structural segregation of the type in · multiple epiphyseal dysplasia normal or mild short...

Journal of Medical Genetics 1988, 25, 521-527

Structural and segregation analysis of the type II

collagen gene (COL2A1) in some heritablechondrodysplasiasPAUL WORDSWORTH*, DONALD OGILVIE*, LINDA PRIESTLEY*,ROGER SMITHt, RUTH WYNNE-DAVIESt, AND BRYAN SYKES*From *the Nuffield Department of Pathology, John Radcliffe Hospital, Oxford OX3 9DU; tthe NuffieldOrthopaedic Centre, Oxford OX3 7LD; and .t2 Dale Close, St Ebbe's, Oxford OX] ITU.

SUMMARY Seventy-seven persons with a variety of heritable chondrodysplasias were screenedfor gross rearrangements of the structural gene encoding the major cartilage collagen, collagenII. None was found. Segregation of the locus (COL2AJ) was studied in 19 pedigrees using threerestriction site dimorphisms (shown by PvuII, HindIII, and BamHI) and a length polymorphismas linkage markers. Discordant segregation between COL2AJ and the mutant locus was seen inpedigrees with multiple epiphyseal dysplasia, autosomal recessive spondyloepiphyseal dysplasiatarda, hypochondroplasia, pseudoachondroplasia, diaphyseal aclasis, and trichorhinophalangealsyndrome. One pedigree with diastrophic dysplasia was weakly concordant. Autosomaldominant spondyloepiphyseal dysplasia tarda and metaphyseal chondrodysplasia (type Schmid)were not informative. We conclude that mutations of the collagen II gene are not a commonfeature of the heritable chondrodysplasias. Since the chondrocyte binding protein, chondrocal-cin, is also encoded at COL2A1 our conclusions apply equally to this gene.

The heritable chondrodysplasias are a very hetero-geneous group of syndromes, the unifying featuresof which are abnormal development of the bonesand joints, frequently associated with shortstature.1 2 The criteria for delineating the recog-nised osteochondrodysplasias were agreed at the1976 Paris conference on nomenclature.3 The de-gree of disability and handicap is very variable but ithas been estimated that as many as 6000 persons arehandicapped throughout life in the United Kingdomalone (population 5-5x 107).4The hypothesis that heritable skeletal dysplasias

could sometimes be the result of abnormalities inextracellular matrix components has been supportedby a combination of biochemical and, recently,genetic linkage evidence in osteogenesis imperfecta(0I).5 6 It has been shown that qualitatively differ-ent mutations at the two structural genes encodingcollagen I, the major species in bone, can give rise toa wide range of 01 phenotypes.7 8 We set out toinvestigate whether those heritable osteochondrody-splasias in which cartilage rather than bone wasprimarily involved could, in an analogous way, beReceived for publication 19 May 1987.Revised version accepted for publication 13 November 1987.

the result of mutations in the structural geneencoding collagen II which is the most abundantspecies in cartilage.

Collagen II (or type II collagen) is one of thethree major fibrillar collagens found in man. Allthree exist in tissues as extracellular, stress resisting,cross linked polymers, the subunits of which aretrimers of a chain polypeptides which form a tighthydrogen bonded triple helix. They perform a vitalstructural role in all tissues. Unlike the other twofibrillar collagens (collagens I and III) which arefound in many different tissues, collagen II has amuch more restricted distribution, being encoun-tered only in hyaline and articular cartilage, thenucleus pulposus, and in the ocular vitreous. Allthree collagen II a chain polypeptides are identicaland are encoded, in man, at a single locus,COL2AJ, on the long arm of chromosome 12.

In the past decade, non-systematic histopatholo-gical and biochemical studies have suggested that inthe chondrodysplasias the primary defect is incartilage synthesis with consequent abnormalities ofendochondral bone.9 A range of cartilage collagenabnormalities has been reported in thespondyloepiphyseallt 11 and spondyloepimeta-

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P Wordsworth, D Ogilvie, L Priestley, R Smith, R Wynne-Davies, and B Sykes

physeal dysplasias, 2 diastrophic dysplasia, 13achondrogenesis and fibrochondrogenesis,1( andthanatophoric dwarfism.14 Not unexpectedly,altered proteoglycan metabolism has been reportedin others, including pseudoachondroplasia, Kniestsyndrome, spondylometaphyseal dysplasia(Kozlowski type), and atypical cases of spondylo-epiphyseal dysplasia.11 However, the complexinterrelationships between the various cartilagematrix components will always make identificationof the primary defect difficult from biochemicalstudies alone.To test the hypothesis that mutations at COL2AJ

cause heritable chondrodysplasias, we have used thecosmid clone cosHcolI which contains the entire30 kb collagen II gene and 7 kb of flankingsequence. '5 In common with the other majorfibrillar collagen genes, the 5 kb of coding sequence

is distributed between 50 to 51 exons. We have usedthis cosmid and subclones derived from it to searchfor gross rearrangements of COL2AJ in multipleoverlapping digests of DNA from chondrodysplasia(CD) patients. In addition, we have used restriction

site and length polymorphisms at this locus as

genetic markers in segregation analysis of CDpedigrees. Using this method, we have alreadyreported independent segregation of COL2AJ andachondroplasia. 16

Patients

Seventy-seven subjects with a variety of chondrody-splasias were identified from four skeletal dysplasiaresearch clinics. All subjects were examined by at

least one of the authors and relevant radiographswere taken, often serially. Nineteen potentiallyinformative pedigrees were investigated. Details ofthe patients and kindreds studied are shown intables 1 and 2. Pedigrees 1-1 (multiple epiphysealdysplasia), 6-1 (pseudoachondroplasia), and 7-1(trichorhinophalangeal syndrome) have featured inprevious clinical reports.t7-20)

Methods

DNA was prepared from frozen peripheral blood,

TABLE 1 Patients studied including their clinical and radiological features.

Disorder Clinical features Radiological features

Multiple epiphyseal dysplasia Normal or mild short stature. Premature osteoarthritis. Small, irregular, delayed epiphyscsMild joint deformities

Spondyloepiphyseal dysplasia tarda Mild, short trunk dwarfism. Premature osteoarthritis Platyspondyly. Severe epiphyseal changes in theparticularly of the hips large proximal joints

Spondyloepiphyseal dysplasia Marked short trunk dwarfism (12(-14(1 cm). Osteoarthritis Platyspondyly. Coxa vara with severe epiphysealcongenita (mild coxa vara) of the hips changes in large proximal joints

Atypical spondyloepiphyseal Abnormal from 2 years. Scoliosis. Severe osteoarthritis Platyspondyly. Epiphyseal changes in hipsdysplasia of hips. Pronounced myopia and knees

Spondylometaphyseal dysplasia Pectus carinatum, scoliosis. Moderate joint laxity. Severe Platyspondyly. Metaphyseal splayingshoulder and hip involvement

Pseudoachondroplasia Severe short limbed dwarfism from 2 years. Marked joint Fragmentation of epiphyses. Mushroom shapedlaxity. Severe premature arthritis of large joints metaphyses. Anterior vertebral beaking.

Platyspondyly

Trichorhinophalangeal syndrome Mild short stature. Minor deformity of intcrphalangeal joints. Brachydactyly. Cone shaped epiphyses. WidenedPear shaped nose. Poor growth of hair metaphyses in femoral necks

Metaphyseal chondrodysplasia Mild lower limb shortness. Bowed femoral necks requiring Mctaphyseal splaying and cupping. Coxa vara.(type Schmid) osteotomy Normal epiphyses

Hypochondroplasia Mild short stature. Normal face. Spinal stenosis in one case Flaring of metaphyses. Mild lumbar interpedicularnarrowing

Diaphyseal aclasis Normal stature. Palpable bony outgrowths around joints Overgrowth of bone adjacent to the growthplate

Diastrophic dysplasia Severe short stature. Joint contractures. Talipes equinovarus. Scoliosis. Small rib cage. Symphalangism. DelayedHitchhiker thumbs. Cauliflower cars epiphyseal ossification. Broad metaphyses

Dyschondrosteosis Mesomelic shortening. Madelung-like deformity. Mild short Madelung-like deformitystature

Chondrodysplasia calcificans punctata Short limbed, short stature. Flat face. Mental retardation Punctate stippling of epiphyses. Metaphyseal(Conradi syndrome) cupping and splaying

Kniest syndrome Pronounced short stature. Joint contractures. Flat face. Much enlarged metaphyses. Flattened vertebrae.Myopia Delayed ossification of femoral heads

Chondroectodermal dysplasia (Ellis- Severe short staturc. Polydactyly. Congenital heart defects Short ribs and long bones. Coned epiphysesvan Creveld syndrome)

Frontometaphyseal dysplasia Bowed long bones. Enlarged supraorbital ridges Frontal skull, bone overgrowth. Failure ofmetaphyseal modelling. Platyspondyly

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Structural and segregation analysis of the type II collagen gene

TABLE 2 Summary of patients and kindreds studied and the results of segregation analysis expressed as lods atzero recombination distance. Where the mode oftransmission was not clearfrom the pedigrees (2-1, 3-1, 3-2, 5-1, 5-2, 11.1)lods were calculated for both autosomal dominant (AD) and autosomal recessive (AR) inheritance. Resultsfrom achondroplasia have already been published'6 and are included for completeness.

Disorder Cases Kindreds Inheritance Lods at 0=000

1 2 3

Multiple epiphyseal dysplasia 25 2 AD -xAR -x

Spondyloepiphyseal dysplasia (tarda) 6 3 AD -00 NI NIAR -xs

Spondyloepiphyseal dysplasia (congenita) 4 2 AD NI NIAR 0-12 -X

Atypical spondyloepiphyseal dysplasia 2 1 AR -Spondylometaphyseal dysplasia 2 2 AD NI NI

AR 0-2 0-12Pseudoachondroplasia 6 3 AR - x - x NITrichorhinophalangeal syndrome 6 1 AD -xMetaphyseal chondrodysplasia (Schmid) 6 2 AD NI NIHypochondroplasia 4 1 AD -xDiaphyseal aclasis 6 1 AD -xDiastrophic dysplasia 2 1 AD NI

AR 0-24Dyschondrosteosis 3Chondrodysplasia calcificans punctata (Conradi syndrome) 2Kniest syndrome IChondroectodermal dysplasia (Ellis-van Creveld syndrome) 1Frontometaphyseal dysplasia 1Achondroplasia 17 3 AD - x -x NI

then restricted, separated by electrophoresis, andblotted onto nitrocellulose filters using our usualmodifications of standard conditions.21 Hybridisa-tion probes were labelled with 32P by either nicktranslation or random primer directed synthesis.Hybridisations were carried out overnight at 42°C ina mixture containing 50% v/v formamide, 5% w/vdextran sulphate, and 200 p.g ml-' heparin afterprehybridisation of the filters with the same mixturewithout dextran sulphate. When the whole cosmidwas used as a probe, hybridisation to highly repeti-

tive sequences was reduced by including 20 [ig ml-ldenatured human DNA in the hybridisation mix.

GROSS REARRANGEMENT OF COL2A1We used a combination of digestion with EcoRI,BamHI, and PvuII to screen for major rearrange-ments in COL2AJ. A partial restriction map of thelocus is illustrated in fig 1. Whereas we agree withthe revisiorn of the original EcoRI map22 bySangiorgi et a123 we disagree on the position ofBamHI sites, both with the original map (substan-

CosH col 1

5.9 4.8 7.3 5.2 9.7 3.9 4.3 EcoRI

1.5 I 6.5 | 6.3 11.11 5.2 | 6-6 4 3 BamHI

2.1 7.0 7.0 531_HidI

ND 5.2 | 7.2 | 6.4 130 | KpnI* m

ND 11-611.7 116111 4.2 112.01 1112.41 PvuII

FIG 1 Partial restriction map ofCOL2AJ . The limits ofthe collagen HI gene are shown by arrows. Starred BamHI,HindIII, and PvuII sites are dimorphic. The solid box denotes the position and limits ofthe length variable segment. NDon the KpnI and PvulI maps refers to unmapped regions ofthe locus.

523


tially) and with the revision of Sangiorgi et al23 in theordering of the 6-5 kb and 6-3 kb fragments. UsingEcoRI and BamHI digestion, the gene is dividedinto fragments of not more than 5 3 kb. In thislaboratory we expect to detect an insertion ordeletion of 200 bp in a 5-3 kb fragment and set thisas the lower size limit of our screen. PvuII dividesthe gene into at least 14 fragments but the map ofPvuII sites is not yet complete. Therefore, althoughfragmentation with this enzyme improves the sensi-tivity of the screen in certain regions, we cannot besure that it does so throughout the gene.

SEGREGATION ANALYSISSegregation of COL2AJ alleles was analysed in thepedigrees using three restriction site dimorphismswithin the gene and a length polymorphism justbeyond its 3' end. We have previously described therestriction site dimorphisms shown by HindlIl andPVUII.24 25 Here we describe one further dimorph-ism shown by BamHI. Fig 2a shows the fragmenta-tion pattern in the three different genotypes. Thevariable BamHI site has been mapped to a position17 kb upstream of the 3' end of the gene (fig 1). Themost recent allele frequency estimates for all threedimorphisms in unrelated English Caucasians areshown in table 3.The length polymorphism was first noticed as an

apparent deletion in one allele in four patients withlethal osteogenesis imperfecta.26 Studies in thislaboratory showed that the 'deletions' were theresult of variation in the length of a DNA segment1-0 to 1-6 kb downstream from the terminationcodon.2' This segment is composed of tandemlyrepeated oligonucleotides of core lengths of 31 and34 bp.27 Although short alleles are much morefrequent in some non-European populations, lengthvariation can be detected in English whites and wehave used this variation, showni by PvulI digestionand hybridisation to E7 (fig 2b), the 3' most EcoRIfragment of cosHcoll, as a further genetic markerfor COL2AL. The map positions of all four markersystems are shown in fig 1.

Results

No rearrangements of COL2AJ were detected inany patient. Segregation of COL2AI using thelinkage markers described above is shown in fig 3and summarised in table 2. Discordant segrega-tion was seen in multiple epiphyseal dysplasia(autosomal dominant, 1.1 and autosomal recessive,1-2 types), spondyloepiphyseal dysplasia tarda(autosomal recessive, 2-1, 4-1), pseudoachondro-plasia (autosomal recessive, 6-1), trichorhinophal-angeal syndrome (7.1), hypochondroplasia (9-1),

FIG 2 (a) Fragments generated by the BamHI sitedimorphism. The three genotypes are shown. Fragmentsizes are in kilobases. (b) Fragments generated by the3' length polymorphism detected by hybridisation withfragment E7 after PvuII digestion. Tracks I and 3 showunresolved 'homozygotes' for alleles oflength 2-5 and2-4 kb respectively. Tracks 2 and 4 show resolvedheterozygotes.

40 to ------....... ''%l __

to to,:w

- 2.

524


P 2 22 1-2 12 2 2 2 2 2 2H211 12 2 2 2 2 12 2??2?

111 2 22 2 2 2 11 11 2 2 2 2 2 222 2 2 22 2 2 2 ? 12

22 22

2.1

P 1-2 11

H 12 12L 12 11

11 11 1 122 22 2212 12 12

1.2

P2 2 1 1H2 2 2822 22L 1 1 1

12 1212 12222222.3 1 1 1

P 12 12H 12 12

1 1222 12

3.1

OT-OP 1-1 1-2L 1-2 1-1

1-1 Ili 1-1

1-1 1I.,

3.2

[}2 2

H 2 22H1 121L1

2 211 2 2

221 2 22 121

2.2 1-

H 1-2 1-2

12 1.2 1-11-1 22 1-2

4.1

P 22 22

Hll 1 2

22 ?21 2 1

5.1 5.2

1 do1ff1P 1-2 1 2H 1-2 28 11 1,1

6.1 L 1- 1-2

O1

H M2-1-2 H- 2-22 -2

~~~~ ~ ~~~~~~~~~~~~~~~B 2 2

6.3 ,, 2 1-2 1-2 Q 22 2 2P~~~ ~ ~ ~ ~ ~ ~ ~ ~ v1-?2 2 2 2

7.1

2-2

P 2 ' 1 1H 1i 22B1 1

L1

2 12 121 2 2 t2

1-11 -1

6.2

p 12 2H 2 1-1B 1.1L 1.1

? 1 21.1

8.2 ,,1*1.

2-2-1 2 1-2 2?

12 1-2

L0. -2

~~~~~~~~~~~~~2221

FIG 3 Pedigrees and COL2A I genotypes. Letters at the top left ofeach pedigree denote the marker svstemused. H=HindlII, B=BamHI, P=PpuII, L=length polymorphism. Details offragmentsizes and allelefrequencies are shown in table .3.

525

(S-122 2 2

1.-.22 2

P 2-2 1-1

p 1-H 1-2 1-1

1-2 2-21-2


TABLE 3 Markers at COL2AJI. Fragment lengths in kilobases generated by the three restriction site dimorphisms (H, P, B)and the allele frequencies estimatedfrom unrelated Caucasian chromosomes. The variants generated by the lengthpolymorphism (L) were shown by PvuII digestion. The apparently mostfrequent allele, designated 1, generates a 2-5 kbfragment, while other alleles, designated 2 in any pedigree, generate shorterfragments. The number ofdistinguishable allelesin the population and theirfrequencies have not been estimated in this study.

Notation Enzyme Allele 1 Allele 2 SE Unrelatedchromosomes

Fragment(s) Frequency Fragment(s) Frequency studied

H HindIll 7-0+7-0 0-47 14-0 0-53 0-03 316P PvuII 3-3 0-48 1-6+1-7 0-52 0-03 334B BamHI 6-3+1-1 0-07 7-4 0-93 0-02 148L PvuII 2-4 <2-4

and diaphyseal aclasis (10.1). In one pedigree withpseudoachondroplasia (6.3), exclusion relied on thedisease being inherited as an autosomal recessivefrom the common ancestor of a consanguineousmarriage, because the patient had inherited differ-ent COL2AJ alleles from his parents. Discordantsegregation in one of the spondyloepiphyseal dys-plasia congenita pedigrees (3-2) depended on therelatively unlikely event that the disorder wasinherited as an autosomal recessive trait. Thekindred was of insufficient size to ascertain themode of inheritance independently.Two families with metaphyseal chondrodysplasia

(type Schmid) (8-1, 8*2) and two with spondylo-epiphyseal dysplasia tarda (autosomal dominant,2.2, 2.3) were uninformative because key personswere homozygous for all markers.

In four kindreds, spondyloepiphyseal dysplasia(congenita) (3-1), spondylometaphyseal dysplasia(5-1 and 5.2), and diastrophic dysplasia (11-1),concordant segregation was observed with weaklypositive lod scores. However, these results only heldif recessive inheritance were operating in thesekindreds.

Discussion

We have used a crude estimate of COL2AI structu-ral integrity so that the absence of observable grossrearrangements of the gene in CD patients shouldnot be taken to mean that the gene does not containthe causal mutation. Gross rearrangements of col-lagen genes seem to be uncommon and only veryrarely would a similar screen have detected muta-tions at the two collagen I loci in osteogenesisimperfecta, even though both fine structure andlinkage data identify these genes as the mutant lociin this disorder.

Discordant segregation in well defined pedigreesis a more secure demonstration that COL2AI is notthe mutant locus. In the absence of special factors,for which no evidence exists, the expected recom-

bination fraction between any of the linkage mar-kers used in the analysis and the physical limits ofthe 30 kb gene is about 0-0003, so that even a singlerecombination event between these markers and theCD gene in a pedigree effectively excludes COL2AJas the mutant locus. Recently, it has been shownthat chondrocalcin, a protein promoting the bindingof chondrocytes to cartilage matrix, is identical tothe C-propeptide of collagen II, which is cleavedfrom the mature collagen molecule during post-translational processing. 8 Since this peptide is alsoencoded at COL2AJ and is entirely containedwithin cosHcoll, our structural and linkage resultson the collagen II gene equally apply to thechondrocalcin gene.

In certain families, it was not possible to deter-mine the inheritance pattern and in these cases weanalysed the data separately for dominant andrecessive transmission rather than relying on pub-lished mechanisms. Pseudoachondroplasia, for in-stance, can be inherited as an autosomal dominantor recessive trait with incomplete distinction be-tween the two mechanisms on clinical groundsalone.21With these reservations we have excluded

COL2AJ as the mutant locus in several CD families.The evidence is now very strong against COL2AI asthe mutant locus in achondroplasia16 and, therefore,our result in hypochondroplasia was not surprisingsince the two are thought to be allelic.29 The strikingabnormalities of cartilage development leading topremature osteoarthritis and deformity make mul-tiple epiphyseal dysplasia a candidate for defects inCOL2AI but our results in two families, one withautosomal dominant and one with recessive inheri-tance, did not support this. Bearing in mind thepresence of collagen II in the vitreous, the ocularinvolvement of spondyloepiphyseal dysplasia sug-gests that COL2AJ might be the mutant locus. Thepedigrees here were not particularly informative butdiscordance was convincingly shown in an atypicalkindred (4.1) where myopia was pronounced.

526


Further work is needed to explore the possibilitythat mutations in COL2AJ are causal in the rarerdysplasias including those that we have studied.Obviously larger kindreds and further genetic mar-kers would be valuable. It was particularly dis-appointing, for instance, that two potentially usefulkindreds with spondyloepiphyseal dysplasia tarda(2.2, 2.3) and two with metaphyseal chondrodyspla-sia (type Schmid) (8-1, 8 2) were not informativebecause the alleles could not be distinguished in keypersons. Diastrophic dysplasia merits further atten-tion, not least because of the collaFen changes whichhave previously been described. The small pedi-gree we studied did not exclude linkage to COL2AIbut many more cases would need to be examined toachieve a significant positive lod score. There is alsodoubt about the method of inheritance of thisdysplasia which is said to be recessive,29 although weknow of no cases of recurrence in sibs from the UK.Perhaps only families with intrinsic evidence ofrecessive inheritance should be studied, a dauntingtask in such a rare disease.

In summary, although COL2A] is a very plausiblecandidate locus in the heritable chondrodysplasias,our results find no evidence to support this and, inseveral cases, strong evidence against it. Certainly,the hypothesis that the different CD phenotypesmight generally be the result of different mutationsin the collagen II gene is now refuted.

We thank the many patients and colleagues for theircooperation in this project. The work was supportedby grants from the Arthritis and RheumatismCouncil, Nuffield Foundation, Medical ResearchCouncil, and the Rehabilitation and Medical Re-search Trust. Lesley Watts typed the manuscript.

References

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2 Wynne-Davies R, Hall C, Apley AG. Atlas of the skeletaldysplasias. Edinburgh: Churchill Livingstone, 1985.Dorst J, Faure C, Giedion A, et al. Nomenclature des maladiesosseuses constitutionelles. Ann Radiol (Paris) 1978;21:253-8.

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6 Sykes B, Ogilvie D, Wordsworth P, Anderson J, Jones N.Osteogenesis imperfecta is linked to both type I collagenstructural genes. Lancet 1986;ii:69-72.

7Bonadio J, Byers PH. Subtle structural alterations in the chainsof type I procollagen produce osteogenesis type II. Nature1985;316:363-5.Prockop DJ, Kivirikko KI. Heritable disorders of collagen. NEngl J Med 1984;311:376-86.

9 Rimoin DL, Sillence DO. Chondroosscous morphologyand biochcmistry in the skelctal dysplasias. Birth Defects1981:,17:249-65.Horton WA, Chou JW. Machado MA. Cartilagc collagenanalysis in the chondrodvstrophics. Coll Relat Res 1985:5:349-54.Stanescu V. Stancscu R. Maroteaux P. Pathogenic mechatnismsin the osteochondrodysplasias. J Bonie Jointt Surg (Ain)1984;66:817-36.

12 Murray LW. Rimoin DL. Type II collagcn abnormaliticsin the spondyloepi- and spondyloepimctaphyscal dysplasias.Am J Humn Genet 1985;37:13A.

3 Stanescu R. Stanescu V. Marotcaux P. Abnormal pattcrn ofsegment long spacing cartilage collagen in diastrophic dysplasia.Coll Relat Res 1982;2:111-6.

14 Hollister DW, Byers PH, Holbrook KA. Gcnetic disorders ofcollagen metabolism. Ads, Humn Geniet 1982:12:66-8.

15 Cheah KSE, Stoker NG. Griffin JR. Grosvcld FG, Solomon E.Identification and characterization of the human al (II) collagengene (COL2AI). Proc Natl Acad Sci USA 1985:82:2555-9.

16 Ogilvie D, Wordsworth P. Thompson E. Sykes B. Evidenccagainst the structural gene encoding typc II collagcn (COL2AI)as the mutant locus in achondroplasia. J Med Geniet1986;23:19-22.

17 Barrie H, Carter C, Sutcliffc J. Multiplc epiphysial dysplasia. BrMed J 1958ji: 133-7.

18 Dennis NR, Renton P. The severe recessive form ofpseudoachondroplastic dysplasia. Pediatr Radiol 1975:3:169-75.

19 Howell CJ, Wynne-Davies R. The trichorhinophalangeal syn-drome. J Bone Joitnt Surg (Br) 1986;68:311-4.

211 Wynne-Davies R, Hall CM. Young ID. Pseudoachondroplasia:clinical diagnosis at different ages and comparison of autosomaldominant and recessive types. J Med Geniet 1986;23:425-34.Sykes BC, Ogilvie DJ, Wordsworth BP. Lethal ostcogenesisimperfecta and a collagen gene deletion. Length polymorphismprovides an alternative explanation. Humn Geniet 1985;70:35-7.

22 Weiss EH, Cheah KSE. Grosveld FG, Dahl HHM. Solomon E.Flavell RA. Isolation and characterisation of a human collagenal(I)-like gene from a cosmid library. Nucleic Ac ids Res1982;10: 1981-94.

23 Sangiorgi FO, Benson-Chanda V, de Wet WJ. Sobel ME.Tsipouras P, Ramirez F. Isolation and partial characterisation ofthe entire human pro-cal(II) collagen gene. Nucleic Acids Res1985;13:22(07-25.

24 Sykes BC. A high frequency Hind III restriction site polymorph-ism within a collagen gene. Disease Markers 1983;1:141-6.

25 Sykes B, Smith R, Vipond S, Paterson C. Cheah KSE. SolomonE. Exclusion of the acl(II) cartilage collagen gene as a mutantlocus in type IA osteogenesis imperfecta. J Med Geniet1985;22:187-9 1.

26 Pope FM, Cheah KSE, Nicholls AC, Price AB, Grosveld FG.Lethal osteogenesis imperfecta congenita and 300 base pairdeletion for an cal (I)-like collagen. Br Med J 1984;288:431-4.

2 Stoker NG. Cheah KSE, Griffin JR, Pope FM. Solomon E. Ahighly polymorphic region 3' to the human type 11 collagengene. Nucleic Acids Res 1985;13:4613-22.

28 van der Rest M, Rosenberg LC. Olsen BR, Poole AR.Chondrocalcin is identical with the C-propeptide of type 11procollagen. Biochem J 1986;237:923-5.

29 McKusick VA. Heritable disorders of the skeleton. St Louis:Mosby, 1972:740-856.

Correspondence and requests for reprints toDr B Sykes, Nuffield Department of Pathology,John Radcliffe Hospital, Level 4, Headington,Oxford OX3 9DU.

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structural segregation of the type in · multiple epiphyseal dysplasia normal or mild short...

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