Ehlers-Danlos syndrome has varied molecular … · generalised dermatochalasis (pendulous or...

40 Med Geniet 1997;34:400-410

Syndrome of the month

Ehlers-Danlos syndrome has varied molecularmechanisms

F Michael Pope, Nigel P Burrows

Ehlers-Danlos syndrome (EDS) is a group ofvariable clinical entities which share a propen-sity to skin fragility, joint laxity, and ligamen-tous fragility or shortening. Tschernogobow,'Ehlers,2 and Danlos3 independently describedunusual bruising, excessive cutaneous extensi-bility, and molluscoid pseudotumours. Theeponym of Ehlers-Danlos syndrome (EDS)was first suggested by Poumeau-Delille andSoulie.4 Spectacular early examples were retro-spectively commented upon by Beighton.5Morris' described a notable British patient whoBeighton re-examined more than 60 yearslater. The proband had classical EDS.Beighton7 not only showed impressive picturesof the so-called "Elastic Lady" but alsoincluded the original lithograph of a Spaniardwith unilateral cutaneous hyperextensibilityinvestigated in Leyden almost 300 years before,who was very probably a somatic mosaic. InEhlers's time there was considerable difficultyin distinguishing clearly between cutis hyper-elastica (overextensible skin), dermatorrhexis(easy splitting of skin), and both localised andgeneralised dermatochalasis (pendulous orredundant skin). To this day the similarities ofsuch physical signs still cause confusion. Inpractice the designations hyperelastic, pendu-lous, and lax skin often overlap and can evenoccur at different times in the same patient. Forexample, EDS skin is hyperelastic early in lifebut later becomes lax or drooping, especially inold age. The systemic implications, complica-tions, and associations of EDS were largelyunrecognised for many years although theassociation of congenital hip dislocations wasdescribed early. In 1960 Mories8 first describeda four-fold increase in fetal prematurity. Healso noticed that the dermis was collagendepleted and elastin rich. Thirdly, he describedcatastrophic lethal arterial bleeding in a 15 yearold adolescent boy with a traumatic arterialtear which was surgically unrepairable becauseof the extreme venous and arterial fragility.Very probably the patients with prematurityhad EDS I/II while the vascular fragility wascaused by acrogeric EDS IV. Subsequent clini-cal, molecular, and genetic progress has beenrapid. For example, Barabas9 first proposedthree distinct subsets. These included classicalforms with prematurity, a milder type with

venous varicosity, and a third, potentiallylethal, form with minor cutaneous and jointchanges but extreme arterial fragility (which wenow call EDS IV). Soon afterwards, Beighton'also clearly described the frequent prematurity.He added an X linked form,' while dividingBarabas's first group into "gravis" and "mitis"variants, which we now call EDS I and IIrespectively, to make five subtypes. He alsodelineated the wide clinical heterogeneity withvarious rheumatological, orthopaedic, surgical,and cutaneous complications and was also fullyfamiliar with such surgical complications asarterial aneurysms, venous varicosities, arterio-venous fistulas, and inguinal, umbilical, andhiatus hernias. He also documented refluxhydronephrosis, bladder neck obstruction, co-lonic diverticulae with perforation, and pleu-roperitoneal rupture (leading to pneumo- orhaemothorax). These complications are nowrecognised as particularly common in EDStypes IV, VI, and VII. In 1972, McKusick'2added two further subtypes (types VI and VII).We now know that the former is caused by lysylunderhydroxylation, while the latter resultsfrom the misprocessing of procollagen (to col-lagen). In 1988 Beighton et al'3 published anInternational Nosology of Connective TissueDisease where nine subcategories of EDS weredefined. Subsequently, he correlated the sub-types with the various biochemical and mo-lecular abnormalities (table 1) and also pub-lished the equivalent Molecular Nosology."'(Med Genet 1997;34:400-410)

Keywords: Ehlers-Danlos syndrome; molecular mecha-nisms

Genetics ofEDS (table 1)Autosomal dominant or autosomal recessiveinheritance occurs in EDS I, II, III, IV, VI, andVII. Compound heterozygosity (see later) isalso very likely. X linked forms have beendescribed but are highly unusual. Autosomaldominant inheritance was first mentioned byKopp.'5 Wiener'" described 12 affected sub-jects in three generations. Stuart'7 and Coe andSilver'8 both described the vertical transmis-sion particularly of EDS type I. Johnson andFalls" published a very large five generationAmerican family of British-Canadian extrac-tion with typical EDS I and with two separate

MRC ConnectiveTissue GeneticsGroup, StrangewaysResearch Laboratoryand Department ofClinical Genetics,Addenbrooke's NHSTrust, Cambridge, UKF M PopeN P Burrows

Correspondence to:Dr Pope, StrangewaysResearch Laboratory, WortsCauseway, Cambridge CB14RN, UK.

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Ehlers-Danlos syndrome has varied molecular mechanisms

homozygotes. They also speculated upon auto-somal recessive inheritance while both Weberand Aitken"0 and Ronchese" separately de-scribed sporadic examples of EDS I withaffected first cousin parents.Autosomal recessive inheritance occurs in

EDS VI although compound allelic heterozy-gosity may be more common than homozygos-ity except in consanguineous or inbred popula-tions. Beighton5 first suggested autosomalrecessive inheritance in an affected brother andsister whose parents and children were normal.Convincing autosomal recessive inheritancealso occurs in procollagen peptidase deficiency.In humans this causes EDS VIIC and inanimals dermatosparaxis. While the animalskin is excessively fragile, in humans there isextreme joint laxity and blepharochalasis/cutislaxa (CL). The original EDS VII patient ofLichtenstein et al'2 had congenital hip disloca-tions and short stature. Skin from EDS VIICpatients, cattle, sheep, and cats show severelydisorganised and misassembled hieroglyphiccollagen fibrils when examined by transmissionelectron microscopy. Homozygosity or doubleheterozygosity has also been postulated in EDSIV." 23-2 The latter patient had a normal colla-gen III profile. The family of Beasley andCohen'6 with joint laxity, lop ears, and an unu-sual face may also be an example of autosomalrecessive inheritance but was unclassifiableclinically. Apparent X linked pedigrees weredocumented by Beighton" in two British fami-lies with a clinical phenotype resembling EDSII. One family contained six affected males andthree possible carrier females in four genera-

tions. Phenotypic variability is far outweighedby biochemical and molecular diversity so thatindividual families are (almost always) unique.An International Working Party was convenedto standardise the nosology"3 and EDS IX (theoccipital horn syndrome) and EDS XI (famil-ial joint instability) are now excluded from theclassification. Lysyl oxidase deficiency is asso-ciated with Menkes syndrome and X linkedcutis laxa which, while allelic, are not a form ofEDS. The lysyl oxidase dependent form ofEDS has not been confirmed.

Molecular abnormalities causing EDS(table 1)In keeping with recent rapid advances, themolecular mechanisms of many EDS subtypesare now well understood. Most disrupt colla-gen fibril assembly by altering certain crucialmolecular components, the anatomical distri-bution of which dictates the consequentmolecular pathology. Thus the molecular com-

position of regions such as skin, vasculature(arteries, veins, and capillaries), pleuroperito-neum, intestinal walls, ligaments, tendons, car-

tilage, and eyes (the cornea and vitreous) maybe severely weakened and disorganised.Many types of EDS have specific mutations,

while in others, such as EDS III, VIII, and X,the abnormality is either unknown or inconsist-ent. Generally, there are two classes ofmutations, one in which structural genes are

faulty and the other in which their processingenzymes are disturbed.'7 Examples of theformer are mutations of the COLlAl, 1A2,3A1, and 5A1 genes and of the latter lysyl

Table 1 Ehlers-Danlos syndrome (cutis hyperelastica) after Beighton et al'3

Histology electronType Synonym McKusick No Specialfeatures microscopy Basic defectEDS I Gravis type AD 130000 Widespread scarring and Cauliflower COL5A1 linked in some

bruising, especially forehead, fibrils families. Mutationschin, and shin. Molluscoid include exon skips and apseudotumours translocation and a

cysteine substitutionEDS II Mitis type AD 130010 Similar but less severe Cauliflower

fibrilsEDS III Hypermobile AD 130020 No cutaneous scars Non-specific Unknown

typeEDS IV Vascular type AD 130050 Nearly always type III collagen Collagen COL3A1 mutations.

IVA acrogeric deficient depletion, Numerous pointIVB acrogeric AR 22535 variation of fibre mutations and exon skips,

size rarely deletionsIVC ecchymotic AD 130050 Risk of arterial rupture highest in

acrogeric subtypesEDS V X linked type XL 305200 Very rare. Not lysyl oxidase

deficient. Resembles EDS I, II,and III

EDS VI Ocular scoliotic AR 225400 Muscular hypotonia, often Non-specific Lysyl hydroxylase pointVIA decreased muscular dystrophy suspected mutations or exon skipslysyl hydroxylase (slow motor milestones) (homozygosity andlevels double heterozygosity)VIB normal Persistent premature scoliosis.levels Arterial rupture in 30%

EDS VII Arthrochalasis AD 130060 Extremely lax joints with Fibrils vary from Types A & B specificA, B, C multiplex congenital hip dislocation. angular in A and exon 6 skips or deletions

congenita Occasionally mild facial cutis B, to of COLlAl, 1A2 eitherlaxa. Some types have skin hieroglyphic in C pNal (I) or pNa2(1)fragility, mandibular hyperplasia retained. Type C

AR 224510 Severe generalised cutis laxa in procollagen peptidasehumans; dermatosparaxis in deficiency. Both pNal (I)cattle, sheep, and cats and pNa2(I) extensions

retainedEDS VIII Periodontitis AD 130080 Allelic variation with variable Non-specific Some are COL3A1type expression others not

EDS IX VacantEDS X Fibronectin AR 225310 Fibronectin deficient Association with FN may

abnormality be coincidental

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Figure 1 Typical clinicalfeatures ofEDS I; the clinical signs ofEDS II are similarmilder. Shown are the characteristic facial scars (A), extreme cutaneous extensibilityand patellar and pretibial scars (C). Broadened feet and hands are also a feature in s

families, as shown in this affected father and child (D).

hydroxylase or procollagen peptidaseciency. Defects of one or other mech-occur in EDS types I, II, IV, VI, and VII.III, in contrast, is highly heterogeneouscally and overlaps with osteogenesis impe(OI), pseudoxanthoma elasticum (PXE)Marfan syndrome (MFS). Candidateinclude COLlA1, 1A2, 3A1, 5A1, filelastin, lysyl oxidase, and lysyl hydroxylas

Clinical features ofEDS types I and ]

(fig 1)The five cardinal clinical elements ofinclude hyperextensible, doughy skin, atrscars, joint hypermobility, connectivefragility, and bruising (table 1). The c&phenotypes of EDS I and II overlap suttially and are probably allelic autosomalnant mutations with variable penetiCompound heterozygosity is also poEDS type I differs from type II only idegree of skin fragility and ligamentousThe skin splits over prominences suchforehead, chin, elbows, knees, andBroadened hands and feet and a widebuild (mesomorphism) are also con

Other notable clinical signs include epicfolds and fibrous nodules of the knees,and Achilles tendons. Even though mitraprolapse is common, aortic dilatation an

ture are not, and arterial fragility is viiunknown. Non-specific features includeneous varicosities, myopia, and late onsetarthritis (OA). Since collagen a (V) (

occur in cartilage and vitreous humour, t

is probably a direct complication rathe:primarily mechanical. In EDS II the c

phenotype is milder and skin fragility mu(

impressive; epicanthic folds, fibrous nodules,and mesomorphism are infrequent or evenabsent.

Collagen type V and the role ofCOLSA1/COL5A2 genesThere is now strong evidence to implicate typeV collagen c 1 chains in the aetiology of EDS Iand II. Clinical features include excessive skinfragility, ligamentous laxity, and relativelybenign prognosis. The collagen is disorganisedand forms so-called cauliflower fibrils.

Like collagens I, II, III, and XI, collagen Vhas an uninterrupted triple helix with N and Cterminal extensions. There are two or possiblythree forms of collagen V proteins, at least twoof which, ul (V) and a2(V), are coded by sepa-rate non-allelic genes. Firstly, on clinicalgrounds, there are very severely disorganisedcollagen fibrils (cauliflowers) (fig 2D). Sec-ondly, abnormalities of both collagen type V c 1chains and the COL5A1 gene have beendescribed in a sporadic EDS I/II patient withincreased skin fragility and joint laxity withcorneal flattening.21 29 Furthermore, transgenichomozygous mutant mice with an in frameexonic deletion in Col5a23° also had fragileskin, dermal thinning, and corneal disorganisa-

but tion as well as severe skeletal and ligamentous(B), deformities. An equivalent rabbit model showssonie similar pathology. Several independent groups

have detected linkage of human COL5A1defi- markers in families with EDS I or II which are

anism therefore allelic.3' 32 Other COL5A1 mutationsEDS have also recently been reported.33-35 In one, aclini- translocation interrupts the COL5A1 gene atrfecta intron 2433 and in the second an exon 65 skip), and was found in a three generation family.34 Agenes third had a mutation of a highly conserved C)rillin, terminal cysteine. There are therefore at leastse. three published COL5A1 mutations with

either EDS I or II phenotypes. In other similar[I families, linkage to COL5A1 has been

excluded36 (Burrows et al, unpublished data).EDS Somehow, collagen type V controls type I col-

rophic lagen fibril packing.37 ' This credibly explainstissue the gross fibrillar disorganisation which typifieslinical EDS I and II and implies that the disorganisa-bstan- tion is a direct effect of the mutant type V col-domi- lagen. Allegedly, collagen type XI analogouslyrance. regulates the thickness of collagen type II fibrilsssible. and collagen V and XI coassociate with or evenin the substitute for one another in vitreous humour.laxity. Corneal shape is also collagen V dependent.as the Mutations of either collagen type XI geneshins. (COLl lAl or COLl A2) can cause variantsbody of the Stickler syndrome (SS)42 43 in humansnmon. and mice.}+ Since COL lAl but not 11A2 isanthic expressed in the vitreous, only mutations of theshins, former cause vitreous pathology. Collagen

1 valve ci2(V) chains coassociate with ci2(XI) chains,43d rup- which may both coassociate with collagen typertually II. It is therefore not surprising that corneal or

cuta- vitreous abnormalities and high myopia coseg-osteo- regate with premature OA in some EDS fami-chains lies. Furthermore, COL5A2 mutations are a

he OA potential cause of arterial fragility,28 suggestingr than the possibility that c2(V) chains and collagenlinical type III interact analogously to ac(V) chainsch less and type I collagen chains. Lastly, several

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Figure 2 Transmission electron micrographs showing variably angular and hiefibrils of (A) EDS VIM, (B) EDS VIIB, (C) EDS VIIC. The distortion ts greatest inEDS VIIC in which every collagen a chain has a persistent N terminal extension. (D)Cauliflowerfibres typical ofEDS IIII. (E) Irregularfibrils ofEDS IV (F) Normal controlshowing (to the left) a uniformly sized, transversely sectioned collagen fibril. Fig 2(A) and(B) are reproduced by permission of the authors8' and the Journal ofMedical Genetics,fig2(B)"' by permission of the British Journal of Dermatology. We thank Professor BrianLake of the Institute of Child Health for providingfig 2(C).

different collagen mutations cause disorderedfibrillogenesis in vivo.46 COL5A1 homozygotesare either exceptionally rare or genetic lethals.Good animal models for COL5A1 mutationwill be essential for the future detailed analysisof the chemical composition and 3Dorganisation of tissues such as articular carti-lage, ligament, cornea, vitreous humour, arter-ies, veins, and capillaries.

Clinical features ofEDS type IV (vascularEhlers-Danlos syndrome) (fig 3)Vascular/arterial EDS differs from other varie-ties by virtue of severe vascular fragility, bothvenous and arterial. It was originally describedby Barabas9 and Sack.47 In ecchymotic EDSthere are often post-haemorrhagic haemosi-derin deposits over the knees, shins, andelbows, which may also complicate EDS I/IIand VIII. Acrogeria is specific to EDS IV and ischaracterised by generally thinned skin; theextremities (hands, feet, and face) are prema-turely aged (fig 3A, B). Other suggestivefeatures include prominent capillaries and kel-

oidal or elastotic scars (often with elastosisperforans serpiginosa). Large eyes, lobelessears, and a Madonna-like face are also typical.48Although short stature is usual, occasional slimand tall people overlap with MFS. Other clini-cal features include acro-osteolysis, diffuse alo-pecia of the scalp, and certain orthopaediccomplications such as congenital talipes or hipdislocations and tendon contractures, particu-larly of the Achilles or extensor tendons of thefeet and toes. Pleuroperitoneal or colonic rup-ture are more non-specific (and may also com-plicate other EDS or MFS phenotypes).Vascular pathology includes aneurysms ofsmall to medium sized arteries such as renal,

* splenic, axillary, brachial, femoral, popliteal,* and internal carotid vessels. The internal

carotid system may be compromised by dilata-'.*** ^ tion, arteriovenous malformations, aneurysms,

or dissections caused by arterial thinning and*,|l -** ^ fragility from collagen III deficiency. Occasion-{ ally the general phenotype is EDS III/BHS.489

For example, we observed a Gly->Ser 637 sub-4 ''3 stitution in a large autosomal dominant

pedigree with generalised joint laxity andpremature osteoarthritis.48 4

In EDS IV, skin histology shows strikingdermal thinning (from one-third to two-thirdsof normal), collagen depletion, and elastin pro-liferation. The mechanism of the latter isunclear (and may be primary rather thansecondary to collagen depletion). Transmissionelectron microscopy typically shows collagenfibril disorganisation with irregularity and abimodal size distribution (fig 2E). Somehowcollagen III/I ratios influence collagen fibrildiameter and interactions and, if mutated or

46~A~ diminished, seriously impair long term arterialroglyphic strength and stability.

Protein chemistryThere is very strong clinical, histological,biochemical, and molecular evidence thatfaulty collagen III causes dermal atrophy andvascular and gastrointestinal fragility.23 24 27Firstly, collagen III predominates in skin bloodvessels and ligaments. It is a unique interstitialcollagen, analogous to types I, II, V, and XI. Ithas a specific role in the strength and stabilityof blood vessels where it exerts its maximumclinical effect. The protein is a homotrimer[cal(III)]3 with two intrachain cysteine crosslinks at the C terminus. Collagen III deficiency,in the main, causes vascular EDS but may alsocause other less specific phenotypes such asEDS III.Two patterns are usual. The first (group 1)

exhibits poorly secreted and overhydroxylatedcollagen III proteins caused by either helicalglycine substitutions or exon skips. Thestoichiometry dictates that only one-eighth ofthe triple helices are normal. The remainingseven-eighths contain either one, two, or threefaulty al1(III) chains which are consequentlyretained intracellularly or poorly secreted anddegraded. Not surprisingly, patients with suchsevere biochemical deficiencies have severelyabnormal clinical phenotypes. The secondabnormal pattern includes null alleles (group2). Here reduced collagen type III secretion

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collagen III, although poorly secreted, is notovermodified. In other words the residualcollagen III molecules are usually normal.Thus, the clinical phenotype is milder, moredifficult to diagnose, and overlaps with EDSIII/BHS or even merges into normality. Simi-larly, the arterial phenotype varies; occasionallythere is premature aortic rupture, internalcarotid or cerebral arterial dilatation, or arterialrupture. In contrast, the more spectacularcarotid-cavernous-sinus aneurysms segregatewith group I mutations. The group 2 (nullallele) phenotype of arterial dilatation andcutaneous striae also overlaps clinically withatypical forms of MFS, from which it may beclinically indistinguishable.

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Figure 3 Various clinicalfeatures of vascular EDS IV (COL3AI mutations). Thin s(A), prematurely aged hands (B), andfacialfeatures typify the acrogeric subtype (D).Sometimes there is a characteristic circinate rash (elastosis perforans serpiginosa) althotthis can occasionally complicate other inherited connective tissue disorders (C).

without intracellular retention or overmol

cation is the norm. The other allele is usu

wild type but occasionally can be mutantcontrast to the secretory (group I) muta

Molecular pathology ofCOL3A1 mutants(fig 4)Since 1991, there have been rapid advances inphenotype/genotype correlations. Generally, 3'mutations are acrogeric whereas the pheno-

'( types of 5' or middle helical exon skips areharder to recognise. Unfortunately, even the

E latter are at risk of arterial rupture which isnevertheless much more frequent in mutationsat the 3' end. Arterial rupture is difficult, if notimpossible, to predict, occurring as early as thesecond decade (rare) to early or late middle age(very common). Like analogous COLlAl,1A2, or 2A1 mutations,7 they are virtuallyprivate to affected families. They include auto-somal dominant glycine substitutions or exonskips. So far, only three large in frame deletionsranging from 0.5 to 3 kb have beenreported.53.55 A smaller 27 bp intraexonic dele-tion was caused by spliced mispairing.56 Alldisrupt the collagen triple helix by a dominantnegative mechanism. Here, each trimer iseither wild type, or contains three, two, or oneabnormal chains in homotrimers such as colla-gens II and III or in heterotrimers such as col-lagen I. Thus, COL3A1 mutations cause severedisturbance with seven-eighths of the proteinmolecules abnormal compared to heterotrim-ers such as type I collagen where between halfand three-quarters are faulty,50 51 dependingwhether the mutation affects COLlAl orCOL1A2. Both homozygosity and heterozy-gosity for various COL3A1 mutations or com-pound heterozygosity with other collagen orextracellular matrix molecules are also theo-

07" retically possible, although to date there havebeen no published examples. Similar consid-erations also apply to COLlAl and COL1A2(OI mutants) in which only occasional doublemutations have been identified. Perhaps suchdouble hits would be catastrophic in wide-spread tissue components such as collagen I orIII. In contrast, double mutations of minoritycomponents like collagen VII are relatively

kin common. Thus, double glycine substitutionsugh cause severe recessive dystrophic epidermolysis

bullosa while in contrast single glycines areeither clinically silent or can cause minor auto-somal dominant epidermolysis bullosa.57

difi- Reliable genetic counselling and prenatalLally diagnosis are available for most vascular EDSIn patients. This requires a combination of histol-

ints, ogy, electron microscopy, and type III collagen

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ModerateEhlers-Danlos IV

diagramatically in fig 4. Other mutants arereferred to in other studies.6 64 Opinions differas to the relevance of collagen III mutations inintracranial and aortic aneurysms.48 65-67 Thereis little doubt that COL3A1 mutations docause such defects but how frequently remainsto be determined.

Collagen type I mutations (fig 5)Collagen type I is a heterotrimer([al(I)]2a2(I)) of two different a chains. Thegenes COLlAl and COL1A2 are located onchromosomes 17 and 7 respectively. Both areinterstitial collagens with uninterrupted(GlyXY) triple helices and globular, removableN and C termini; other fibrillar collagensinclude types II, III, V, and XI, all with crossbanded fibrils. Mutations of COLlAl andCOL1A2 produce distinctive but overlappingclinical phenotypes which are position, type,and domain related.'1"2 Mutations of either Nterminal extensions each cause EDS VII (withdistinctive clinical features) while, in contrast,helical and C terminal mutations cause 01. Asexpected, OI and EDS VII overlap clinicallyand biochemically.68 69 In EDS VII, cutaneousfragility and ligamentous laxity predominate,while in OI the bones are abnormally fragile.OI families with loose ligaments and fragile,delicate blood vessels and skin are uncommon,while otherwise typical EDS families some-times have bone fragility/osteoporosis. Theclinical phenotype is strictly dictated by muta-tional position and EDS VII is an especiallyfocused example. Its mutations all causeskipping of exon 6 of either the COLlAl orCOL1A2 gene.27 5o51 By contrast, mutationswhich cause OI are more diffusely distributedbetween exons 7 and 52. In general the pheno-type worsens with higher exon numbers (3' endor C terminal locations). EDS VII is caused bytwo distinct but related mechanisms. Eitherthere is a structural abnormality of thepeptidase cleavage site or the cleaving enzymeis faulty.

14 24 37 Milder

-27/54 Ehlers-Danlos IV

33 48

5 10 20 25 30 35 40 45 50

Figure 4 Map of recently published COL3AI mutations. (A) Glycine substitutionsarranged by residue number and (B) exon skips or large deletions (studies ofKuivaniemi etal" and Pope et a8).

protein or COL3A1 gene analysis. Prenataldiagnosis by amniocentesis, chorionic villusbiopsy, or termination of pregnancy is poten-tially hazardous in affected females owing tothe inherent fragility of cervical, uterine, or

abdominal blood vessels. On the other hand,opinions vary as to the safety or dangers ofpregnancy in this subset.58 59

Two recent papers48 60 summarise the variouspublished COL3A1 mutations in vascular EDSIV. The 41 published mutations are illustrated

EDS type VII (fig 6E)There are two distinctive clinical phenotypes,one overlapping with EDS I/II and the otherwith congenital cutis laxa. The former is mildand autosomal dominant, while the latter issevere and autosomal recessive. EDS VIIA andB result from structural mutations of type Icollagen,22 while EDS VIIC is caused by theenzyme deficiency. Clinical signs include shortstature and excessive premature ligamentouslaxity (such as congenital dislocation of thehips). There may also be excessive cutaneousfragility reminiscent of EDS I/II, or mild cutislaxa (CL). Spinal and ligamentous deformitiessuch as kyphoscoliosis are relatively infrequent.In contrast to EDS I/II, in EDS VIIA and Bcollagen fibril morphology is only marginallyabnormal and the fibres are angular intransverse section rather than forming cauli-flowers (fig 2A, B). On the other hand, EDSVIIC shows grossly distorted, hieroglyphicfibrils.

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Normal allele

{I AJ4 A

I.V.S.5 I.V.S.6

COLIA 1

GTAG exon 6'AG Weil et al 1989a

GT AG exon 6*A GT AG exon D'Alesslo et al 1991

COLIA 2

GTI-AG exon 6 |GC* AG exon 7 Weil et al 1988

exonGGT AG| exon 6 GC* AG exon7 Hoet al 1994

GTexon 6'A | Weil et

1989b

GT AG exon 6 ]*CT AG exon 7 Chlodo et al 1992

5GT- exon 6 'AT Weil 1990

OGT AG exon 6 *AT AG Nicholls et al 1991

exon5GT -AG exon 6 *AT AG axon Watson etal 1992

exonG5 GT AG exon 6 *AT- AG Lehman et al 1994

exon5GT -AG| exon 6 'AT -AG exon 7 Vasan et al 1990

Figure 5 Diagrammatical representation of various published exon 6 splice junctionmutations causing EDS VIIA or B. Coding sequences (exons) are boxed while non-codingsequences (introns) are shown as lines. The first two bases (splice donor) and last two bases(splice acceptor) of the intron sequence are always GT orAG respectively. Mutations are

denoted by asterisks.

Deletion of the peptidase cleavage siteSince collagen type I is a heterotrimer, loss ofeither the pNa 1 or pNa2 cleavage sites can

cause EDS VII. When pNal sequences are

uncleaved three-quarters of collagen type Iheterotrimers possess either one or two persist-ent pNa chains. With pNa2 defects, half thetype I collagen retains abnormal pNa2 se-

quences while the rest become normal a2chains. In both circumstances collagen fibrilassembly is compromised and normal head to

tail packing of individual triple helical mole-cules is severely disrupted. The persistent pNsequences seriously distort gap regions. Suchmisassembled fibrils are visible by transmissionelectron microscopy, which shows slightlyirregular to angulated structures in cross

section. When thicker than 10 nm, both initialnucleation and subsequent fibril growth are

seriously distorted by the presence of persistentpNa sequences374" 7' which are longer than theaggregating a chains. Exon skips also disrupttype I collagen a chains since both the pepsinand peptidase cleavage sites are deleted.Protein electrophoresis of pepsinised a chainstherefore shows elongated molecules withabnormally persistent pN sequences in addi-tion to the correctly cleaved normal a chains.Such abnormal pN proteins can also bepurified from NaCl extracted tissues. Procolla-gen "snapshots" also show inefficient process-ing patterns. Following the original descrip-tions of Lichtenstein et af22 of faulty conversionof procollagen to collagen in EDS VII, persist-ent pNa 1 and pNa2 components were sepa-rately demonstrated by Steinmann et ao0 71 andCole et al.72 73 Subsequently, various missplic-ing mutations of exon 6 sequences caused byfaulty splice acceptor or donor sequences havebeen discovered. Most are near to the obligate

G of the 5' intron acceptor GT sequence inintron 6 (fig 5).7484

Mutations ofprocollagen peptidase (PP)PP deficiency of cows was first described byLapiere et al.85 The animals had excessivelyfragile skin (dermatosparaxis), which was agenetic lethal. Transmission EM of the dermisshowed numerous bizarrely shaped collagenfibrils which formed flanged rods instead ofnormal cylinders. In transverse section theseappeared as hieroglyphs while in longitudinalsection they were severely disaggregated andtwisted. Biochemical analysis showed abnor-mally elongated collagen pNal and a2 chainswhich were extractable with salt or acidsolutions, implying abnormal cross linking. Atthe time, procollagen was thought to have sin-gle N terminal extensions and no C terminaladditions. In contrast to EDS type VIIA and B(see above), every component of type I triplehelix are abnormal, that is, contains bothpNal(l) and pNa2(1) chains. Consequently,no normal collagen triple helices are formed:instead fibrils assembled form [pNa(I)] 2pNa2(I) triple helices. Consequently, fibrillarpacking is very seriously disrupted (fig 2C).The equivalent human phenotype was notidentified until recently.85.89 Various otheranimal models such as sheep90 and Himalayancats9' were described previously, but includedvery convincing clinical and structural data,and the expected hieroglyphic fibrils andderanged biochemistry. The human clinicalphenotype unexpectedly showed prematurecutis laxa (CL) with blepharochalasis. So farthree examples have been published, two withexceptionally clear biochemical data and thethird with excellent illustrations of the clinicalphenotype.87.89 Both the French and Americandescriptions included disturbed procollagenprocessing, pNal (1) or a2(1) molecules, hiero-glyphic fibres, and deficient enzyme levels. Thephenotype of Smith et al88 included prematuredelivery, soft, fragile, and easily bruised skin,and patchily sagging skin, while in the patientof Nusgens et al,87 skin fragility, bruising, andgeneralised osteoporosis were notable. Rear-don et al,89 in contrast, described severe prema-ture generalised CL with blepharochalasis,redundant skin, and fragility. Every patient hadtypical hieroglyphic fibrils. Transient CL alsooccurs with the milder EDS VII type A and Bphenotypes in which skin fragility varies. Eventhough less disrupted than dermatosparaxis,the fibrils of EDS VII A and B can be manipu-lated to produce hieroglyphics under suitableexperimental conditions.92 Plainly the faultyangular fibrils characteristic of exon 6 deletions(which cause EDS VII A and B) are one step inthe progression from normal cylinders tohieroglyphs. Theoretically both more localisedand extensive mutations within and aroundexon 6 of the N propeptide coding region couldproduce a wider spectrum of fibrillar angularityand clinical features which are maximised inthe propeptidase enzyme deficiency of animalsand humans.

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A V. C

IE

Figure 6 Various physicalfeatures ofEDS III (A, B), EDS VI (C, D), and EDS VII (E). In practice a degree of laxity(A, B) can complicate any of the EDS subtypes but is a sole feature in EDS III. Scoliosis is a consistent feature ofEDS VI,sometimes complicates EDS VII, and rarely occurs in EDS I, II, and III. The criss cross lines shown in (D) are typical ofeither EDS VI or EDS VII and a slight facial cutis laxa is particularly characteristic ofEDS VII (E). At other times thelatter shows cutaneous fragility very similar to that offig 1.

Other enzyme abnormalities causingEDSOther crucial post-translational modificationsof fibrillar collagen triple helices include enzy-matic hydroxylation of certain lysines and exci-sion of the C propeptides in addition totrimming by the specific N propeptidases.Lysyl oxidase then promotes cross links bydecondensing hydroxylysines to aldehydes.Mutated enzymes cause very specific forms ofEDS. Thus, lysyl hydroxylase (LH) deficiencycauses EDS VIA93-'04 while procollagen pepti-dase deficiency causes EDS VIIC85-87 (see alsoabove). The role of lysyl oxidase is morecontroversial'05 106 and so far the diseasescaused by C terminal propeptidase mutationsare unknown. Since the C propeptide is essen-tial for chain association, homozygous enzymemutations might be genetically lethal. Bonemorphogenic protein 1 (BMP1) and the Cpropeptidase are now known to beidentical.'O7 108

Ehlers-Danlos syndrome type VI (lysylhydroxylase deficiency) (fig 6C)The disorder was first recognised as unique byMcKusick,'2 although Beighton et all3 hadobserved autosomal recessive inheritance in afamily with severe spinal deformities, ligamen-tous laxity, ocular fragility, and retinal detach-ment. There is extreme ligamentous laxity withsevere ocular fragility, retinal detachment, andscleromalacia. The severe ligamentous prob-lems present in early infancy usually withmotor delay caused by severe muscular hypoto-

nia. Sometimes muscular dystrophy is(wrongly) suspected but nearly all affectedchildren eventually walk normally. The diffi-culties are caused by excessive ligamentouslaxity which young muscles cannot adequatelycontrol. In adult life or middle age, there maybe aortic dilatation or arterial rupture.Krane et al3 and Pinnell et al4 then

discovered underhydroxylation of lysine anddeficient collagen lysyl hydroxylase in twoaffected sisters with severe infantile hypotonia,premature scoliosis, and ocular fragility. Othersignificant features included soft, hyperextensi-ble, easily scarred skin and premature ruptureof the membranes. There are two similar clini-cal phenotypes, type VIA with enzyme defi-ciency and type VIB with normal enzyme.There may also be a third, predominantly ocu-lar, variant.

Biochemistry and molecular pathology(table 2)Krane et at3 and Pinnell et al4 measuredenzyme activity using tritium labelled chickprocollagen as substrate. Nearly 20 years later,both the chick and human genes were clonedand sequenced.95 96 LH or procollagen lysyl 2oxoglutarate 5 dioxygenase requires copper,Fe", and 02 as cofactors. EDS VI is caused byeither deficient or faulty enzymes with variableKm or V max.97 98 The mutations are oftencomplex and include Alu-Alu compoundrecombinations and homozygous or doubleheterozygous exon skips.98-'02 Homozygousdeletions tend to cluster in inbred families. The

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Table 2 Lysyl hydroxylase mutations (the gene has 19 exons)

Ha et al 9 Double heterozygote 3 base pair deletion Glu (-) 532 andGly 678 to Arg

Hautala et al 9 Homozygous 7 exon duplication rearrangement at Alu/Alu in two sibs

Hyland et al 1'0 Homozygous stop codon Arg 319-X:CGA-TGAPousi et al "' As for Hautala et al9 but in an unrelated female

hydroxylysine content of defective tissuesvaries between 5% and 50% being lowest inskin, bone, and tendon. Collagen I is more

underhydroxylated than collagen III, while col-lagens II, IV, and V are normally hydroxylated.This implies more than one hydroxylase withvariable specificity for other collagen types, so

that other clinical phenotypes are also likely.Underhydroxylation of collagen al (1) anda2(1) chains causes overmigration and impairscondensation cross linking of adjacent hy-droxylysines which can be measured inurine.'03 104 Urine sampling and type I collagena chain electrophoresis are simple and usefulscreens for enzyme deficiency.

The C terminal propeptide (BMPI)deficiencyIt is now apparent that the C terminalpropeptidase (CPP) is identical to bonemorphogenetic protein 1 (BMP 1). The latter ishomologous to the Drosophila pattern forma-tion genes TLD and TRI with analogues ininsects and sea urchins, such as the BP 10 andSpAN genes which are TGFO activators.'07Homozygous enzyme deficiencies would eitherbe genetic lethals or severely crippling inmechanical terms. Persistence ofpC extensionswould severely impair collagen triple formationjust as persistent pN sequences interfere withmolecular packing. Errors of the pC extensionswould seriously disrupt both helical windingand chain association; quite possibly collagentriple helices would not form at all. The effectsof C propeptidase deficiency should be worse

than N propeptidase deficiency which causes

EDS VIIA-C. Holmes et ar' studied thestepwise removal of pC or pN propeptidesbefore collagen fibrillogenesis. Retention ofeither extension by faulty processing inhibitsfibril formation as do helical mutations of thefully processed molecule.

Heterozygotes for CPP deficiency might alsohave impaired fibrillogenesis. Homozygositycould easily cause severe clinical phenotypes,such as lethal 01, and certain severe chondro-dysplasias or EDS variants either as allelichomozygotes or as double heterozygotes incombination with other ECM propeptidemutations such as pC I, II, III, V, or XI abnor-malities respectively.

EDS variants ofunknown cause

This applies to EDS types III, VIII, and X inwhich the causes are non-specific (EDS III),variable (EDS VIII), or represented by a singleone-off example (EDS VIII).

EDS type III (fig 6 A, B)Unlike EDS types I and II there is no

significant cutaneous scarring despite obviousjoint hypermobility and doughy skin. This

clinical subtype merges with the so-calledbenign hypermobile syndrome (BHS) butwhich has normal skin texture. This milderclinical phenotype can also segregate in typicalEDS I/II families probably owing to incom-plete penetrance. Similar clinical features mayalso accompany COL3A1 mutations," suchas MFS and SS. Heterozygotes for autosomalrecessive disorders such as PXE and lysylhydroxylase deficiency (EDS VI) have verysimilar clinical phenotypes.109 There can also beoverlap (clinical and biochemical) with vascu-lar EDS (see above).

EDS type VIIIFirst described by Stewart et all'" this pheno-type is clinically, biochemically, and allelicallyheterogeneous. Distinguishing clinical featuresinclude chronically inflamed, heavily pig-mented, discrete, pretibial plaques and prema-ture periodontal recession. There is collagendegradation as judged by gum resorption andthe cutaneous inflammation. In EDS VIII noconsistent biochemical or structural changesare detectable...... Certain mutant collagensmay be abnormally proteinase susceptible.Alternatively, faulty collagenase inhibitors suchas TIMP might be implicated, although all ofour EDS VIII patients have normal TIMP pro-files. We have observed both vertical and hori-zontal transmission of early adult gum reces-sion, sometimes accompanied by collagen IIIdeficiency. Similar features may accompanyEDS I, II, and IV. Others have also noted thisphenotype.111-114 The distinctive clinicalphenotype'06 is therefore biochemically andgenetically heterogeneous. Histology of theskin lesions shows a granulomatous collagendegeneration resembling necrobiosislipoidica. '15

Ehlers-Danlos syndrome with fibronectindeficiency (EDS X)Only one family with this phenotype has beendescribed."6 This included four affected sibs inone generation but with normal parents, whichMcKusick later classified as autosomal reces-sive. The phenotype includes thin, fragile, eas-ily scarred skin, joint hypermobility, and exces-sive bruising and therefore resembles EDSII/III. Platelet aggregation was abnormal butcorrectable with normal fibronectin. In otherEDS families a variety of clotting abnormalitieshave been described which are too inconsistentto be directly related."7 118

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4 Poumeau-Delille GA, Soulie P. Un cas d'hyperlaxitecutanee et articulaire avec cicatrices atrophiques etpseudo-tumeurs molluscoides (syndrome d'Ehlers-Danlos). Bull Soc Med Hopitaux de Paris 1934;50:593-5.

5 Beighton P. Ehlers-Danlos syndrome. London: HeinemannMedical Books, 1970.

6 Morris M. Diseases of the skin. 4th ed. Plate XL. London:Cassell & Co, 1908.

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