Identification and Localization of the Gene for EXTL, a Third Member of the Multiple Exostoses Gene Family Carol A

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Identification and Localization of the Gene for EXTL, a Third Member of the Multiple Exostoses Gene Family Carol A Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH Identification and Localization of the Gene for EXTL, a Third Member of the Multiple Exostoses Gene Family Carol A. Wise, I Gregory A. Clines, I Hillary Massa, 2 Barbara J. Trask, 2 and Michael Lovett I I Department of Otorhinolaryngology, Molecular Biology, and Oncology and the McDermott Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 7.5235-8.591; 2Department of Molecular Biotechnology, University of Washington, Seattle, Washington 9891.5 Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by multiple bony outgrowths from the juxtaepiphyseal region of long bones. In a small proportion of cases, these exostoses progress to malignant chondrosarcomas. Genetic linkage of this disorder has been described to three independent loci on chromosomes 8q24.1 (EXTI), llpll-13 (EXT2), and l?p (EXT-3). The EXTI and EXT2 genes were isolated recently and show extensive sequence homology to each other. These genes are deleted in exostoses-derived tumors, supporting the hypothesis that they encode tumor suppressors. We have identified a third gene that shows striking sequence similarity to both EXTI and EXT2 at the nucleotide and amino acid sequence levels, and have derived its entire coding sequence. Although the mRNA transcribed from this gene is similar in size to that from EXTI and EXT2, its pattern of expression is quite different. We have localized this gene by fluorescence in situ hybridization to metaphase chromosomes and by whole genome radiation hybrid mapping to chromosome Ip36.1 between DIS458 and DIS511, a region that frequently shows loss of heterozygosity in a variety of tumor types. This gene, EXTL (for EXT-like), is therefore a new member of the EXT gene family and is a potential candidate for several disease phenotypes. [The sequence data described in this paper have been submitted to GenBank under accession no. U67191] Hereditary multiple exostoses (EXT) is an autosomal this disease (Hennekam 1991; Wicklund et al. dominant disorder of bone growth characterized by 1995). the presence of exostoses, bony outgrowths capped Multiple exostoses have been observed as part by cartilage that emerge primarily from the juxtae- of the clinical profile in patients with Langer- piphyseal regions of the long bones (Solomon Giedion contiguous gene syndrome, which is a de- 1961). Some exostoses may be apparent at birth but letion of 8q24.1 (Cook et al. 1993), and a recently continue to increase in size and number during delineated contiguous gene syndrome that is a de- growth until the closure of the growth plates (Hen- letion of 11p11-p13 (Bartsch et al. 1996). Consistent nekam 1991). These outgrowths may limit joint with these observations, linkage analysis in EXT movement or cause pain as a result of compression families has revealed that this is a multigenic disor- of nerves. In these cases surgical intervention is of- der (Cook et al. 1993), identifying genetic loci on ten necessary, and many patients require multiple chromosomes 8q24.1 (EXT1) (Cook et al. 1993) and surgeries throughout the course of the disease. EXT 11p11-13 (EXT2) (Wu et al. 1994; Wuyts et al. is estimated to have a prevalence of one in 50,000 to 1995). Genetic linkage has also been reported to a one in 100,000 in Western populations (Hennekam third locus on chromosome 19p (EXT3) (Le Merrer 1991; Cook et al. 1993). In a few percent of cases the et al. 1994). The EXT1 gene was cloned recently and exostoses are transfomed to malignant chondrosar- shown to harbor mutations in affected members comas or osteosarcomas, the most severe form of from two linked families, and to span chromosomal breakpoints identified in two multiple exostoses pa- tients (Ahn et al. 1995). Recently, we utilized a combination of posi- 3Corresponding author. E-MAIL [email protected]; FAX (214) 648-1666. tional cloning approaches to isolate the EXT2 gene l 0 @GENOME RESEARCH 7:10-I 6 9 997 by Cold Spring Harbor Laboratory Press ISSN 10.54-9803/97 $5.00 Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press IDENTIFICATION AND LOCALIZATION OF EXTL (Stickens et al. 1996). A chromosome l 1-specific using the BLASTN and BLASTX programs (Altschul cDNA library (Del Mastro et al. 1995) was con- et al. 1990). Three ESTs were identified, yo78g12, structed and screened with YACs encompassing the y186d04, and yf56h05, which are similar but not previously defined EXT2 region. A candidate gene identical to EXT1 and EXT2. These EST sequences from this library was identified from its sequence overlap with each other and thus appear to repre- similarity to the EXT1 gene. Subsequent studies sent a third gene, EXTL. The EST sequences demonstrated that this gene maps precisely within yo78g12.r1 and yf56h05.s1 represent the 5' and 3' the genetically defined region of 11p11-13 and ends of overlapping cDNA clones isolated from a within a small deletion that cosegregates with the normalized neonatal brain cDNA library (Soares et disorder in one family. Moreover, the transcript size al. 1994). The DNA sequence between these two and expression of this candidate EXT2 gene closely cDNA ends was derived by first amplifying human resemble that of EXT1. Mutational analysis of EXT2 placental cDNA in the PCR with primers specific to in a linked family identified a 4-bp deletion segre- each EST. The resulting 2-kb PCR product was gating in affected individuals, which is predicted to cloned and sequenced, revealing an open reading result in protein truncation. frame of 852 bp before reaching a stop codon. In Although the functions of these genes are not order to derive more 5' coding sequences, primers clear, both EXT1 and EXT2 are believed to encode were designed from this sequence and used to ex- tumor suppressors, based on the observation of loss tend the sequence by rapid amplification of cDNA of heterozygosity (LOH) of nearby markers in spo- ends (RACE). The cDNA was extended an additional radic and exostoses-derived chondrosarcomas 1980 bp by this method, resulting in a composite (Hecht et al. 1995; Raskind et al. 1995). LOH has sequence of 4009 bp that contains a 2031-bp open also been found at polymorphic loci on chromo- reading frame extending to a stop codon at nucleo- some 19p, the putative locus for EXT3, in tumors tide 2895. The conceptual translation product en- derived from patients whose disease is not linked to coded by this cDNA shows significant similarity to either EXT1 or EXT2 (Hecht et al. 1996). A separate both EXT1 and EXT2 proteins (Fig. 1). There are 328 study detected LOH of polymorphic markers linked (49%) identical amino acids between the EXTL cod- to EXT1 and EXT2 in 44% of tumors with no LOH at ing sequence and EXT1. If one includes conserva- 19p (Raskind et al. 1995). These observations have tive changes, the homology reaches 72%. By com- supported the proposal that the EXT genes fit the parison, the EXT1 and EXT2 proteins contain 22% classic profile of tumor suppressors (Knudson 1971), identical amino acids and have 49% homology wherein a mutation in one gene causes bony pro- when conservative amino acid changes are in- trusions, and mutation or loss of the normal allele cluded. None of the three EXT genes show signifi- causes progression to tumors as a result of lack of cant homology to known proteins or protein mo- tumor suppression (Ahn et al. 1995; Hecht et al. tifs, although each has a clustering of hydrophobic 1995; Raskind et al. 1995; Stickens et al. 1996). amino acids at the amino terminus, which could At the time that we identified the EXT2 gene we comprise a leader sequence. Overall, the three pre- also detected an additional sequence within the EST dicted proteins are most similar, with fewer gaps, at database (Lennon et al. 1996) that was similar, but the carboxyl terminus. not identical, to EXT1 and EXT2. We have now ex- In order to determine the chromosomal loca- tended the sequence of this EST by rapid amplifica- tion of EXTL, primer sets were designed from the tion of cDNA ends (RACE). Northern blot analysis cDNA sequence. These primers were used in the PCR indicates that this gene's transcript size is similar to to amplify DNAs from a panel of human X rodent EXT1 and EXT2, but its expression pattern is differ- monochromosomal somatic cell hybrids (Coriell In- ent. We have mapped this gene (which we have stitute). The results indicated that this gene is lo- called EXTL for EXT-like) to chromosome lp36, cated on chromosome 1. Identical results were ob- thus excluding it as a candidate for EXT3. Interest- tained with primer sets derived from 3' cDNA se- ingly, this region of the genome is frequently de- quences (data not shown). These mapping results leted in a variety of tumor types and is thus believed exclude EXTL as a candidate for EXT3, which has to contain one or more tumor suppressor loci. been genetically linked to chromosome 19p. The chromosomal localization was confirmed and refined in two ways. First, the St. Louis yeast RESULTS artificial chromosome (YAC) library (Burke et al. EXT1 and EXT2 cDNA sequences were searched 1987), which had been pooled in a PCR-amplifiable against the expressed sequence tag (EST) database format, was screened with EXTL-specific primers. GENOME RESEARCH ~ 1 1 Downloaded from genome.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press WISE ET AL. EXTL ................ ~MQSWRRRKSLWLALSASWLLLVLr,GGFSLI,RI2tLppRPR ........... PGASQGWPRWLDAELLQSFSQPG 62 with signals also evident at I1. ::1 ::1. :.:: I1..:11: : I ....
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