GILLIAN A. WALLIS BONE GROWTH

Here today, bone tomorrow The effects of mutations of the type X collagen gene in mice and man establish a role for this extracellular matrix protein during endochondral bone formation.

In 1943, FE Stephens [l] reported on the inheritance of an ‘achondroplasia’ phenotype in more than 40 in- dividuals in four generations of a Mormon family. The disorder in this family was subsequently termed meta- physeal chondrodysplasia type Schmid (MCDS) on the basis of the autosomal dominant inheritance of short stature, genu varum (bow legs), coxa vara (a deformity of the femoral neck in which the angle between the neck and shaft is decreased) and the waddling gait of affected members [z] , Now, iifty years and three gen- erations later, the mutation causing the disorder in this family has been found within the gene encoding type X collagen [3]. Further, the development of a spondy- lometaphyseal dysplasia (SMD) phenotype by transgenic mice expressing a dominant-negative mutant form of type X collagen [4] firmly establishes that this collagen has a role in skeletal morphogenesis (Fig. 1). Type X collagen consists of three identical a-chains. It is a member of a class of short-chain collagens in which the triple helical domain is approximately half the length of that found in the classical fibrillar collagens. The molecule consists of a non-collagenous amino-terminal domain, a triple helical domain characterized by repeating Gly-X-Y triplets and a large, globular carboxy-terminal domain, The chick, bovine, mouse and human genes encoding the type X collagen a-chains have been cloned and sequenced and show a considerable degree of sequence conservation in the collagenous as well as non-collagenous domains [5], The carboxyterminal domain of the al (X) chain, as for other collagen types, is thought to be required for the intracellular aggregation and precise alignment of the three a-chains so that triple helix formation pro- ceeds in a carboxy-terminal to amino-terminal direction. In addition, the carboxy-terminal domain of the molecule is retained extracellularly, where it is probably important for the macromolecular aggregation of the molecules to form lattice-like networks [G] , Interest in type X collagen has centred around its spe- cific function during endochondral bone formation. En- dochondral ossification is the process whereby the car- tilaginous model of the axial and appendicular skeleton of higher vertebrates is replaced by bone and marrow, with accompanying longitudinal growth. This process is initiated during embryogenesis when chondrocytes in the centre of the cartilage anlage proceed through the devel- opmental stages of proliferation, maturation and hyper- trophy (Fig. 2) (reviewed in [7]). The hypertrophic car- tilage in the centre of the anlage is first calcified and then replaced by primary bone that, through remod- elling, is replaced by secondary bone and bone marrow.

Fig. 1. Consequences of mutations within the type X collagen gene in (a) mice and (b) humans. The mice are litter mates at day 21 after birth; one is normal and the other is a hemizygous hunch-backed mutant. (Photograph courtesy of 0 Jacenko). The father, son and daughter in the family picture have metaphyseal chondrodysplasia type Schmid but the mother is unaffected. The affected individuals are short in stature and the genu varum is particularly evident in the son and daughter. (Photograph courtesy of Cl Scott).

This process radiates outward with the development of growth plates at the ends of these tissues. ‘Qpe X colla- gen is exclusively and transiently synthesized by the hyper- trophic chondrocytes of the growth plate and represents a considerable proportion of collagen synthesized by these chondrocytes (reviewed in [8] ). The specific expression of type X collagen suggests that it plays an important role in the transition of cartilage to bone during endochondral ossification.

Direct evidence that type X collagen is required for nor- mal bone development has recently been obtained first, by the generation of transgenic mice expressing a mutant

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Diaphysis

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Reserve zone

Proliferative zone

Zone of maturation

Upper hypertrophic zone

lower hypertrophic zone

,Calcification of cartilage

-Invading capillary -0steoblast

-Calcified trabecula

rammatic representation of a long bone in the latter stages of embryonic development. The ossification centres cartilage model of the bone have migrated outward towards the ends of the bone. Secondary ossification

within the epiphyseal cartilage and blood vessels have penetrated the calcified cartilage matrix. On the right, ded to show the organization and differentiation of the chondrocytes.

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form of type,,X’collagen and second, from the study of naturally occurring mutations in MCDS patients [3,4,9]. For the generation of the transgenic mice, four mini-gene constructs of the chick al(X) gene were prepared. The constructs contained 1600 or 4700 base pairs of upstream non-translated sequence in combination with cMNAs con- taining either a 21 codon or 293 codon deletion within the triple helical domain, The deletions were constructed in such a way that the reading frame of the coding sequences for the triple helical and carboxyterminal domains was preserved. On the basis of the homology between the mouse and chick carboxyterminal domains, it was ex- pected that the chick and the endogenous mouse al(X) chains would associate, thereby preventing assembly of functional homotrimeric molecules. The hybrid molecules would be expected to be either degraded or u.nable to function effectively in the matrix. The constructs were microinjected into mouse embryos and fourteen transgenic lines, with a phenotype that included, to a greater or lesser extent, thoracolumbar kyphosis and growth retardation, were established. These transgenic lines represented all four constructs, each of which had an independent integration site demonstrating that the phenotype was not caused by disruption of an

endogenous gene. In most instances, the phenotype was only apparent in mice post-weaning and when they be- came more mobile. At this stage, about 15-20 % of the mice developed progressive hunching of the back and gradual limb paresis and died within 4 days of the onset of symptoms. Histological analysis of the genotypically posi- tive mice revealed in all a compression of the growth plate, particularly in the region of chondrocyte hypertrophy, and a reduction in the size and number of newly formed bony trabeculae, although mineralization appeared normal. An unexpected and as-yet unexplained observation was that in the 15-20 % of mice with the severe phenotype, the bone marrow showed a predominance of mature erythro- cytes but a paucity of leukocytes, and the sizes of the thy- mus and spleen were reduced. Tissue-specific expression of the transgenes was confirmed by northern blot anal ysis. Immunohistochemistry, using monoclonal antibod- ies specific for chick-type X collagen, demonstrated that the transgene product co-localized with regions showing histological defects. These data strongly imply that the skeletal defects observed in the mice were caused by dis- ruption of the endogenous type X collagen matrix by the transgene product. The precise mechanism whereby this process occurs, however, remains to be determined.

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The skeletal and growth abnormalities observed in the type X collagen transgenic mice are similar to the defor- maties characteristic of certain human forms of SMD and metaphyseal chondrodysplasia. The search for a compara- ble human disease caused by a mutation within the type X collagen gene (COLIC&U) has recently borne fruit with the descriptionsof a 13 base-pair deletion within the coding re- gion of this gene that segregates with the MCDS pheno- type in the large Mormon family [3]. The mutation results in a frameshift that alters the last 60 amino acids of the 160 amino-acid carboxy-terminal domain. The frameshift also introduces a premature stop codon so that the carboxy terminal domain is nine amino acids shorter than normal. The molecular consequences of this mutation on Ibe syn- thesis and structure of the type X collagen molecule are as-yet not known. The most likely effect of the mutation is that the altered structure of the carboxy-terminal do- main prevents association of the mutant molecules with either normal or other mutant a-chains during trimer for- mation, thereby leading to a reduction in the amount of type X collagen deposited in the matrix. However, it is also possible that the mutant @ l(X) chain is unstable and rapidly degraded, or that the mutant chains are able to as- sociate with other a-chains but the specific interactions of the nucleation complex are altered in such a way Ithat the structure of the molecule is abnormal. These structurally abnormal molecules could either be rapidly degraded or deposited in the matrix where they would alter the specific properties of the matrix. We have recently identified two further mutations within the carboxyterminal domain of type X collagen in individuals with MCDS. In these cases the mutations are single base-pair changes that lead to the substitution of conserved residues [9]. The possible molecular consequence of these mutations are similar to those described above for the deletion mutation.

In some instances of MCDS we (unpublished data) and others [3] have been unable to 6nd any type X collagen mutations, despite extensive examination of the coding regions of the gene. This may, of course, be because the disorder is heterogeneous, but it is equally likely that mutations causing a decrease in the amount of type X collagen mRNA may have escaped detection. A further class of mutations that have not a-yet been identified in human forms of chondrodysplasia comprises those that alter the structure of the triple helical region of the type X collagen molecule. Such mutations may have a more severe phenotype than MCDS and resemble more closely that of the transgenic mice. Equally, such mutations may be lethal during embryonic development, or result in a phenotype that is primarily haematological rather than skeletal in presentation.

By examining the consequences of the type X collagen mutations in both mouse and man, it is now possible to

hypothesize with some degree of certainty as to the role of this matrix molecule during skeletal development. One hy- pothesis is that the lattice-like structure of type X collagen replaces the more densely packed fibrillar type II collagen network and provides stability and room for the chondro- cytes to hypertrophy. This hypothesis is in keeping with the proposed structure of type X collagen, the histologi- cal findings and the time of onset of symptoms in both transgenic mice and in individuals with MCDS [lo]. In nei- ther the mouse model nor in MCDS did mineralization per se appear abnormal, although in both there appeared to be a reduction of mineral in the regions of the trabecu- lae. It is possible that, following the death of the hyper- trophic cells, type X collagen could provide a temporary matrix for the attachment of osteoblasts responsible for the deposition of trabecular bone in this region.

References 1.

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STEPHENS FE: An achondroplastic mutation and the nature of its inheritance. J Hered 1943, 34:2229-2235. BEIGHTON P: Inherited disorders of the skeleton. 2nd edn. Edinburgh: Churchill Livingston; 1989. WARMAN ML, ABOTT M, API-E SS, HEFFERON T, MCINTOSH I, COHN DH, HECHT JT, OLSEN BR, FRANCOMANO CA: A type X coUa- gen mutation causes S&mid metaphyseal chondrodysplasia. Nature Genet, in press. JACENKO 0, Lu VALLE P& OLSEN BR: Spondylometaphyseal dys- plasia in mice carrying a dominant-negative mutation in a matrix protein specific for cartilage-to-bone transition. Nature 1993, 365:56(;1.

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KONG RYC, KWAN KM, LAu ET, THOMAS JT, BOOT-~FORD RP, GRANT ME, CHEAH KSE: Intron-exon structure, alternative use of the promoter and expression of the mouse collagen X gene, COLIOAI. Eur J Biocbem 1993, 213:99-111. KwAN APL, CUMMINGS CE, CHAPMAN JA, GRANT ME: Macromolec- ular organisation of chicken type X collagen in vitro. J Cell Biol 1991, 14597-604.

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POOLE AR: The growth plate: cellular physiology, cartilage assembly and mineralization. In Cartilage: Molecular meets. Edited by Hall BK, Newman SA Boca Raton: CRC Press; 1991. GORDON MK, O&EN BR: The contribution of couagenous pro- teins to tissue-specific matrix assemblies. Curr Opin Cell Biol 1990, 2:833$838.

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WALUS GA, RASH B, SWEETMAN WA, THOMAS JT, SUPER M, EVANS G, GRANT ME, BOOT-HANDFORD R: Amino-acid substitutions of conserved residues in the carboxy-terminal domain of the al(X) chain of type X collagen occur in two unrelated fam- ilies with metaphyseal chondroclysplasia type Schmid. Am J Hum Genet, in press. L~CHMAN RS, RIMON DL, SPRANGER J: Metaphyseal chondrodys- plasia, S&mid type. Clinical and radiographic delineation with a review of the literature. Pediatr Radiol 1988, 18:93-102.

Gillian A. Wallis, Department of Biochemistry and Molecular Biology, University of Manchester, Oxford Road, Manchester Ml3 9PT, UK. -.,: ..-