Heparan sulfate abnormalities in exostosis growth plates

Hereditary multiple exostoses (HME), a condition associated with development and growth of bony exostoses at the ends of the long bones, is caused by germline mutations in the EXT genes. EXT1 and EXT2 function as glycosyltransferases that participate in the biosynthesis of heparan sulfate (HS) to modify proteoglycans. HS proteoglycans, synthesized by chondrocytes and secreted to the extracellular matrix of the growth plate, play critical roles in growth plate signaling and remodeling. As part of studies to delineate the mechanism(s) by which an exostosis develops, we have systematically evaluated four growth plates from two HME and two solitary exostoses. Mutational events were correlated with the presence/absence and distribution of HS and the normally abundant proteoglycan, perlecan (PLN). DNA from the HME exostoses demonstrated heterozygous germline EXT1 or EXT2 mutations, and DNA from one solitary exostosis demonstrated a somatic EXT1 mutation. No loss of heterozygosity was observed in any of these samples. The chondrocyte zones of four exostosis growth plates showed absence of HS, as well as diminished and abnormal distribution of PLN. These results indicate that, although multiple mutational events do not occur in the EXT1 or EXT2 genes, a complete loss of HS was found in the exostosis growth plates. This functional knockout of the exostosis chondrocytes' ability to synthesize HS chains further supports the observations of cytoskeletal abnormalities and chondrocyte disorganization associated with abnormal cell signaling.     

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.     

Hereditäre multiple Exostosen

Hereditary multiple exostosis (HME), a disorder inherited in an autosomal dominant manner, is characterized by multiple projections of bone, mainly at the extremities. The risk of malignant transformation of the exostoses is estimated to be up to 2%. The most common underlying cause of the disease involves mutations in either the EXT1 or the EXT2 gene. We report on the clinical and molecular findings in a family affected with HME.A mother and her three children from different partnerships, all clinically diagnosed with HME, were referred for genetic counseling. Subsequently, molecular analysis of the EXT1 gene was performed according to standard procedures. We identified a mutation in the EXT1 gene in all four affected family members (delA in codon 133). This mutation has not been previously described and is suggested to cause the disease in this family. Identification of disease causing mutations in patients with HME and their relatives can help to improve the clinical management of tumor prevention, early tumor detection, and orthopedic therapy.     

Manifestations of hereditary multiple exostoses

The solitary osteochondroma, a common pediatric bone tumor, is a cartilage-capped exostosis. Hereditary multiple exostosis is an autosomal dominant disorder manifested by the presence of multiple osteochondromas. Linkage analysis has implicated mutations in the EXT gene family, resulting in an error in the regulation of normal chondrocyte proliferation and maturation that leads to abnormal bone growth. Although exostoses are benign lesions, they are often associated with characteristic progressive skeletal deformities and may cause clinical symptoms. The most common deformities include short stature, limb-length discrepancies, valgus deformities of the knee and ankle, asymmetry of the pectoral and pelvic girdles, bowing of the radius with ulnar deviation of the wrist, and subluxation of the radiocapitellar joint. For certain deformities, surgery can prevent progression and provide correction. Patients with hereditary multiple exostosis have a slight risk of sarcomatous transformation of the cartilaginous portion of the exostosis.     

Clinical outcome and genotype in patients with hereditary multiple exostoses

Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder with a wide spectrum of clinical manifestations. In 52 out of 60 individuals from HME+ families, exostoses became clinically apparent. In this study, the clinical and radiological outcome of these 52 HME patients (19 families) was investigated by medical history, clinical examination, and radiographs. In addition to correlating phenotype with genotype, a linkage/exclusion analysis was performed in 35 HME patients. We found several correlations between HME genes (EXT1, EXT2) and phenotype. Compared to EXT2‐linkage, female individuals with EXT1‐linkage were smaller in stature. Patients with EXT1‐linkage and patients with undetermined linkage (EXT?) were more severely affected, underwent more surgeries, and showed a higher number of exostoses at follow‐up. Moreover, we found an increased phenotype risk for limb shortening for EXT1‐ and EXT?‐linkage. This study corresponds to data of other investigators who showed that EXT1 mutations are associated with a more severe phenotype than other EXT forms. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1541–1551, 2007     

Cervical myelopathy in hereditary multiple exostoses

Spinal cord compression due to cervical exostoses is a rare but recognized complication of hereditary multiple exostosis (HME), an autosomal dominant disorder. This disease, also called multiple osteochondromatosis, is characterised by osteocartilaginous exostoses, typically involving the juxtaepiphyseal regions of long bones. Complications such as transformation to sarcoma (1 to 5%) or neurological compression (of the spinal cord, 1 to 9%) can arise during the course of the disease. We report the case of a 64-year-old man with progressive difficulties in walking over many years, ascribed to congenital rachitism. A diagnosis of HME was not made until late in the disease course. Investigations revealed cervical myelopathy due to vertebral exostosis as well as multiple exostoses in other sites. His gait was not improved after surgical decompression. A better knowledge of this disease could have prevented this neurological complication.     

Hereditary multiple exostoses: a case report

Hereditary multiple exostoses (HME) is a dominantly inherited skeletal disorder which alters enchondral bone during growth and is characterized by exostoses of the juxta-epiphyseal regions. These exostoses are benign cartilaginous neoplasms that consist of a pedicle of normal bone covered with proliferating cartilage cells. Pathologic, clinical, and radiographic findings are discussed and a case of a nine-year-old male is reviewed.     

Compound heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs and long bones

Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1(+/-) and compound Ext1(+/-)/Ext2(+/-) mice. Like Ext2(+/-) mice described previously (Stickens et al. Development 132:5055), Ext1(+/-) mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1(+/-)Ext2(+/-) exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1(+/-)Ext2(+/-) chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1(f/+)Col2Cre and Ext1(f/+)Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.     

Clinical and radiographic analysis of osteochondromas and growth disturbance in hereditary multiple exostoses

Hereditary multiple exostoses (HME) is traditionally described as a skeletal dysplasia. However, the discovery that the EXT family of tumour suppressor genes are responsible for HME suggests that it is more appropriate to classify HME as a familial neoplastic trait. In a clinical and radiographic analysis of paired bone length and exostoses number and dimensions in a HME cohort, the local presence of osteochondromas was consistently associated with growth disturbance. In particular, an inverse correlation between osteochondroma size and relative bone length (p<0.01) was found. These data suggest that the growth retardation in HME may result from the local effects of enlarging osteochondromas rather than a skeletal dysplasia effect. This study provides the first clinical rationale for ablation of rapidly enlarging exostoses to reduce growth disturbance.     

Familial nephropathy and multiple exostoses with exostosin-1 (EXT1) gene mutation

Glomerular deposition of fibrillar collagen is a characteristic finding of genetically distinct conditions, including nail-patella syndrome and collagen type III glomerulopathy. A case of familial nephropathy in which steroid-sensitive nephrotic syndrome and glomerular deposits of fibrillar collagen are associated with multiple exostoses due to mutation of the EXT1 gene is described. This gene encodes a glycosyltransferase required for synthesis of heparan sulfate glycosaminoglycans. There is deficiency of heparan sulfate and perlecan, together with accumulation of collagens, in the matrix of EXT1-associated osteochondromas. Similar glomerular basement membrane abnormalities could offer an explanation for both the renal ultrastructural changes and steroid-sensitive nephrotic syndrome.     

The type 2 diabetes associated rs7903146 T allele within TCF7L2 is significantly under-represented in Hereditary Multiple Exostoses: Insights into pathogenesis

Hereditary Multiple Exostoses (HME) is an autosomal-dominant disorder characterized by benign cartilage tumors (exostoses) forming near the growth plates, leading to severe health problems. EXT1 and EXT2 are the two genes known to harbor heterozygous loss-of-function mutations that account for the vast majority of the primary genetic component of HME. However, patients present with wide clinical heterogeneity, suggesting that modifier genes play a role in determining severity. Our previous work has pointed to an imbalance of β-catenin signaling being involved in the pathogenesis of osteochondroma formation. TCF7L2 is one of the key ‘gate-keeper’ TCF family members for Wnt/β-catenin signaling pathway, and TCF7L2 and EXT2 are among the earliest associated loci reported in genome wide appraisals of type 2 diabetes (T2D). Thus we investigated if the key T allele of single nucleotide polymorphism (SNP) rs7903146 within the TCF7L2 locus, which is strongly over-represented among T2D cases, was also associated with HME. We leveraged genotype data available from ongoing GWAS efforts from genomics and orthopedic centers in the US, Canada and Italy. Collectively 213 cases and 1890 controls were analyzed and, surprisingly, the T allele was in fact significantly under-represented in the HME patient group [P = 0.009; odds ratio = 0.737 (95% C.I. 0.587–0.926)]; in addition, the direction of effect was consistent within each individual cohort. Immunohistochemical analyses revealed that TCF7L2 is differentially expressed and distributed in normal human growth plate zones, and exhibits substantial variability in human exostoses in terms of staining intensity and distribution. In summary, the data indicate that there is a putative genetic connection between TCF7L2 and EXT in the context of HME. Given this observation, we suggest that these loci could possibly modulate shared pathways, in particular with respect to β-catenin, and their respective variants interplay to influence HME pathogenesis as well as T2D.     

EXT1 regulates chondrocyte proliferation and differentiation during endochondral bone development

Multiple Hereditary Exostoses (MHE) is an autosomal dominant skeletal disorder most frequently caused by mutations in the EXT1 gene. MHE affects proper development of endochondral bones, such that all affected individuals present with exostoses adjacent to the growth plate of long bones, while some individuals exhibit additional bone deformities. EXT1 functions as a heparan sulfate (HS) co-polymerase, and when defective causes improper elongation of glycosaminoglycan side chains on core proteins of HS proteoglycans. Although analysis of heterozygous EXT1-deficient mice has failed to reveal any significant gross morphological variations in skeletal development, significant alterations in molecular signaling occur in the developing long bones. Our results indicate that defects in EXT1 and the resulting reduction in HS lead to enhanced Indian Hedgehog diffusion causing an increase in chondrocyte proliferation and delayed hypertrophic differentiation.     

The Taniguchi classification in cases of multiple cartilaginous exostoses]

27 patients treated surgically at Child Orthopaedic Clinic of Pomeranian Medical Academy between 1974-1996 for multiple cartilaginous exostosis (Aclasia Diaphysealis Keith) were classified into three groups according to the Taniguchi classification. This classification is based on whether multiple cartilaginous exostoses are present on distal forearm. Group I--no involvement of the distal forearm (n = 2), in group II involvement of the distal forearm without shortening of either bone (n = 7) was stated. Group III consists of members with involvement of the distal forearm with shortening the radius or the ulna (n = 18). Groups were compared with regard to: number of lesions, distribution of exostoses in different body areas, age of onset of the Keith disease, height of children, presence of valgus deformity of the ankle, dislocation of the radial head and presence of exostoses around hip area. This classification should be useful in estimating severity of Keith disease, identifying cases at high risk for complications like dislocation of the radial and malignant transformation.     

Disturbed growth in height in multiple cartilaginous exostoses (author's transl)]

Basing on the hypothesis that reduced body height in patients with multiple cartilaginous exostoses would be mainly accounted for by shorter extremities, not by a shorter trunk, the authors clinically examined 19 exostosis patients in respect of thigh, lower leg, upper and lower arm, as well as height of the seated patient. The dimensions were compared by the method of matched pairs, with 19 volunteers without diseased skeleton, who corresponded with one of the exostosis patients in respect of age, sex and height of seated patient. Results were evaluated according to Wilcoxon's test. This showed a statistically significant reduction in length of the extremities in adults with multiple cartilaginous exostoses.     

骨髓间充质干细胞种植Ⅰ型胶原支架材料修复关节软骨缺损的初步研究

目的研究骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)种植在Ⅰ型胶原支架材料(type Ⅰ collagen-glycosaminoglycan,CG)上,软骨定向诱导后修复关节软骨缺损的可能性.方法将来源于10只成年实验犬骨髓的贴壁细胞培养传代至第3代,收集后以2×106密度种植于直径9 mm,厚3 mm(干样品尺寸)干热交联处理(dehydrothermal treatment,DHT)的CG材料中,软骨诱导培养基诱导培养21 d.观察每日细胞-材料复合体直径与初始直径的百分比,反应其收缩性.Ⅱ型胶原及平滑肌肌动蛋白(smooth muscle actin,SMA)免疫组织化学染色观测体外软骨形成情况.将体外诱导培养的细胞-材料复合体植入实验犬膝关节软骨缺损模型,12周后取材观察.结果诱导培养过程中细胞材料复合体直径随时间延长而下降.21 d后,细胞-材料复合体收缩至初始直径的64.4%±0.3%;组织学见:材料的孔隙收缩,新合成的基质使细胞-材料复合体的多数区域变为实体组织;Ⅱ型胶原及SMA染色阳性.植入实验犬膝关节软骨缺损12周后,犬膝关节功能恢复,关节软骨缺损处有软骨样组织填充.结论将MSCs种植于CG材料中,经软骨诱导培养后并植入软骨缺损后能形成含有Ⅱ型胶原的软骨样实体组织.     

Chondroid chordomas and low-grade chondrosarcomas of the craniospinal axis. An immunohistochemical analysis of 17 cases.

The classification of cartilaginous neoplasms of the craniospinal axis is controversial. Indeed, the very existence of chondroid chordomas has recently been questioned. In an effort to clarify the direction of differentiation of cartilaginous neoplasms of this region, 17 neoplasms obtained from 17 patients with cartilaginous tumors of the craniospinal axis were examined by immunohistochemistry with a panel of antibodies. The panel included antibodies to cytokeratin (CK), epithelial membrane antigen (EMA), vimentin (VIM), S-100 protein, carcinoembryonic antigen (CEA), and type II collagen. Areas with cartilaginous differentiation were present in all 17 neoplasms. These areas were characterized by a matrix of amorphous blue ground substance with lacunae that contained enlarged and slightly atypical cells. This cartilaginous matrix stained strongly for type II collagen. Thirteen of the 17 neoplasms had a biphasic growth pattern in which areas with conventional chordoma were admixed with areas with cartilaginous differentiation. The cells within the cartilaginous components of these 13 neoplasms stained for CK (10 of 12 cases), EMA (10 of 13 cases), VIM (12 of 12 cases), S-100 protein (seven of 12 cases), and CEA (two of nine cases). Similarly, the conventional chordoma components of these same 13 neoplasms stained for CK (12 of 12 cases), EMA (13 of 13 cases), VIM (12 of 12 cases), S-100 protein (nine of 12 cases), and CEA (two of nine cases). The hyaline-appearing areas between the cords and sheets of cells of the conventional chordoma components of these 13 tumors also stained with type II collagen. These 13 tumors with both neoplastic cartilage and conventional chordoma were classified as chondroid chordomas. One of the 17 cases was composed entirely of neoplastic cartilage; however, the cells within the matrix of the cartilage of this neoplasm stained with the epithelial markers (CK and EMA). Based on the presence of epithelial differentiation within this otherwise cartilaginous neoplasm, it was also classified as a chondroid chordoma. In contrast, the remaining three cases without histologic evidence of chordoma differentiation did not stain for CK or EMA, but they did stain for S-100 protein (three of three cases) and VIM (three of three cases). These three tumors were therefore classified as chondrosarcomas. For purposes of comparison, 19 conventional chordomas without cartilage and 29 peripheral chondrosarcomas were also stained. The 19 conventional chordomas stained in a pattern similar to the conventional chordoma components of the chondroid chordomas, whereas the 29 peripheral chondrosarcomas stained in a pattern similar to the three chondrosarcomas of the craniospinal axis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Skeletal growth patterns in hereditary multiple exostoses: a natural history

Hereditary multiple exostoses (HME) is a commonly inherited musculoskeletal condition and is associated with a diminished stature. We demonstrated that adults with HME were significantly shorter when compared with a control group (P<0.001); preadolescents, however, were significantly taller than predicted (P=0.01). This was reflected by their height centile; 58% of the adults were under the 25th centile, whereas 53% of the preadolescence group were above the 75th centile. Stature was more severely affected in patients with an EXT1 mutation (P=0.008). This study illustrates a novel age-related growth pattern associated with HME, which is also affected by genotype.     

Pseudoaneurysm of the popliteal artery caused by exostosis of the femur: case report and review of the literature

A 13-year-old boy with a solitary exostosis of the left femur was seen with a pseudo-aneurysm of the popliteal artery. When left leg pain occurred 3 months earlier, radiographic examination revealed an exostosis with a cartilage cap. Serial radiographic examination demonstrated gradual disruption of the cartilage cap of the exostosis as the pseudoaneurysm developed. An exostosis with an irregular surface was found at surgery. A literature review disclosed 39 similar cases in which loss of the cartilage cap was considered as one of the causes of the aneurysm formation. Considering the clinical course of our patient, however, we believe that exostoses lose their cartilage caps by pressure destruction due to the aneurysms. It is highly probable that loss of the cartilage does not cause the aneurysms.     

遗传性多发性骨软骨瘤临床及影像学特征观察与分析

目的: 探讨遗传性多发性外生骨软骨瘤 (hereditarymultipleexostosis, HME) 的临床及影像学特征, 以说明发病部位特点及规律。方法: 分析经临床诊断及病理证实的 2个HME家系。结果: 男性患者占 51. 6%, 女性患者占 48. 4%; 其临床表现有随遗传代次增加而加重的现象; 肿瘤好发部位位于长骨的干骺段, 颅面骨没有发现。结论: HME的发病率无性别差异, 其临床表现有遗传递增性, 发病部位具有选择性。该研究有利于进一步探索其发病机制及基因型与表现型的关系。