II

Allan J Richards, Sam Martin, Alan C Nicholls, J Barrie Harrison, F Michael Pope, Nigel P Burrows

Abstract lagen to the more abundant types Ehlers-Danlos syndrome (EDS) is a het- resulted in altered diameters.13-15 Also, erogeneous group ofconnective tissue dis- corneal collagen , where orders. Recently mutations have been represents a much larger proportion (15-20%) found in the genes for type V collagen in a of the total fibrillar collagen,'6 17 are smaller MRC Connective small number of people with the most than in other expressing tissues, Tissue Genetics common forms of EDS, types I and II. where type V collagen is less abundant (2-5%). Group, Department of Here we characterise a COL5A2 mutation Not all EDS I/II pedigrees are linked to Pathology, University in an EDS II family. Cultured dermal COL5A1 or COL5A218 (N P Burrows, unpub- of Cambridge, Tennis fibroblasts obtained from an affected sub- lished results) and other candidate genes Court Road, abnormal type V include those for '9 and X.2' Cambridge CB2 IQP, ject synthesised collagen. UK Haplotype analysis excluded COLSAl but While it is preferable initially to perform A J Richards was concordant with COL5A2 as the haplotype analysis in multigeneration families, S Martin disease locus. The entire open reading for sporadic cases or small families it is often A C Nicholls frame of the COL5A2 cDNA was directly unhelpful. Here we describe a COL5A2 muta- J B Harrison sequenced and a single base mutation tion in a small family which showed the clinical F M Pope detected. It substituted a glycine residue phenotype of EDS II (NPB). Electron micro- N P Burrows within the triple helical domain (G934R) scopic analysis of dermal collagen structure in MRC Connective of a2(V) collagen, typical of the dominant this family showed only minor variability in the Tissue Genetics negative changes in other , which collagen fibril size and shape (data not shown). Group, Institute of cause various other inherited connective Cultured skin fibroblasts were labelled with '4C Medical Genetics, tissue disorders. All three affected family proline and the collagens synthesised were University Hospital of members possessed the single base analysed by SDS polyacrylamide gel electro- Wales, Heath Park, V from the Cardiff CF4 4XN, UK change, which was absent in 50 normal phoresis (fig 1). Type collagen cell F M Pope chromosomes. line migrated more slowly than that from a (J7Med Genet 1998;35:846-848) control. Similar observations have been madehttp://jmg.bmj.com/ Departmnent of in cases of where Dermatology, Keywords: COL5A2; mutation; Ehlers-Danlos syn- mutations in either COLlAl or COL1A2 Addenbrooke's NHS drome cause overmodification ofboth al (I) and a2(I) Trust, Hills Road, in this case it was Cambridge CB2 2QQ, collagen .2' Thus, VUKTr N P Burrows Mutations in the genes for type V collagen COL5A1 1 1 (COL5AI and COL5A2) have recently been D9S18181 2 on September 29, 2021 by guest. Protected copyright. Correspondence to: reported'-4 in cases of the commonest forms of Dr Burrows. COL3A1 2 1 Ehlers-Danlos syndrome (EDS types I and II). D2S389 3 4 Other EDS also have defects which result Received 15 Decemiber 1997 types 2 Revised version accepted for in abnormal collagen molecules. Fibrillar publication 6 March 1998 collagens are the most abundant components and fibrils may be hetero- typic, being composed of more than one colla- COL5A1 gen type.5 6 The fibrillar collagen family D9S1818 11.2i c; includes the homologous types highly I, II, III, COL3A1 - (V1 V, and XI proteins, each with a long uninter- D2S389 4W___ rupted triple helix of Gly-X-Y repeats.7 Al- 2 1v though type V and XI collagens are quantita- _

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Figure 3 Sequence analysis. The mutant (M) and normal (N) cDNA and genomic sequence obtainedfrom various family members or control (as indicated) is shown. The same sense primer was usedfor each sequencing reaction. The DNA and corresponding amino acid sequences are wrinten adjacent to the autoradiographs. The heterozygous GIC base change which converts glycine 934 to arginine is indicated, as is the position of the intron within the genomic sequence. http://jmg.bmj.com/

likely that there was a mutation in either results (fig 4) and that this change was absent COL5A1 or COL5A2. in 50 chromosomes from 25 unrelated con- Haplotype analysis was undertaken using trols. markers (COL5A1, D9S1818, COL3A1, and This alteration is typical of many mutations D2S389) within or close to either COL5A1 or in other fibrillar collagens which cause inher-

COL5A2.8 22-25 The haplotypes (fig 2) ex- ited disorders, such as osteogenesis imperfecta, on September 29, 2021 by guest. Protected copyright. cluded COLMAM, but were concordant with the chondrodysplasias, and EDS type IV.26 COL5A2 as the disease locus. While the family This, along with the absence of this change in was too small to generate a significant lod normal subjects, provides strong evidence that score, the combination of protein profile with it is the causative mutation in this EDS II fam- haplotype analysis strongly implied a causative ily. Only a few mutations of COLMAM and mutation in COL5A2. The cDNA for the complete coding sequence of a2(V) collagen 1 1 N2I 1 r ki (accession NoY14690) was reverse transcribed =M and amplified from RNA as 11 overlapping products. Each of these were directly se- quenced. A single base mutation was seen, which converted an obligatory glycine within the triple helical region of the molecule to 11I|11 l l * * I arginine (fig 3). This change was confirmed in each affected family member by amplification and sequencing (fig 3) of a 500 bp genomic DNA product. Each affected subject contained the same G to C base change (data are only shown for III.2), and an intron was positioned between bases 3600 and 3601 of the cDNA sequence (fig 3). The mutation abolished a BsrI Figure 4 Restriction enzyme digestion. Genomic DNA restriction enzyme site which normally re- from each family member (as indicated) was amplified and moved 80 bp from one end ofthe 500 bp prod- incubated with the restriction enzyme BsrI. Electrophoresis on a 2% agarose gel was used to separate the products, uct. Incubation of the amplified genomic DNA along with undigested DNA (C) and DNA ofknown sizes with this enzyme confirmed the sequencing used as markers (M). 848 Richards, Martin, Nicholls, et al

COL5A2 have been characterised in EDS I/II 4 Michalickova K, Susic M, Willing MC, Wenstrup RJ, Cole WG. Mutations of the a2(V) chain of type V collagen patients.' 4 Three of these affect the impair matrix assembly and produces Ehlers-DanlosJ Med Genet: first published as 10.1136/jmg.35.10.846 on 1 October 1998. Downloaded from C-propeptide and possibly inhibit assembly of syndrome type I. Hum Mol Genet 1998;7:249-55. 5 Mendler M, Eich-Bender SG, Vaughan L, Winterhalter the mutant collagen with normal a chains. KH, Bruckner P. contains mixed fibrils of Others affect the correct splicing of exons cod- collagen types II, IX and XI._J Cell Biol 1989;108:191-7. for of the 6 Birk DE, Fitch JM, Babiaz JP, Doane KJ, Linsenmayer TF. ing part triple helix. These, like the Collagen fibrillogenesis in vitro: interaction of types I and V mutation described here, will have a dominant collagen regulates fibril diameter. J Cell Sci 1990;95:649- as will coassemble with 57. negative effect, they 7 Van Der Rest M, Garrone R. The collagen family of normal collagen a chains. proteins. FASEBJ 1991;5:2814-23. COL5A2 is also a candidate for Stickler 8 Linsenmayer TF, Gibney E, Igoe F, et al. Type V collagen: syn- molecular structure and fibrillar organization of chicken drome as a2(V) coassembles with al (XI) at1 (V) NH2-terminal domain, a putative regulator of in the vitreous27 and a mutation in corneal fibrillogenesis. Jf Cell Biol 1 993;121:1181-9. collagen 9 Fichard A, Kleman JP, Ruggiero F. Another look at collagen COLI lAl was previously shown to cause that V and XI molecules. Matrix Biol 1995,14:515-31. disorder.28 However, a detailed ophthalmologi- 10 Burgeson RE, El Adli FA, Kaitila II, Hollister DW. Fetal membrane collagens: identification of two new collagen o cal examination of the proband from this EDS chains. Proc NatlAcad Sci USA 1976,73:2579-83. family showed no abnormality of the vitreous, 11 Elstow SF, Weiss JB. Extraction, isolation and characteriza- tion of neutral salt soluble type V collagen from fetal calf thereby excluding . How- skin. Collagen Rel Res 1983;3:181-94. ever, the cornea showed keratoconus, a devel- 12 Broek DL, Madri J, Eikenberry EF, Brodsky B. Characteri- zation of the tissue form of type V collagen from chick opmental forward bulging of the central area of bone.JfBiol Chem 1991;260:555-62. the cornea. A previous study of44 patients with 13 Marchant JK, Hahn RA, Linsenmayer TF, Birk DE. Reduc- tion of type V collagen using a dominant-negative strategy keratoconus showed that 50% had clinical alters the regulation of fibrillogenesis and results in the loss signs suggestive of EDS, indicating a possible of corneal-specific fibril morphology. JfCell Biol 1996;135: 1415-26. link between these disorders.28 These corneal 14 Li Y, Lacerada DA, Warman ML, et al. A fibrillar collagen changes confirm observations seen in a trans- gene, COLl lAl, is essential for skeletal morphogenesis. Cell 1995;80:423-30. genic mouse model with a targeted deletion of 15 Garofalo S, Metsaranta M, Ellard J, et al. Assembly of carti- exon 6 from col5a2.3° Heterozygous mice, lage collagen fibrils is disrupted by overexpression of normal type II collagen in transgenic mice. Proc NatlAcad although clinically normal, had abnormal Sci USA 1993;90:3825-9. ultrastructural appearance of corneal collagen 16 Tseng SCG, Smuckler D, Stern R. Comparison of collagen types in adult and fetal bovine corneas. _7 Biol Chem 1982; fibrils. Because of the corneal abnormality of 257:2627-33. the index case here, it is likely that in humans 17 McLaughlin JS, Linsenmayer TF, Birk DE. Type V collagen synthesis and deposition by chicken embryo corneal fibro- type V collagen is also an important compo- blasts in vitro. _7 Cell Sci 1989;94:371-9. nent of the cornea. 18 Greenspan DS, Northrup H, Au KS, et al. COL5Al: fine genetic mapping and exclusion as candidate gene in Two other candidate genes for EDS I/II are families with nail-patella syndrome, tuberose sclerosis 1, those which code for decorin'9 and tenascin hereditary hemorrhagic telangiectasia and Ehlers-Danlos syndrome type II. Genomics 1995;25:737-9. X.20 In sporadic cases of EDS I/I, or small 19 Danielson KG, Baribault H, Holmes DF, Graham H, families in which exclusion of candidate genes Kadler KE, Iozzo RV. Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility..7 is not possible, variation in clinical signs may Cell Biol 1997;136:729-43. provide important clues for determining which 20 Burch GH, Gong Y, Liu W, et al. Tenascin-X deficiency http://jmg.bmj.com/ is associated with Ehlers-Danlos syndrome. Nat Genet 1997; gene is mutated. In this instance the index case 17:104-8. presented with keratoconus. Unfortunately, as 21 Bonadio J, Byers PH. Subtle structural alterations in the the two chains of type I procollagen produce osteogenesis imper- other affected subjects were not fecta type II. Nature 1985;316:363-6. available for ophthalmic examination, we do 22 Lee B, Vitale E, Superti-Furga A, Steinmann B, Ramirez F. not know if it with in G to T transversion at position +5 of a splice donor site segregates EDS this fam- causes skipping of the preceding exon in the type III ily. However, there has been one previous procollagen transcripts of a patient with Ehlers-Danlos

of flattened corneas a syndrome type IV. _7 Biol Chem 1991;266:5256-9. on September 29, 2021 by guest. Protected copyright. report associated with 23 Cutting GR, McGinniss MJ, Kasch LM, Tsipouras P, COL5A1 mutation.2 It is possible, therefore, Antonarakis SE. Physical mapping by PFGE localizes the that abnormal COL3A1 and COL5A2 genes to a 35-kb region on human corneal morphology may indi- chromosome 2. Genomics 1990;8:407-10. cate the presence of COL5A1 and COL5A2 24 Dib C, Faure S, Fizames C, et al. A comprehensive genetic map of the human genome based on 5,264 microsatellites. mutations in other EDS patients. Nature 1996;380:152-4. 25 Schuler GD, Boguski MS, Stewart EA, et al. A gene map of We would like to thank Mr M P Snead for ophthalmic examina- the human genome. Science 1996;274:540-6. tion of the proband and Mrs M Laidlaw for assistance with tis- 26 Kuivaniemi H, Tromp G, Prockop DJ. Mutations in fibrillar sue culture. NPB was supported by a grant from the Wellcome collagens (type I, II, III and XI, fibril-associated collagen Trust (type IX), and network-forming collagen (type X) cause a (044116). spectrum of diseases of bone, cartilage and blood vessels. Hum Mutat 1997;9:300-15. 1 Wenstrup RJ, Langland GT, Willing MC, Dsouza VN, Cole 27 Mayne R, Brewton RG, Mayne PM, Baker JR. Isolation and WG. A splice-junction mutation in the region of COL5A1 characterization of the chains of type V/type XI collagen that codes for the carboxyl propeptide of pro-au (V) chains present in bovine vitreous. _7 Biol Chem 1993;268:9381-6. results in the gravis form of Ehlers-Danlos syndrome (type 28 Richards AJ, Yates JRW, Williams R, et al. A family with I). Hum Mol Genet 1996;5:1733-6. Stickler syndrome type 2 has a mutation in the COLI lAI 2 Nicholls AC, Oliver JE, McCarron S, Harrison JB, gene resulting in the substitution of glycine 97 by valine in Greenspan DS, Pope FM. An exon skipping mutation of a al (XI) collagen. Hum Mol Genet 1996,5:1339-43. type V collagen gene (COL5A1) in Ehlers-Danlos 29 Robertson I. Keratoconus and the Ehlers-Danlos syndrome: syndrome. iMed Genet 1996;33:940-6. a new aspect of keratoconus. MedJAust 1975;1:571-3. 3 De Paepe A, Nuytink L, Hausser I, Anton-Lamprecht I, 30 Andrikopoulos K, Liu X, Keene DR, Jaenisch R, Ramirez F. Naeyaert JM. Mutations in the COL5A1 gene are causal in Targeted mutation in the col5a2 gene reveals a regulatory the Ehlers-Danlos syndromes I and II. Am J Hum Genet role for type V collagen during matrix assembly. Nat Genet 1 997;60:547-54. 1995;9:31-6.