Multiple hereditary exostosis, EXT genes, and skeletal development

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1 Washington University School of Meicine Digital Open Access Publications Multiple hereitary exostosis, EXT genes, an skeletal evelopment Lina J. Sanell Washington University School of Meicine in St. Louis Follow this an aitional works at: Part of the Meicine an Health Sciences Commons Recommene Citation Sanell, Lina J.,,"Multiple hereitary exostosis, EXT genes, an skeletal evelopment." The Journal of Bone an Joint Surgery.91,Supplement (2009). This Open Access Publication is brought to you for free an open access by Digital It has been accepte for inclusion in Open Access Publications by an authorize aministrator of Digital For more information, please contact

2 58 COPYRIGHT Ó 2009 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED Multiple Hereitary Exostosis, EXT Genes, an Skeletal Development By Lina J. Sanell, PhD Multiple hereitary exostosis is an autosomal ominant inherite isease in which osteochonral growths occur on the periphery of bones. These growths are comprise of bone surroune by a cap of cartilage. A small number of these exostoses procee to a low-grae chonrosarcoma 1,2. Although the isease can occur spontaneously, it has been estimate that 80% of affecte iniviuals have a positive family history 2. Research on the genetics of multiple hereitary exostosis over the past thirty years has been prouctive. Avances in the unerstaning of the isease have parallele the methoological avances that have occurre in the fiel of molecular genetics. Initially, it was recognize that multiple hereitary exostosis is often inherite an that large families were available for genetic mapping. As techniques for gene mapping improve, regions of the chromosomes involve were ientifie, localize, an eventually subjecte to DNA sequencing. The genes ientifie, the exostosins, were foun to encoe known enzymes whose function within the isease coul be reasonably preicte. Mouse moels were create, an the hypothesize function of these genes was verifie. Many surprises were encountere along the way, which serve to uncover important biological principles. The unerstaning of human multiple hereitary exostosis is a paraigm for the power of combining moern molecular biology, genetics, an clinical science. The Genetics of Multiple Hereitary Exostosis In the early 1990s, the clearly autosomal ominant inheritance of multiple hereitary exostosis was recognize by clinicians an DNA techniques became available to localize the inheritance patterns in the DNA. By genetic linkage analysis, Hecht et al. an Le Merrer et al. were able to localize the inheritance patterns of these families to three chromosomal locations: 8q24.1, 11p11-13, an 19p 2,3.Thesegeneswere ientifie as tumor-suppressor genes, an loss of heterozygosity in these regions was associate with transformation into chonrosarcoma 2,4. The genes that cause multiple hereitary exostosis were calle exostosins an name EXT1, EXT2, an EXT3. EXT1 an EXT2, corresponing to the chromosomal localizations on chromosomes 8 an 11, respectively, were ientifie by positional cloning 5,6, whereas EXT3 has not yet been ientifie an its linkage to patients with multiple hereitary exostosis has been questione 7. Base on DNA sequence homology, genome screens have uncovere aitional members of the gene family, three EXT-like (EXTL) genes, bringing the total number of similar genes to six. However, only EXT1 an EXT2 have been associate with both familial multiple hereitary exostosis an spontaneous multiple hereitary exostosis, an more than 80% of unrelate patients who have been teste have a mutation in one of these two genes 2,4. Function of EXT Genes The EXT1 an EXT2 genes encoe two glycosyltransferase subunits of the heparan sulfate (HS)-synthesizing system that elongates HS chains to specific proteins belonging to a class calle proteoglycans. The HS chain is a linear glycosaminoglycan mae up of alternating D-glucuronic aci (GlcAc) an N-acetyl-D-glucosamine (GlcNAc) subunits. The synthesis of HS chains is a very complex post-translational event, initiate with a protein linkage through serine. The HS chains are elongate with the alternating GlcAc an GlcNAc resiues via a complex of enzymes that inclues the EXT1 an EXT2 glycosyltransferases. The length of the chains can vary an appears to be cell specific. Mutation in the glycosyltransferase genes, usually by causing a frame-shift in protein elongation or missense in amino aci coe, creates truncate forms of the enzymes that those genes encoe 6,8, leaing to lower enzyme activity an less HS chain synthesis. In fact, chonrocytes isolate from persons with multiple hereitary exostosis contain less enzyme 9. The connection between HS-synthesizing glycosyltransferases an the multiple hereitary exostosis genes was mae by two inepenent stuies. EXT1 was emonstrate to encoe for a protein that coul rescue HS biosynthesis in an HS-eficient mutant cell line 10. EXT2 was ientifie as an HS co-polymerase purifie from bovine serum 11. Mutations in EXT1 an EXT2 result in the formation of clinically inistinguishable exostoses. While both enzymes are able to transfer GlcNAc an GlcAc, they are not functionally reunant Disclosure: The author i not receive any outsie funing or grants in support of her research for or preparation of this work. Neither she nor a member of her immeiate family receive payments or other benefits or a commitment or agreement to provie such benefits from a commercial entity. J Bone Joint Surg Am. 2009;91 Suppl 4:58-62 oi: /jbjs.i.00391

3 59 Fig. 1 Function of HS proteoglycans. Synecans are transmembrane or extracellular matrix proteoglycans mae up of alternating GlcAc an GlcNAc carbohyrate resiues attache to a protein backbone. A: Bining of growth factor ligans (black squares) to HS chains (circles). B: Distribution of morphogen (black circle) attache to HS chains (white circles) of proteoglycan 22. (Reprinte, with permission, from: Naanaka S, Kitagawa H. Heparan sulphate biosynthesis an isease. J Biochem. 2008; 144:8.) in vivo; instea, EXT1 an EXT2 seem to be a complementary pair that forms a stable enzyme complex in vivo. The EXT1/ EXT2 complexes have consierably higher glycosyltransferase activity than either EXT1 or EXT2 alone 10. The proucts of the EXTL genes are able to transfer GlcNAc 12,13 an are thought to be involve in transferring the first GlcNAc resiue to the linkage region to initiate HS synthesis. While the roles of these enzymes have not been completely etermine in vivo, at least EXTL2 an EXTL3 may have aitional biological functions in the regulation of HS synthesis an the etermination of chain length but are not necessary for initiation an elongation of HS chains. Function of Heparan Sulfate The HS chains of these proteoglycans are responsible for a variety of functions primarily involving carbohyrateprotein interactions. Heparan sulfate chains are foun on a variety of proteoglycans, incluing the large proteoglycan versican, foun primarily in bloo vessels; perlecan, the major proteoglycan in basement membranes an many other tissues, incluing the eveloping limb an chonrocytes; an smaller proteoglycans calle synecans. Synecans are foun in most tissues an occur in a family of four proteins (Fig. 1, A) 14 ; they are foun at the cell surface boun into the membrane as receptors or in the extracellular matrix. At the cell surface or in the extracellular matrix, the proteoglycansactasligansorco-receptorswherethehs chains are necessary for receptor recognition an bining 15.HS proteoglycans are known to be necessary for signaling of fibroblast growth factors (FGFs), vascular enothelial growth factor (VEGF), an transforming growth factor-beta (TGF-b) an are involve in the graient formation of morphogens such as hegehog or bone morphogenetic proteins (BMPs) (Fig. 1, B). HS proteoglycans also influence the formation of amyloi fibrils in the brain an help to incorporate lipoproteins into cells in the liver. The istribution an function of proteins that contain HS chains are extensive, an not all have been well characterize. Since these proteoglycans occur throughout the boy, it is unclear why mutations in the genes coing for HS chains cause multiple hereitary exostosis, a phenotype apparently restricte to bone. Lessons from Developmental Biology: Drosophila an Mice Most of the insight into the function of HS proteoglycans an the role of the EXT genes has come from stuies in Drosophila an mice, as a result of genetic manipulations. In Drosophila, there are three orthologs of mammalian EXT genes, EXT1 (tout-velu), EXT2 (sister of tout-velu), an EXTL3 (brother of tout-velu). The phenotypes resulting from muta-

4 60 Fig. 2 Hypothesis for creation of exostoses from ysregulate chonrocytes. Chonrocyte nests in the Ext2 1/ heterozygote. Top panel: a: Normal growth occurs until embryonic ay 14.5 of long-bone evelopment. b: Due to ysregulate growth-factor signaling, isolate chonrocytes in the periphery of the eveloping cartilage anlagen unergo terminal ifferentiation by proliferation an hypertrophy. c: When expose to the vasculature from the passing growth plate, cells unergo enochonral bone evelopment. Bottom panel: A: Safranin O staine longituinal section of a rib of the Ext2 1/ mouse, showing noules (arrows) that are forcing the perichonrium to bulge out. B: Safranin O staine transverse section through the rib, showing a single isplace chonrocyte (arrow) near the perichonrium (P). C: Higher magnification of safranin O staine longituinal section, showing a noule. D: Low magnification of alcian blue (cartilage) an alizarin re (bone) staining of a rib of an Ext2 1/ mouse, showing a large noule (arrow). Scale bars: 200 mm ina;50mm in B an C; an 300 mm ind. Reprouce an aapte with permission from: Stickens D, Zak BM, Rougier N, Esko JD, Werb Z. Mice eficient in Ext2 lack heparan sulfate an evelop exostoses. Development. 2005;132:5062, Reprinte with permission. tions in these genes provie insight into the primary functions of HS proteoglycans. In all of these mutant phenotypes, the morphogenetic graients of the Drosophila proteins Hegehog (mammalian homolog hg), Wingless (mammalian homolog Wnt), an Decapentaplegic (mammalian homolog BMP) were interrupte, causing these proteins to accumulate in front of the mutant cells 16. All three protein pathways are involve in both early embryonic evelopment an mammalian skeletal evelopment. Targete eletion of the Ext1 gene in mice (mouse genes are inicate with lower case letters) abolishes the synthesis of HS 17 an isrupts signaling of FGFs, TGF-b, Wnts, an BMPs an results in early embryonic lethality aroun the time of gastrulation. Mice that are heterozygous for the Ext1 eletion show somewhat increase chonrocyte proliferation an elaye hypertrophic ifferentiation, probably ue to increase Inian Hegehog (Ihh) 18. Mice that are hypomorphic for the Ext1 allele (which means they have a mutation with a reuce level of gene activity) survive to a later embryonic stage, with expane growth plates an an expane range of Inian Hegehog signaling. These results inicate that, in mice, one of the critical roles of HS is to establish a graient of Inian Hegehog signaling to inuce the proper ifferentiation of chonrocytes in the growth plate. Deletion of the Ext2 gene was expecte to result in the same phenotype as eletion of the Ext1 gene, because human mutations in EXT1 an EXT2 result in the same phenotype.

5 61 However, as Stickens et al. showe, this is not the case 19. Inactivation of the Ext2 gene results in early embryonic lethality similar to the Ext1 eletion, but compare with Inian Hegehog-null mice, which ie mi-gestation, the Ext2-null mice ie earlier with efects in the extra-embryonic structures an gastrulation. This early emise coul imply that fibroblast growth factor (fgf) signaling is involve; however, while fgfr1 an fgf8 cannot be rule out, fgf4 an fgf2 mutant mice ie shortly after implantation, earlier than the time at which Extnull mice ie. The Ext-null phenotype also oes not coincie with the phenotype of members of the BMP or Wnt pathway families. Consequently, it is possible that multiple pathways, or an as yet uniscovere pathway, are moulate by the Ext genes. Aitional interesting moels use by evelopmental biologists to stuy Ext genes inclue Caenorhabitis elegans (a nematoe 20 ) an zebrafish 21. Alternate Hypotheses for Initiation of Exostoses The hypothesis that haploinsufficiency of an EXT gene (i.e., one allele inactivate by a mutation, with the resulting reuce gene prouct not sufficient for a normal phenotype) reuces enzymatic activity of the glycosyltransferases, causing formation of exostoses, is not the only plausible explanation for this conition. An alternate hypothesis for the initiation of exostoses has been presente by Stickens et al. 19.Thishypothesis involves the interaction of the FGF an BMP-TGF-b pathways. As note above, FGF signaling pathways epen on HS. In long-bone growth plates, FGF signaling shortens proliferative cell columns, both by ecreasing chonrocyte proliferation irectly an by suppressing Ihh expression. BMPs antagonize the effects of FGF signaling an are necessary for chonrocyte ifferentiation. Therefore, mutations in the EXT1 an EXT2 genes ecrease HS synthesis, which reuces FGF signaling an leas to efects in chonrocyte ifferentiation. Whether the premature ifferentiation of chonrocytes leas to the formation of exostoses or whether the processes are istinct is not known. However, a moel that combines the two possibilities into a unifying hypothesis is presente. If, ue to a slightly erange signaling system, some chonrocytes near the perichonrium ifferentiate into hypertrophic chonrocytes (characterize by type-x collagen synthesis), as the growth plate grows past this nest of hypertrophic chonrocytes, vascular elements of the growth plate are expose to the hypertrophic chonrocyte nest an bone formation is initiate (Fig. 2, upper panel; the neste chonrocytes from the Ext2 1/2 mice are shown in Fig. 2, lower panel). This coul result in growth at a 90 angle from the normal bone growth as seen in an exostosis, an this hypothesis coul explain the low penetrance of the phenotype an the variable istribution of exostoses. Conclusions While the exact mechanism by which unerprouction of HS proteoglycan chains causes exostosis formation is not fully unerstoo, the synergism of molecular biology, genetics, an biochemistry combine with the power of animal moels an the observations of astute clinicians has prouce a superior unerstaning of the inheritance an biology of multiple hereitary exostosis. The pursuit of knowlege about this isease has in turn stimulate the evelopment of molecular biology, impacting the unerstaning of both normal evelopment an other isease processes. The efforts focuse on unerstaning this particular isease have provie an outstaning paraigm for the roles of HS chains in regulating biological processes, the elineation of the unerlying causes of other cartilage an bone iseases, an some of the factors that enable early embryo survival. Finally, because the EXT genes have been shown to be tumor-suppressor genes an HS proteoglycans are intimately involve in angiogenesis an cancer, stuy of multiple hereitary exostosis coul even she light on the mechanisms of cancer evelopment. n Lina J. Sanell, PhD Department of Orthopaeic Surgery, Washington University School of Meicine, MS 8233, 660 South Eucli Avenue, St. Louis, MO aress: Sanelll@wuosis.wustl.eu References 1. Schmale GA, Conra EU 3r, Raskin WH. The natural history of hereitary multiple exostoses. J Bone Joint Surg Am. 1994;76: Hecht JT, Hogue D, Strong LC, Hansen MF, Blanton SH, Wagner M. Hereitary multiple exostosis an chonrosarcoma: linkage to chromosome II an loss of heterozygosity for EXT-linke markers on chromosomes II an 8. Am J Hum Genet. 1995;56: Le Merrer M, Legeai-Mallet L, Jeannin PM, Horsthemke B, Schinzel A, Plauchu H, Toutain A, Achar F, Munnich A, Maroteaux P. A gene for hereitary multiple exostoses maps to chromosome 19p. 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