Molecular Exclusion of Mutations in EXT1 and EXT2 as the Cause of Metachondromatosis T475 Molecular Exclusion of Mutations in EXT1 and EXT2 as the Cause of Metachondromatosis Steven Mumm1, Margaret Huskey1, William H. McAlister2, Michael P. Whyte3 1Division of Bone & Mineral Diseases, Washington University School of Medicine, St. Louis, MO, USA, 2Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO, USA, 3Research Center, Shriners Hospitals for Children, St. Louis, MO, USA. Figure 1 Figure 2 Figure 3 Discussion Abstract: Pedigrees of Metachondromatosis Families 1 and 2 Metachondromatosis and hereditary multiple exostoses (HME) are distinct skeletal Langer-Giedion syndrome (OMIM #150230; also called trichorhinophalangeal syndrome type II) is a dysplasisas, yet share some similarities. They are both autosomal dominant disorders contiguous gene deletion syndrome including TRPS1 and EXT1. Patients have clinical features of featuring exostoses (cartilage-capped bony protrusions from the metaphyseal region of long both trichorhinophalangeal syndrome type I (dysmorphic facial features including large ears and a bones) and often affect the hands (Maroteaux 1971). However, the protuberances tend to bulbous nose, sparse hair, and skeletal defects) due to deletion of TRPS1, and HME due to Metachondromatosis (OMIM# 156250) is a rare, autosomal dominant, skeletal point toward the adjacent joint in metachondromatosis, whereas they point away from the deletion of EXT1. We were careful to look for polymorphisms in EXT1 and EXT2 for our dysplasia featuring multiple metaphyseal juxtaepiphyseal exostoses joint in HME. These exostoses develop soon after birth and increase in size and number until metachondromatosis patients to exclude the possibility of complete deletion of either gene. For (characteristically pointing toward the adjacent joint, and often involving the bones of closure of the growth plate. family 1, we did not find polymorphisms in EXT1. Further, the affected boys have mild facial the hands and feet), metaphyseal striated enchondromas (see figure), periarticular features resembling TRPS and sparse hair, suggesting the possibility that they have a form of ossification, and femoral head deformities resembling avascular necrosis. These HME often exhibits shortening and bowing of the forearm and short stature, and sometimes Langer-Giedion syndrome. However, routine karyotyping for 2 patients, and high-resolution banding result in pain and deformities, and often require surgical removal. At least 22 cases (0.5-2.0% of cases) the exostoses develop into chondrosarcoma. HME is a genetically studies of the TRPS1/EXT1 locus on chromosome 8, showed no abnormalities (Herman et al. have been reported, although lack of large families has precluded identification of heterogenous disorder caused by mutations in either the EXT1 or EXT2 genes, which show 1997). Further studies are needed to resolve these findings. We plan to look at additional SNPs in the genetic defect through linkage analysis. We have clinically evaluated two considerable homology (Bernard et al. 2001, Stickens et al. 1996). A third locus (EXT3) has EXT1, TRPS1, and neighboring genes, and will likely perform high-resolution copy number analysis families with multigenerational metachondromatosis. been identified on chromosome 19, but no causal gene has yet been identified (Wuyts and using gene chips. Van Hul, 2000). EXT1 and EXT2 encode glycosyltransferases involved in heparin sulfate biosynthesis (Simmons et al. 1999). It has been proposed that the EXT1/2 proteins are Eventual identification of the metachondomatosis gene and protein function should shed new light Boy in Family 1 at 4-5/12 years Left knee in this boy in Metachondromatosis Family 1 The second digit shows a shows a broad and slightly short Family 2 at age 2-11/12 involved in the synthesis of glycosaminoglycan, which regulates the diffusion of IHH into the on the mechanism for endochondral bone development. Metachondromatosis shares clinical similarities with hereditary multiple exostoses broad exostosis in the radius that has some linear areas of years shows small perichondrium and up-regulates PTHrP. PTHrP then signals, via the PTH/PTHrP receptor on base of the proximal sclerosis and lucencies as does the exostoses in the distal (HME, OMIM #133700 & #133701), which is also an autosomal dominant disorder; phalanx. distal ulna. Small focal calcifications femur and proximal fibula. prehypertrophic chondrocytes, preventing additional differentiation of chondrocytes. HME is caused by loss-of-function mutations in the EXT1 and EXT2 genes. Like at the proximal first and distal third There are small areas of Therefore, mutations in EXT1/2 prevent proper diffusion of IHH, and disrupt the negative A small calcification is at and fourth metacarpals are seen. sclerosis and lucencies in metachondromatosis, HME has cartilage-capped exostoses, primarily in the the base of the middle the metaphyses and the feedback loop on PTHrP expression. The result is perturbed bone development, i.e. phalanx laterally in the fibular epiphysis. juxtaepiphyseal region of the long bones. However, in HME, the exostoses point boy of Family 2 at 2-11/12 exostoses. away from the adjacent joint. Accordingly, we examined EXT1 and EXT2 for years of age References mutations in our 2 families with metachondromatosis. One proband from each was Based on the similarities between HME and metachondromatosis, we hypothesized that the defect in metachondromatosis may also disrupt the signaling events between IHH and studied using genomic DNA isolated from blood leukocytes. All 11 exons for EXT1 Bassett GS, Cowell HR. Metachondromatosis. Report of four cases. J Bone Joint Surg Am. 1985; 67:811-4. and 15 exons for EXT2, including the entire coding region and adjacent mRNA The tips of the noses are mildly bulbous and the hair is fine and sparse. PTHrP, during bone development. Accordingly, we screened EXT1 and EXT2 as candidate genes for metachondromatosis in our 2 families. However, from this study reported here, it Beals RK. Metachondromatosis. Clin Orthop. 1982; 169:167-70. splice sites, were amplified by PCR and sequenced in both directions. DNA Patients 1 (center) and 2 (right) had their first haircuts at the age of 2 and now seems that mutations in EXT1 and EXT2 do not account for metachondromatosis. We did Bennett LB, Roach ES, Bowcock AM. A locus for paroxysmal kinesigenic dyskinesia maps to human chromosome 16. sequence was examined using VectorNTI-AlignX software. No mutations in EXT1 or Neurology 2000; 54: 125-30. have them every 3 –4 months and 6 months, respectively. Patient 3 (left) had not find any mutations in EXT1 or EXT2 that would cause our families’ bone disease. The EXT2, that would explain metachondromatosis, were found (approximately 95% of Bernard MA, Hall CE, Hogue DA, Cole WG, Scott A, Snuggs MB, Clines GA, Ludecke HJ, Lovett M, Van Winkle WB, his first haircut at the age of four years and has had one annual haircut since. presence of polymorphisms (with one exception, discussed below) in EXT1 and EXT2 Hecht JT. Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes. this analysis is complete). Metachondromatosis Family 2 exclude the possibility of complete deletion of either gene. Cell Motil Cytoskeleton. 2001; 48:149-62. Clines GA, Ashley JA, Shah S, Lovett M. The structure of the human multiple exostoses 2 gene and characterization of homologs in mouse and Caenorhabditis elegans. Genome Res. 1997; 7:359-67. Herman TE, Chines A, McAlister WH, Gottesman GS, Eddy MC, Whyte MP. Metachondromatosis: report of a family with The metachondromatosis gene encodes a protein necessary for endochondral bone facial features mildly resembling trichorhinophalangeal syndrome Pediatr Radiol. 1997; 27:436-41. development, including shaping (defects result in exostoses) and ossification Figure 4 Horton WA. Abnormalities of Bone Structure, Chapter 154 In Emery and Rimoin’s Principles and Practice of Medical (defects result in enchondromas). We hypothesize that the metachondromatosis Genetics, 4th Edition, Vol 3, Churchill Livingstone, London, 2002, pp 4152-5153. protein plays a role in the regulation of the critical pathways, including PTHrP and This boy in family 2 at 7-9/12 years of age shows widening The boy of Family 1 at 5-2/12 years of age shows wide and Ikegawa S, Nagano A, Matsushita T, Nakamura K. Metachondromatosis: A report of two cases in a family. Nippon and deformity of the femoral necks and intertrochanteric shortened femoral necks plus epiphyseal changes of Perthes The histologic features consist of small fragments of bone Seikeigeka Gakkai Zasshi. 1992; 66:460-6. IHH, essential in chondrocyte differentiation and endochondral bone formation. areas with areas of sclerosis and lucencies consistent with greater on the left. There are areas of sclerosis and lucencies and cartilage consistent with multiple small enchrondromata. in the femoral necks and acetabulae. Karaplis AC. Embryonic development of bone and the molecular regulation of intramembranous and endochondral bone osteochondromatous proliferations. formation, Chapter 3, In: Principles of Bone Biology, 2nd Edition, JP Bilezikian, LG Raise and GA Rodan, Eds., 2002, Academic Press, San Diego, pp 33-58. This is consistent with the clinical history of Kennedy LA. Metachondromatosis. Radiology. 1983; 148:117-8. metachondromatosis. Kozlowski K, Scougall JS. Metachondromatosis: report of a case in a 6 year old boy. Aust Paediatr J. 1975; 11:42-5. Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am J Hum Genet. 1996; 58:1347-63. Lachman RS, Cohen A, Hollister D, Rimoin DL. Metachondromatosis. Birth Defects Orig Artic Ser. 1974; 10:171-8. Lathrop GM, Lalouel JM, Julier C, Ott J. Multilocus linkage analysis in humans: detection of linkage and estimation of Patients and Methods recombination. Am J Hum Genet. 1985; 37:482-98. Introduction Lüdecke HJ, Ahn J, Lin X, Hill A, Wagner MJ, Schomburg L, Horsthemke B, Wells DE. Genomic organization and promoter structure of the human EXT1 gene. Genomics. 1997; 40:351-4. Maroteaux P. La Metachondromatose Z Kinderheilkd. 1971; 109:246-61. Metachondromatosis (OMIM# 156250) is a rare, autosomal dominant disease of children Patients Methods Family 2 Individual V-7: Family 2 Individual V-3: Family 2 Individual V-2: Mumm S, Jones J, Finnegan P, Henthorn PS, Podgornik M, and Whyte, MP. Denaturing Gradient Gel Electrophoresis featuring multiple metaphyseal juxtaepiphyseal exostoses (characteristically pointing 10x H and E undecalcified edited. 20x Bone Masson undecalcified edited. 10x undecalcified H and E. Analysis Of The Tissue Non-Specific Alkaline Phosphatase Isoenzyme Gene In Hypophosphatasia. Molecular Genetics and Metabolism. 2002; 75:143-53. toward the adjacent joint and often involving the bones of the hands and feet), We have ascertained 2 kindreds with metachondromatosis at the Research Center at All exons and adjacent mRNA splice junctions (including at least 20 bp of intronic Ott J. Computer-simulation methods in human linkage analysis. Proc Natl Acad Sci USA 1989; 86:4175-4178 sequence) of EXT1 and EXT2 were amplified by PCR, using leukocyte genomic DNA metaphyseal striated enchondromas, periarticular ossification, and frequent femoral head Shriners Hospital for Children, St. Louis. The families are from Missouri and Indiana and are Peters JM, Barnes R, Bennett L, Gitomer WM, Bowcock AM, Garg A. Localization of the gene for familial partial deformities resembling avascular necrosis (Fig 1, Maroteaux 1971, Horton 2002, unrelated based on pedigree analyses from family histories (Fig 2). [isolated with Gentra PureGene Kit, Qiagen, (Valencia, CA)], and sequenced in both lipodystrophy (Dunnigan variety) to chromosome 1q21-22. Nat Genet. 1998; 18:292-5. Spranger 2002). These skeletal defects result in pain and deformities, which often require directions, for one metachondromatosis patient for each family. Simmons AD, Musy MM, Lopes CS, Hwang LY, Yang YP, Lovett M. A direct interaction between EXT proteins and Results glycosyltransferases is defective in hereditary multiple exostoses. Hum Mol Genet. 1999; 8:2155-64. surgical removal or correction. At least 22 cases have been reported worldwide, although Metachondromatosis Family 1 Spranger, J. W. Brill, P. W. Poznanski. A. Bone Dysplasias: An Atlas Of Genetic Disorders Of Skeletal Development, 2nd the lack of large affected families has precluded identification of the genetic defect via PCR used a 40ul reaction volume, including 1X AmpliTaq Gold master mix (Applied Biosystems, Foster City, CA, 8ul 1.2uM primer pairs, 6.4ul 50% glycerol, and 4ul diluted Edition, Oxford University Press, pp 560-561, 2002. linkage analysis approaches (Lachman et al. 1974, Beals 1982, Vanek 1982, Kennedy This first family was reported for its clinical and radiograph findings in 1997 (Herman et al. o We amplified by PCR and then sequenced all the exons and adjacent mRNA splice junctions These include 2 synonymous polymorphisms in the coding region (c.1065C>T, p.C355C in Stickens D, Clines G, Burbee D, Ramos P, Thomas S, Hogue D, Hecht JT, Lovett M, Evans GA. The EXT2 multiple (1/10) genomic DNA. PCR conditions included an initial denaturation at 96 C for 5 min, for both EXT1 and EXT2 for one affected individual from each of our two metachondromatosis exon 3; c.1761G>A, p.E587E in exon 9) and a SNP in IVS 6 (-51G>A from exon 7). The 1983, Dorst 1983, Bassett and Cowell 1985, Kozlowski 1975. Ikegawa et al. 1992, 1997). The family was noted to have facial features mildly resembling trichorhinophalangeal o o o exostoses gene defines a family of putative tumour suppressor genes. Nat Genet. 1996; 14:25-32. followed by 35 cycles of 30 sec at 94 C, 45 sec at 60 C, and 45 sec at 72 C, followed by a families. We were careful to examine intronic sequence flanking the exons to reveal any presence of these polymorphisms precludes the possibility of complete deletion of EXT1 as Herman et al. 1997). At the Research Center at Shriners Hospitals for Children, St. Louis, syndrome (mildly bulbous noses; fine, sparse hair) (Fig 3). This is a o Terwilliger JD. A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more 4-generation kindred with 3 affected boys with three different mothers originating from a final extension at 72 C for 10 min. The primer pairs were either from VariantSEQr (Applied potential mRNA splicing mutations; this always included at least 20 bp of intervening the cause of metachondromatosis in this family. polymorphic marker loci. Am J Hum Genet. 1995; 56:777-87. we have ascertained and clinically evaluated 2 families with metachondromatosis. single father (Fig 2). This man likely represents a founder for the metachondromatosis Biosystems, Foster City, CA) or were designed by us. Some annealing temperatures were sequence, but often included up to 100bp. We also sequenced extensive regions of the 5' and Tomfohrde J, Silverman A, Barnes R, Fernandez-Vina MA, Young M, Lory D, Morris L, Wuepper KD, Stastny P, Menter A, specific for the primer set and were determined by gradient PCR. Primer sequences and et al. Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q. Science. 1994; mutation, because neither of his parents is affected by history. One of his sisters (indicated 3' UTRs. DNA sequence chromatograms were carefully examined visually, and the 264:1141-5. Like metachondromatosis, hereditary multiple exostoses (HME) is a rare autosomal annealing temperatures are available on request. The PCR amplicons were purified using EXT2 by quarter-filled circle in the pedigree) has rheumatoid arthritis and may be unaffected sequencing text files were aligned to control sequence to identify any defects. EXT2 spans about 150 kb of genomic sequence on chromosome 11p12-p11, and contains Vanek J. Metachondromatosis, 3 case reports with hereditary occurrence Beitr Orthop Traumatol. 1982; 29:103-7. dominant disease featuring similar exostoses at the juxta-epiphysieal region of the long the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA). No mutations in either gene (EXT1 and EXT2) were identified in either family that would despite hip and knee replacements (further clinical characterization is underway). The a total of 16 exons that includes 2 alternatively spliced exons (labeled 1a and 1b; others are Weeks DE, Ott J, Lathrop GM SLINK: a general simulation program for linkage analysis. Am J Hum Genet 1990; 47:A204 bones (Wuyts and Van Hul 2000). However, the protuberances tend to point away from 3 affected boys have metachondromatosis symptoms and radiographic findings of account for their bone disease. Several apparent polymorphisms were found, as reported labeled 1-14) and 2 polyadenylation signals in exon 14 (OMIM*608210; Clines et al. 1997; (Supplement) DNA sequencing reactions were performed in 20 ul reaction volumes that included 1X Big the adjacent joint in HME, whereas they point toward the joint in metachondromatosis. metachondromatosis including striated metaphyseal enchondroma-like lesions, juxta- below. Wuyts and Van Hul 2000). For the patient from family 1, a common polymorphism was Wise CA, Clines GA, Massa H, Trask BJ, Lovett M. Identification and localization of the gene for EXTL, a third member of Dye buffer (Applied Biosystems, Foster City, CA), 4 ul Big Dye v3.1 (Applied Biosystems, the multiple exostoses gene family. Genome Res. 1997; 7:10-6. These exostoses develop soon after birth and increase in size and number until closure of epiphyseal exostoses, and findings of Legg-Calve-Perthes disease (Fig 1). The 3 affected found in IVS 6 (A/T at -20 from exon 7; refSNP ID: rs11037882). One apparent Foster City, CA), 2 ul (3.2 pmol/ul) sequencing primer, and 5ul purified DNA from the PCR Wuyts W, Van Hul W. Molecular basis of multiple exostoses: Mutations in the EXT1 and EXT2 genes. Hum Mutat. 2000; the growth plate. HME often exhibits shortening and bowing of the forearm and short boys from this kindred were evaluated (M.P.W.) as in-patients at the Research Center at o polymorphism was detected in exon 1 for the patient from family 2. This was a single reactions. Cycling conditions included an initial denaturation at 96 C for 1 min, followed by EXT1 15:220-7. Shriners Hospital for Children, St. Louis, MO. o o EXT1 is located on chromosome 8q24, spans about 300 kb of genomic sequence, and nucleotide change (G>A) early in the 5'UTR of exon 1. This change is likely not disease- stature, and sometimes (0.5-2.0% of cases) the exostoses develop into chondrosarcoma. 25 cycles of denaturation at 96 C for 10 sec, annealing at 50 C for 5 sec, and extension at o causing since it is not in the coding region (the ATG start codon is in exon 2) and is reported HME is a genetically heterogenous disorder, being caused by mutations in either the 60 C for 4 min. Sequencing primers for the VarientSEQr primer sets were M13 forward or contains 11 exons (OMIM *608177; Ludecke et al. 1997; Wuyts and Van Hul 2000). For the Metachondromatosis Family 2 patient from family 1, no polymorphisms were detected in the exons or the intronic sequence. as a known SNP (refSNP ID: rs3923808). No known mutations causing HME are located in EXT1 or EXT2 genes, which show considerable homology (Bernard et al. 2001, Stickens reverse. Other amplicons were sequenced with a single primer that was used for PCR. Therefore, based on this result alone, we can not rule out complete deletion of EXT1 as the exons 1, 1a, or 1b (Wuyts and Van Hul 2000). et al. 1996). These genes encode glycosyltransferases involved in heparin sulfate Sequencing reactions were purified with the DyeEx 2.0 Spin Kit (QIAGEN, Valencia, CA) Our second family was also ascertained and evaluated at Shriners Hospital for Children, St. cause of metachondromatosis in this family. This is of particular interest because the affected Therefore, no mutations were found in EXT2 that would explain the bone disease in either Acknowledgments biosynthesis (Simmons et al. 1999). and run on a 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA). DNA Louis in 1998, with additional DNA samples acquired in April, 2004. This is a large, 5- boys from this family showed mild facial features of trichorhinophalangeal syndrome type II, family. The presence of at least one polymorphism for each patient/family rules-out the sequence was analyzed visually and with VectorNTI AlignX software. generation kindred exhibiting autosomal dominant inheritance of metachondromatosis in at which is a contiguous gene deletion syndrome including EXT1 (see the Discussion). possibility of a complete deletion of EXT2 as the cause of metachondromatosis for these Francis H. Gannon, MD at the Armed Forces Institute of Pathology provided the histology report. Vivienne Lim created this Because of the clinical similarities between metachondromatosis and HME, we screened poster with the help of Dawn Russell and Bruce Marren, Graphic Arts Department, Shriners Hospitals for Children. Supported by least 4 generations. (Fig 1, Fig 2). Three of the affected children are patients at Shriners Three apparent polymorphisms in EXT1 were detected in the patient from family 2. families. Shriners Hospitals for Children, The Clark and Mildred Cox Inherited Metabolic Bone Disease Research Fund and The EXT1 and EXT2 as candidate genes in our 2 metachondromatosis families. Hospital, St. Louis. Hypophosphatasia Research Fund.