Homozygous Gly530Ser substitution inCOL5A1 causes mild classical Ehlers-Danlos syndrome

American Journal of Medical Genetics 109:284–290 (2002)

Homozygous Gly530Ser Substitution in COL5A1Causes Mild Classical Ehlers-Danlos Syndrome

C. Giunta,1 L. Nuytinck,2 M. Raghunath,3 I. Hausser,4 A. De Paepe,2 and B. Steinmann1*1Division of Metabolism and Molecular Pediatrics, University Children’s Hospital, Zurich, Switzerland2Center of Medical Genetics, Ghent University Hospital, Ghent, Belgium3Department of Dermatology, University of Munster, Munster, Germany4EM Laboratory, Department of Dermatology, University of Heidelberg, Heidelberg, Germany

Skin hyperelasticity, tissue fragility withatrophic scars, and joint hypermobilityare characteristic for the classical type ofEhlers-Danlos syndrome (EDS). The diseaseis usually inherited as an autosomal do-minant trait; however, recessive mode ofinheritance has been documented in tenas-cin-X-deficient EDS patients. Mutations inthe genes coding for collagen a1(V) chain(COL5A1), collagen a2(V) chain (COL5A2),tenascin-X (TNX), and collagen a1(I) chain(COL1A1) have been characterized in pa-tients with classical EDS, thus confirmingthe suspected genetic heterogeneity. Recen-tly, we described a patient with severeclassical EDS due to a Gly1489Glu substitu-tion in the a1(V) triple-helical domain whowas, in addition, heterozygous for a disease-modifying Gly530Ser substitution in thea1(V) NH2-terminal domain [Giunta andSteinmann, 2000: Am. J. Med. Genet. 90:72–79; Steinmann and Giunta, 2000: Am. J. Med.Genet. 93:342]. Here, we report on a 4-year-old boy with mild classical EDS, born tohealthy consanguineous Turkish parents;the mother presented a soft skin, while thefather had a normal thick skin. Ultrastruc-tural analysis of the dermis revealed in thepatient the typical ‘‘cauliflower’’ collagenfibrils, while in both parents variable mod-erate aberrations were seen. Mutation re-

vealed the presence of a homozygousGly530Ser substitution in the a1(V) collagenchains in the patient, while both parentswere heterozygous for the same substitu-tion. An additional mutation in either theCOL5A1 and COL5A2 genes was excluded.Furthermore, haplotype analysis with poly-morphic microsatellite markers excludedlinkage to the genes coding for a3(V) col-lagen (COL5A3), tenascin-X (TNX), throm-bospondin-2 (THBS2), and decorin (DCN).These new findings support further ourprevious hypothesis that the heterozygousGly530Ser substitution is disease modifyingand now suggest that in the homozygousstate it is disease causing.� 2002 Wiley-Liss, Inc.

KEY WORDS: classical EDS; a1(V) col-lagen; fibril formation; mis-sense mutation

INTRODUCTION

Patients affected with the classical type of Ehlers-Danlos syndrome (EDSI/II [Beighton et al., 1998;Steinmann et al., 2002]) present with marked jointhypermobility and hyperelastic skin with atrophic,hemosiderotic scars and molluscoid pseudotumors. Ithas been proposed that the skin abnormalities resultfrom the disorganization and enlargement of theheterotypic collagen fibrils containing collagens I, III,and V, some of which present as characteristic ‘‘cauli-flower-like’’ fibrils that are sparse and scattered widelyapart [Vogel et al., 1979; Hausser and Anton-Lam-precht, 1994]. Amousemodel lacking exon 6 ofCOL5A2presents similarly disorganized and enlarged dermalcollagen fibrils and phenotypically resembles classicalEDS [Andrikopoulos et al., 1995]. Therefore, it has beenproposed that collagen V plays a crucial role in contro-lling the formation of the heterotypic fibrils and inregulating their diameter [Birk et al., 1990; Linsen-mayer et al., 1993; Marchant et al., 1996].

Grant sponsor: Swiss National Science Foundation; Grantnumber: 32-59445.99; Grant sponsor: Fund for ScientificResearch, Flanders; Grant number: G.0090.99N.

*Correspondence to: Prof. B. Steinmann, Division of Metabo-lism and Molecular Pediatrics, University Children’s Hospital,Steinwiesstrasse 75, CH-8032 Zurich, Switzerland.E-mail: [email protected]

Received 11 July 2001; Accepted 31 January 2002

DOI 10.1002/ajmg.10373

� 2002 Wiley-Liss, Inc.

Haploinsufficiency of COL5A1 is a common cause ofclassical EDS [Schwarze et al., 2000; Wenstrup et al.,2000], while missense and splice-site mutations ineither COL5A1 and COL5A2 genes account only for alimited number of cases [Nicholls et al., 1996; Torielloet al., 1996; Wenstrup et al., 1996; Burrows et al., 1998;De Paepe and Nuytinck, 1998; Michalickova et al.,1998; Richards et al., 1998; Giunta and Steinmann,2000; Steinmann and Giunta, 2000]. The findingof mutations in COL1A1 [Nuytinck et al., 2000] andTNX [Burch et al., 1997; Schalkwijk et al., 2001] inpatients with classical EDS, as well as the exclusion oflinkage to COL5A1 and COL5A2 in several families[Greenspan et al., 1995; Loughlin et al., 1995], indicateslocus heterogeneity [Steinmann et al., 2002].

A clinically distinct recessive form of classical EDScaused by deficiency of tenascin-X (TNX) has also beendescribed [Burch et al., 1997; Schalkwijk et al., 2001].Furthermore, deficiency of other proteins involved inthe formation and/or regulation of collagen fibrils, suchas decorin and thrombospondin-2, may account forclassical EDS in some families. In fact, mice harboringa targeted disruption of the genes coding for decorin[Danielson et al., 1997] and thrombospondin-2[Kyriakides et al., 1998] have fragile skin with re-duced tensile strength. Furthermore, the ultrastruc-ture of their dermis is remarkable for the presence ofabnormally large and irregularly shaped fibrils withoutcauliflower appearance intermingled with thin collagenfibrils.

In a previously reported case we showed evidencethat the heterozygous Gly530Ser substitution in thea(1) V collagen chain is a disease-modifying mutation[Giunta and Steinmann, 2000; Steinmann and Giunta,2000]. The data presented here confirm our previoushypothesis and suggest further that homozygosity forGly530Ser causes classical EDS.

MATERIALS AND METHODS

Clinical Data

The propositus (C.B.) is a 4-year-old boy born tohealthy first-cousin parents from Turkey (Fig. 1). Therewas no obvious family history for connective tissuedisorders. He presented with moderate skin hyperelas-ticity, marked joint laxity, and joint hypermobility,especially in his fingers. The skin had a doughy texture.He had multiple hematomas on the arms and the lowerlegs (Fig. 2), posttraumatic hyperpigmentation on thechest, and several scars on the forehead with palpableindurated bumps in the subcutis. He had a moderatepectus excavatum and bilateral genua recurvata.Furthermore, he had antimongoloid palpebral fissuresand epicanthus. He was hyperopic (R/L, þ12.5/þ12.5)and astigmatic. At age 2 months he underwent abilateral inguinal hernia operation. The patient’smother

Fig. 1. Pedigree. The parents are first cousins. The arrow indicates thepatient affected with mild classical EDS; his two brothers, born prema-turely at the 26th week of gestation, died of unknown causes.

Fig. 2. Multiple hematomas and a doughy appearance are present in the lower legs (A and C) and the arms (B and C). [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

EDS Caused by Homozygous Gly530Ser in COL5A1 285

was born with a clubfoot, which spontaneously remitted;she presented with soft skin and hypermobile fingers.She had three abortions in early pregnancy and gavebirth to two boys born prematurely in the 26th week ofgestation, because of premature rupture of the mem-branes, who died of an unknown cause 2weeks and a fewhours after birth, respectively (Fig. 1). The patient’sfather had normal-appearing and thick skin.

Ultrastructural Study

Skin biopsies from the upper arm of the proband andhis parents was processed for transmission electronmicroscopy accordingly to the method previously repor-ted [Hausser and Anton-Lamprecht, 1994].

Collagen Analysis

Cultured dermal fibroblasts were established from askin biopsy obtained from the proband and his parentsand were maintained under standard conditions. Cellswere radiolabeled, and at the end of the incubation, themedium and cell layer were harvested separately.Collagen samples were prepared by digestion withpepsin, precipitated with ethanol, separated by 5%sodium dodecylsulfate-polyactylamide gel electrophor-esis (SDS-PAGE), and visualized by fluorography[Steinmann et al., 1984].

Mutation Analysis

Genomic DNA was isolated from peripheral bloodleukocytes and confluent cultured fibroblasts usingcommercially available kits (Qiaquick, Qiagen andDneasy, Invitrogen).

Total RNA was isolated from cultured dermal fibro-blasts using TRIZOL Reagent (Invitrogen Life Tech-nologies, Carlsbad, CA) following the manufacturer’sprotocol. An overnight incubation of fibroblast cultureswith cycloheximide (100 mg/ml) was performed prior toRNA extraction in order to prevent nonsense-mediatedmRNA decay [Wenstrup et al., 2000].

Mutation analysis in COL5A1 was performed onreverse transcription-PCR (RT-PCR) products by acombination of single-strand conformation polymorph-ism (SSCP) [Orita et al., 1989] and conformation-sensitive gel electrophoresis (CSGE) [Ganguly et al.,1993] and by direct sequencing. First-strand copy DNA(cDNA) synthesis and PCR amplification were per-formed as previously described [Giunta and Stein-mann, 2000]. Sequences were originated using the Big-Dye Terminator cycle sequencing kit (Applied Biosys-tem, Foster City, CA), and the analysis was performedon the ABI Prism 310 automated DNA sequencer(Applied Biosystem).

Frequency of Gly530Ser was assessed in a normalCaucasian population, by means of either amplificationrefractory mutation system (ARMS) PCR [Giunta andSteinmann, 2000] or restriction enzyme analysis of the286-bp region between introns 12 and 13 of COL5A1with the endonuclease BbvI (NEB, Beverly, MA).

The sequence encoding the entire N-terminal, thea-helical, and the C-terminal regions of COL5A2 were

amplified by RT-PCR in 13 overlapping fragments.Mutation analysis of COL5A2 was performed using acombination of CSGE and SSCP analyses. Fragmentsshowing an aberrant migration shift were subsequentlysequenced.

Haplotype Analysis

Haplotype analysis was undertaken using highlypolymorphic microsatellite markers with a minimumexpected heterozygosity �0.58, which map as the can-didate disease loci COL5A3, TNX, THBS2, and DCN toa �5-centimorgan interval. Microsatellite markers forTNX (D6S1548 and D6S1562), THBS2 (D6S281 andTBP.PCR1/2), and DCN (D12S322 and D12S351) wereselected by searching the Genome Database and theGeneMap’99 database (GB4 map). For DCN, an intra-genic marker with low maximum heterozygosity (DCN-A/�B) was additionally used. For COL5A3, genotypeswere generated by using the microsatellite markerD19S413, which maps, as COL5A3, to the �3.6-centi-morgan interval between WI-8049 and WI-7557[Imamura et al., 2000]. PCR-based genotyping wasgenerated by means of the ABI 310 automated se-quencer and the Genscan and Genotyper software.

RESULTS

Ultrastructure

The biopsy of the patient revealed thin dermis with aheterogeneous morphology; some areas showed smallcollagen bundles and big, morphologically normalelastic fibers. In other areas the overall architectureof the dermis was disturbed with the elastic fibersclumping together in groups, while other parts of thedermis were free of elastic material, indicating a veryloose structure of the tissue. Electron microscopyshowed small collagen bundles with numerous abnor-mal fibrils, cauliflower-like in the cross sections andtwisted, broad, and unraveled in the longitudinal sec-tions (Fig. 3a). Some collagen fibers consisted entirelyof irregular collagen material as aggregation of bizarre,irregularly shaped fibrils (Fig. 3b).

Ultrastructural analysis of the father’s dermisshowed collagen bundles consisting of mainly normalcollagen fibrils with uniform circular cross section, afew fibrils with irregular size and contours, and a verylow amount of composite fibrils with enlarged cauli-flower-like cross sections and twisted longitudinalsections (Fig. 3c). These findings were suggestive of avery mild hypermobile EDS phenotype. The electronmicroscopic findings of the mother’s dermis revealedpatches of either normal collagen bundles or looselyarranged bundles containing either single or severalabnormally large collagen fibrils, as well as fibrils withcauliflower-like cross sections (Fig. 3d). This was sug-gestive of a mild classical EDS phenotype, which wasmore severe than that of her husband.

Biochemical Data

Radiolabeled collagens retained by cultured dermalfibroblasts and secreted into the culture medium were

286 Giunta et al.

separated and treated with pepsin and subjected toSDS-PAGE and fluorography. In the proband and hisparents we detected normal migration patterns of achains for collagen V, as well as for collagens I and III.

Mutation Analysis

Mutation analysis of RT-PCR products covering thecoding sequence of the N-propeptide, the entire triple-helical region, and part of the C-propeptide of COL5A1detected a homozygous G-to-A transition in exon 13(nucleotide position 1588), which converted a glycine atposition 530 of the NH2-terminal domain to a serine(Fig. 4). The result was confirmed by sequencing thePCR-amplified genomic DNA from the proband (Fig. 4).

Restriction enzyme analysis of PCR-amplified genomicDNA with the endonuclease BbvI showed the presenceof the substitution in the proband’s unaffected fatherandmother (not shown). In both parents heterozygosityfor the G-to-A transition in exon 13 was furtherconfirmed by cycle sequencing (Fig. 4).

ARMS PCR and restriction fragment length poly-morphism (RFLP) analysis with the BbvI restric-tion endonuclease revealed that Gly530Ser has arelative frequency of 2% (2/101) in a normal Caucasianpopulation.

Haplotype Analysis

To clarify whether any of the following genes,COL5A3, TNX, THBS2, and DCN, were involved in

Fig. 3. Ultrastructure of dermis of the patient and his parents. A:Patient: section of the dermis showing irregularly shaped (cross section)and twisted (longitudinal sections) collagen fibrils that are aggregated in atangle pattern in this particular area, whereas in others the findings areless pronounced. B: Patient: cross section of the dermis showing abnormalcollagen fibrils with enlarged cauliflower outlines (arrows). C: Mother:

collagen fibrils are less irregular in shape than in the patient, but abnormalcollagen fibrils do occur with enlarged diameters and cauliflower contours(arrows). D: Father: abnormal collagen fibrils with slightly enlargeddiameter and irregular contour (arrow) occur within otherwise regularlyshaped fibrils.

EDS Caused by Homozygous Gly530Ser in COL5A1 287

the disease, haplotype analysis was performed. Lack ofhomozygosity for microsatellites closely flanking theCOL5A3, TNX, THBS2, and DCN genes excluded themas the disease loci (Fig. 4).

DISCUSSION

Collagen V is widely distributed in vertebrate tissuesas (a1(V))2a2(V) heterotrimer and forms with collagensI and III heterotypic fibrils in the extracellular matrix.A considerable body of evidence indicates that collagenV plays a crucial role in controlling the formation of theheterotypic fibrils and in regulating their diameter. Infact, the high concentration of collagen V in the chickcorneal stroma is required for the small, uniform fibril-lar diameter observed in this tissue, as opposed to othercollagen I-containing tissues, where fibrillar diametersare larger [Birk et al., 1990]. Using a dominant-negative strategy to decrease secretion of collagen V,thus perturbing the collagen I/V ratio within the fibrils,Marchant et al. [1996] have demonstrated that collagenV indeed regulates fibril diameter in the cornea. Abnor-malities in collagen fibril structure, namely increaseddiameter and irregularity of shape, have been welldocumented in the classical type of EDS [Vogel et al.,1979; Burrows et al., 1998; De Paepe et al., 1997;Michalickova et al., 1998; Richards et al., 1998].Furthermore, a mouse model lacking exon 6 of Col5a2

has composite dermal collagen fibrils, disorganized andenlarged, and similar to those in human EDS, they aresparse and scattered widely apart [Andrikopoulos et al.,1995]. In contrast, the Dcn�/� transgenic mouse[Danielson et al., 1997] and the Thbs-2�/� mouse[Kyriakides et al., 1998] present abnormally large andirregularly shaped fibrils that are homogeneouslyintermingled with thin collagen fibrils. A remarkablefinding is the absence of cauliflower-like fibrils, whichare typical for a collagen V defect. Despite clinicalfindings consistent with a mild classical EDS, tenascin-X-deficient patients do not present the above-describedultrastructural abnormalities of the collagen fibrilshape [Burch et al., 1997; Schalkwijk et al., 2001].Thus, the ultrastructural findings in the patient areconsistent with the lack of linkage to the TNX gene.

In contrast to the major fibrillar-forming collagens,the a chains of collagen V (Fig. 5) bear considerableNH2-terminal non-triple-helical extensions [Moradi-Ameli et al., 1994]. Immunofluorescence analysis combi-ned to immunoelectron microscopy data suggest thatthe triple-helical domain of collagen V molecules lieswithin the mature heterotypic fibril, while its shorttriple-helical domain serves as an extension to displacethe large globular tyrosine-rich domain on the fibrilsurface, where it modulates fibrillar growth [Linsen-mayer et al., 1993]. The NH2-terminal domain is highlyconserved at the amino acid level between man, rodent,

Fig. 4. Haplotypes showing lack of homozygosity for the microsatellites that flank the COL5A3, TNX, THBS, and DCN genes. Sequencing of theCOL5A1 RT-PCR product bearing the heterozygous (father and mother) and the homozygous (affected son) G-to-A transition in exon 13, which leads toGly530Ser, are shown. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5. Heterotrimer (a1(V))2a2(V). The a1(V) chain is shown in black, while the a2(V) chain is shown in gray. The organization of the NH2-terminaldomain of the a1(V) chain is shown; it consists of a globular tyrosine-rich region, an interrupted minor triple-helical domain (where the Gly530Sersubstitution is localized), and a short noncollagenous region corresponding to the flexible hinge-like region, according to Linsenmayer et al. [1993].

288 Giunta et al.

and chicken, especially the short interrupted triple-helical region, which contains the conserved glycine atposition 530 (Fig. 6). This suggests that a substitutionof serine for glycine at position 530 may impair thecorrect folding of the region and thus alter its function.Indeed, we found severe abnormal fibrils in the patienthomozygous for the Gly530Ser substitution and fewerand more mildly abnormal collagen fibrils in hisheterozygous parents.

A recently described patient who was compoundheterozygous for two mutations in the a1(V) collagenchain, Gly1489Glu in the triple-helical domain andGly530Ser in the NH2-terminal domain, presented withamore severe classical EDS phenotype than his affecteddaughter, who carries only the Gly1489Glu substitu-tion. Accordingly, the patient’s unaffected mother anddaughter, as well as a control individual carrying onlythe Gly530Ser substitution, presented with thin andsoft skin as the only clinical manifestation [Giunta andSteinmann, 2000; Steinmann and Giunta, 2000].

The patient described here has a mild form of theclassical type of EDS; he presents moderate skin hyper-elasticity, marked joint laxity, and joint hypermobility,especially of the fingers. Despite multiple hematomasand posttraumatic pigmentation, he did not have thetypical atrophic scars. After mutation analysis of thethree family members, both parents were carefullyexamined clinically. Interestingly, the mother had ahistory of clubfoot and two episodes of prematurerupture of the membranes, and presented with soft skinand hypermobile fingers that were not noticed before.However, the father was inconspicuous in this regard.The clinical discrepancy between the parents isunknown, but may be explained by the fact that femalestend to have softer connective tissues than males or bythe presence of a different genetic makeup among them.

In the majority of individuals with the classical formof EDS, the biochemical analysis of procollagens and

collagens is uninformative. This is so because mostcases are due to haploinsufficiency of a1(V) collagenEDS [Schwarze et al., 2000; Wenstrup et al., 2000],which is not easily detectable by the commonly usedbiochemical analyses. The few described cases ofglycine substitutions within the triple-helical domainof collagen V show only very subtle biochemical changeswith regard to the relative amounts and electrophoreticmobility of the a1(V) and a2(V) chains, respectively[Michalickova et al., 1998; Giunta and Steinmann,2000]. In contrast, ultrastructural changes are easilyvisible in all these cases, such as increased and irregu-larly shaped diameter of the collagen fibrils. It istherefore not surprising that in the patient, homozy-gous for Gly530Ser, no biochemical alterations werefound despite the abnormal ultrastructure, and evenlesser so in his heterozygous parents (Fig. 3).

Although the number of normal individuals tested forheterozygosity for the Gly530Ser substitution is rela-tively small, the data suggest a frequency of homo-zygous individuals of �1:10,000. This incidence seemsto be rather high, however, since clinical expressionmay be variable, not all homozygous individuals maycome to medical attention or biochemical investigation.This is supported by the variable expression observedin the mother and father of the patient, although theycarry the same heterozygous substitution. At present,we are studying normal individuals with a somewhatunusual soft skin for heterozygosity.

These findings demonstrate that heterozygosityfor the Gly530Ser substitution is associated with mildultrastructural abnormalities and variably subtleclinical signs and is disease modifying, while homo-zygosity for this mutation is associated with classicalEDS.

Additional studies are required to determine themolecular mechanism underlying the Gly530Ser sub-stitution.

Fig. 6. Sequence identities (%) between the human NH2-terminal domain and that of the hamster, the rat, the mouse, and the chicken (BLASTP 2.0.14and CLUSTALW 1.74 at http://www.ch.embnet.org/software/). The black segment corresponds to the tyrosine-rich region, the gray one to the minor triplehelix, and the dashed one to the hinge-like domain (not to scale). The star indicates the conserved glycine at position 530, which is substituted in thepatient.

EDS Caused by Homozygous Gly530Ser in COL5A1 289

ACKNOWLEDGMENTS

We are grateful to Dr. P. Schulz, Department ofDermatology, University of Munster, Munster, Ger-many, for helpful clinical information. We thank A.Schwarze, K. Wettinck, and M. Van Thielen forexcellent technical assistance and Dr. G. Matyas, Dr.K. Oexle, and Prof. A. Superti-Furga for interestingdiscussions during manuscript preparation. This studywas supported by grant 32-59445.99 from the SwissNational Science Foundation (to B.S.) and by grantG.0090.99N from the Fund for Scientific Research,Flanders (to A.D.P.).

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