The Structure of the Human Multiple Exostoses 2 Gene and Characterization of Homologs in Mouse and Caenorhabditis elegans

Hereditary multiple exostoses (EXT) is an autosomal dominant condition characterized by multiple cartilage-capped projections originating from the epiphyseal plates of endochondral bones (Jaffe 1943; Solomon 1961). The prevalence of this disorder has been estimated at between 1/50,000 and 1/100,000 (Hennekam 1991; Cook et al. 1993; Schmale et al. 1994). The development of exostoses appears to be correlated with growth of the epiphyseal plate, with new exostoses rarely appearing after epiphyseal plate closure (Hennekam 1991). Exostoses usually form at the ends of long bones but can also occur on the hands, feet, pelvis, ribs, and scapula. Shortened stature is sometimes seen in this disorder and is possibly attributable to these benign tumors interfering with the epiphyseal growth plates and reducing overall bone growth (Hennekam 1991). Exostoses also frequently impinge on adjacent nerves, vessels, tendons, and joints and thus require surgical removal. A serious complication of this disorder is the occasional transformation to chondrosarcoma or osteosarcoma that occurs in 0.9%–2.9% of cases (Voutsinas and Wynne-Davies 1983; Wicklund et al. 1995). Genetic linkage analysis in EXT families has identified three loci responsible for hereditary multiple exostoses on chromosomes 8q24.1 (EXT1), 11p11–13 (EXT2), and 19p (EXT3) (Cook et al. 1993; Le Merrer et al. 1994; Wu et al. 1994; Wuyts et al. 1995). To date, the exostoses that are caused by disruption of these genes appear to be indistinguishable; no specific phenotypic differences have been observed in patients from different linkage groups (Le Merrer et al. 1994). Multiple exostoses has also been observed as one phenotype in two contiguous gene deletion syndromes that encompass EXT1 or EXT2 separately, confirming the hypothesis that haploinsufficiency for the products of these genes results in the disruption of bone growth (Ludecke et al. 1991; Parrish et al. 1991; Bartsch et al. 1996). Loss of heterozygosity (LOH) studies using polymorphic markers that flank EXT1, EXT2, and EXT3 indicate that these chromosomal regions are lost in chondrosarcomas that develop in genetically linked families (Hecht et al. 1995, 1997; Raskind et al. 1995). In addition, LOH for loci flanking EXT1 and/or EXT2 has been detected in 7 of 17 sporadic chondrosarcomas (Raskind et al. 1995), and LOH at both EXT1 and EXT2 has been observed in a single chondrosarcoma that was derived from an EXT2-linked individual (Hecht et al. 1995). Together, these findings suggest that the EXT genes may act as tumor suppressors in developing skeletal tissues, in addition to playing a role in bone growth and modeling. The chromosome 8 EXT1 gene was cloned by an approach involving analysis of chromosomal breakpoints (Ahn et al. 1995). The EXT2 gene was identified recently by our collaborative group using a new positional cloning strategy (Stickens et al. 1996) and subsequently was cloned independently by Wuyts et al. (1996). The two genes encode very similar, novel proteins, the functions of which are unknown. However, the presence of a putative signal sequence composed of a hydrophobic core at the amino terminus of both proteins indicates that these proteins may be either secreted or embedded within the cell membrane (Wise et al. 1997). A third member of the EXT family (named EXT-L for EXT-like) also has been identified by our group (Wise et al. 1997). This gene has been localized to chromosome 1p36.1 and is thus not a candidate for the chromosome 19-linked EXT3 gene. Currently, no evidence exists that this gene is associated with any skeletal disorder, but it lies within a chromosomal region that frequently exhibits LOH in other types of malignancies (Lothe et al. 1995; Nagai et al. 1995; Ezaki et al. 1996). Previously, we have described the EXT2 cDNA sequence and a limited characterization of 8 exons (Stickens et al. 1996). However, the efficient detection of mutations in EXT2-linked families requires a more thorough description of the genomic structure of EXT2, including precise exon/intron boundaries. To this end, we have subcloned a yeast artificial chromosome (YAC) clone, which contains the entire EXT2 coding region, into cosmids and have used primer-directed DNA sequencing to identify all of the exon/intron boundaries in the gene. The lengths of the introns were also determined by long-range PCR using cosmid clones and genomic DNA to define a gene of 108 kb that contains 16 exons and utilizes alternative exons and alternative polyadenylation signals. One approach to unraveling the function of the EXT2 gene is to study its homologs in other species. Here, we describe the cDNA sequence of the mouse EXT2 gene that we have mapped to chromosome 2. We also describe two EXT homologs in Caenorhabditis elegans. The detection of EXT homologs in this nematode worm indicates that these genes do not function exclusively as regulators of bone growth and opens the way for a functional analysis of these genes in lower organisms. In addition, the description of the mouse EXT2 sequence should enable transgenic knockout experiments to be initiated to further elucidate gene function.