Mutation Surveyor Demo

Aims: Multiple exostoses (MO), also referred to as hereditary multiple exostoses (HME), is an autosomal dominant inherited skeletal disorder that has been found to be associated with mutations in the EXT1 and EXT2 genes. IntroductionHereditary multiple exostoses (HME), also referred to as multiple exostoses (MO), is an autosomal dominant, inherited skeletal disorder characterized by the development of multiple benign cartilage-capped tumors that appear as outgrowth from the metaphyses of the long bones. In 1814, the first HME patient was described by Boyer (Peterson,; Ryckx et al., ).

With a recorded prevalence of 1 in 50,000 in the western population (Schmale et al., ), osteochondromas are the most common benign bone tumors. The incidence of HME is greater in males than females, and the mutations exert incomplete penetrance (Legeai-Mallet et al., ). Patients with HME typically do not have symptoms, unless the increasing osteochondroma results in pressure in the adjacent muscles, tendons, nerves, or blood vessels, which leads to clinical manifestations that include pain, joint deformity, limited range of motion, and shortened stature (Jones et al.,; Wu et al., ).

Although HME is a benign tumor, malignant transformation into chondrosarcoma occurs in 1–2% of patients (Jennes et al.,; Tian et al., ).The molecular mechanisms underlying HME are still not clear, but previous studies have reported that the majority of patients have mutations in the exostosin-1 ( EXT1) and exostosin-2 ( EXT2) genes, which are located on chromosomes 8q24.1 and 11p13, respectively (Wu et al.,; Wuyts et al., ). To date, over 650 mutations in EXT1 and EXT2 have been identified, most of which are nonsense, frameshift, or splice site and which result in the synthesis of truncated EXT protein with dysfunctional activity (Jennes et al.,; Ciavarella et al., ).With the rapid development of DNA sequencing technologies, exome sequencing has already been applied in the investigation of genetic diseases, revealing novel and pathogenic genetic variants within whole exomes and/or targeted sequences (Huang et al.,; Liu et al., ). In this study, a Chinese family with three affected individuals was investigated by examining all exons of the EXT1 and EXT2 genes, to discover pathogenic mutations related to the disorder. A novel mutation c.1056GT (p.Gln352His) was identified in exon 2 of the EXT1 gene that resulted in structural changes of EXT1 protein.

Subjects and clinical evaluationIn this report, three generations of a family were studied , including three individuals with HME. The proband (III-2) was a 12-year-old male who was admitted to Zhejiang Provincial People's Hospital (Hangzhou, China) due to a deformity of the left leg, which led to limitation of physical activity.

After a careful examination and through inquiry of medical history, the proband was diagnosed with HME. The subject manifested MO of the proximal and distal tibiae, distal femur, and proximal fibula.

Venous blood samples (5 mL) were drawn from seven family members (I-2, II-1, II-2, II-3, II-4, III-1, and III-2) using EDTA-anticoagulant vacuum blood collection tubes. Informed consent was obtained from all subjects recruited in this study, which was approved by the Ethics Committee of Zhejiang Provincial People's Hospital.FIG. Pedigree of a family with Hereditary multiple exostoses (HME). The black arrow notes the proband (III-2). Empty symbols indicate unaffected individuals, filled symbols indicate affected individuals, and oblique lines indicate deceased.FIG. Clinical features of the proband with HME. (A, B) Multiple osteochondromas on the femurs, tibiae, and fibulae of the legs (indicated by white arrows).

(C) Osteochondromas on proximal humerus (indicated by white arrows). (D) Osteochondromas on the proximal femurs (indicated by white arrows). Mutation screeningGenomic DNA was extracted from peripheral blood leukocytes with standard procedures using the AxyPrep™-96 Blood Genomic DNA Kit (Axygen) according to the manufacturer's instructions.

All exons of the EXT1 and EXT2 genes were amplified by polymerase chain reaction using the primers listed in. The DNA sequences of the amplified EXT1 and EXT2 coding regions were analyzed as previously described (Cao et al., ).

Mutation Surveyor Demo software version 4.0 was used to analyze the sequence data using reference sequences from the National Center for Biotechnology Information (NCBI) ( EXT1: NM000127.2; EXT2: NM000401) as control sequences. A novel EXT1 mutation was identified in a Chinese family of HMETo reveal the genetic determinant of HME in the family, each exon of the EXT1 and EXT2 genes was sequenced in the proband. No mutations were discovered in the EXT2 gene; however, a mutation in exon 2 of the EXT1 gene was detected (c.1056GT), which was predicted to lead to an aa substitution. We also analyzed other family members, including affected individuals (II-1 and II-3) and unaffected individuals (I-2, II-2, II-4, and III-1) by sequencing the EXT1 and EXT2 genes. Affected family members were found to carry the same mutation as the proband, but unaffected individuals did not possess it. Therefore, we conclude that the novel mutation in EXT1, c.1056GT, is the molecular and pathophysiological mechanism that causes HME in this family.

In silico analyses and homology studyThe EXT1 gene consists of 11 exons, and the heterozygous mutation (c.1056GT) was found to be located in exon 2. To understand the potential impact of the c.1056GT mutation on EXT1 function, an in silico analysis was performed. The mutation (c.1056GT) results in the substitution of a glutamine (Gln) residue with a histidine (His) at position 352. The normal EXT1 protein is 746 aa and contains an exostosin domain (aa 110–396) and a glycosyltransferase domain (aa 480–729). Gln352, located in the exostosin domain, is highly conserved among various vertebrate species , suggesting that it plays an important role in the function of the EXT1 protein.FIG.

Identification of a novel mutation (c.1056GT) in the EXT1 gene. (A) A novel missense mutation was identified in the EXT1 gene of the proband. (B) No mutations were present at the same location in unaffected family members. (C) Gln352 is highly conserved in many vertebrate species.

DiscussionThe exact etiology of HME is still under investigation, but research has shown that both environmental and genetic factors are involved in the pathogenesis of this disorder (Eggen and Natvig,; Raldi et al., ). Genetic studies have indicated that mutations appearing in the EXT1 and EXT2 genes are implicated in HME, being responsible for 70–95% of the cases. Mutations in EXT1 account for 56–78% of HME cases, whereas mutations in EXT2 are detected in 21–44% of cases (Jennes et al., ). Patients with HME who have mutations in the EXT1 gene tend to present more severe symptoms and a greater risk for malignant transformation than those with mutations in the EXT2 gene (Francannet et al.,; Alvarez et al., ).In this study, we investigated a Chinese family with HME and performed a sequence analysis of the full coding regions of both EXT1 and EXT2.

A novel disease-causing mutation (c.1056GT) was identified in the EXT1 gene, and an in silico analysis revealed that the missense mutation substituted a Gln residue with a His at aa position 352 (p.Gln352His). This mutation is located in the exostosin domain which is highly conserved in many vertebrate species indicating its importance for the function of EXT1 protein. Previous studies of mutant EXT1 protein have provided evidence that missense changes in the exostosin domain reduce synthesis of heparin sulfate (HS) and result in the development of HME (McCormick et al., ).

HS is an important component of extracellular matrix glycoprotein, which is involved in regulation of cell adhesion, receptor and ligand binding, and signal transduction (Esko and Lindahl, ). It also plays a crucial role in balancing the growth and differentiation of growth plate chondrocytes, and its dysfunction may play a role in generation of HME (Koziel et al.,; Anower et al.,; Jochmann et al., ).This study provides novel genetic data regarding the expanding mutational spectrum of EXT1 and increases understanding of the pathogenesis of HME. In addition, the study provides important information for genetic counseling and prenatal diagnosis of this family. References. Alvarez CM, De Vera MA, Heslip TR, et al.

( 2007) Evaluation of the anatomic burden of patients with hereditary multiple exostoses. Clin Orthop Relat Res 462:73–79.,. Anower EKMF, Matsumoto K, Habuchi H, et al. ( 2013) Glycosaminoglycans in the blood of hereditary multiple exostoses patients: half reduction of heparan sulfate to chondroitin sulfate ratio and the possible diagnostic application. Glycobiology 23:865–876.,.

Cao L, Liu F, Kong M, et al. ( 2014) Novel EXT1 mutation identified in a pedigree with hereditary multiple exostoses. Oncol Rep 31:713–718.,.

Ciavarella M, Coco M, Baorda F, et al. ( 2013) 20 novel point mutations and one large deletion in EXT1 and EXT2 genes: report of diagnostic screening in a large Italian cohort of patients affected by hereditary multiple exostosis. Gene 515:339–348.,.

Eggen S, Natvig B ( 1991) Variation in torus mandibularis prevalence in Norway. A statistical analysis using logistic regression. Community Dent Oral Epidemiol 19:32–35.,. Esko JD, Lindahl U ( 2001) Molecular diversity of heparan sulfate. J Clin Invest 108:169–173.,. Francannet C, Cohen-Tanugi A, Le Merrer et al.

Mutation Surveyor Software

( 2001) Genotype-phenotype correlation in hereditary multiple exostoses. J Med Genet 38:430–434.,.

Huang XF, Xiang P, Chen J, et al. ( 2013) Targeted exome sequencing identified novel USH2A mutations in Usher syndrome families. PLoS One 8:e63832.,. Jennes I, Pedrini E, Zuntini M, et al. ( 2009) Multiple osteochondromas: mutation update and description of the multiple osteochondromas mutation database (MOdb). Hum Mutat 30:1620–1627.,.

Jochmann K, Bachvarova V, Vortkamp A ( 2014) Heparan sulfate as a regulator of endochondral ossification and osteochondroma development. Matrix Biol 34:55–63.,. Jones KB, Datar M, Ravichandran S, et al.

( 2013) Toward an understanding of the short bone phenotype associated with multiple osteochondromas. J Orthop Res 31:651–657.,. Koziel L, Kunath M, Kelly OG, et al. ( 2004) Ext1-dependent heparan sulfate regulates the range of Ihh signaling during endochondral ossification. Dev Cell 6:801–813.,. Legeai-Mallet L, Munnich A, Maroteaux P, et al.

( 1997) Incomplete penetrance and expressivity skewing in hereditary multiple exostoses. Clin Genet 52:12–16.,.

Liu H, Wu S, Duan L, et al. ( 2015) Identification of a novel EXT1 mutation in patients with hereditary multiple exostosis by exome sequencing. Oncol Rep 33:547–552.,. McCormick C, Duncan G, Goutsos KT, et al. ( 2000) The putative tumor suppressors EXT1 and EXT2 form a stable complex that accumulates in the Golgi apparatus and catalyzes the synthesis of heparan sulfate. Proc Natl Acad Sci U S A 97:668–673.,. Peterson HA ( 1989) Multiple hereditary osteochondromata.

Clin Orthop Relat Res 222–230.,. Raldi FV, Nascimento RD, Sa-Lima JR, et al.

( 2008) Excision of an atypical case of palatal bone exostosis: a case report. J Oral Sci 50:229–231.,. Ryckx A, Somers JF, Allaert L ( 2013) Hereditary multiple exostosis.

Acta Orthop Belg 79:597–607.,. Schmale GA, Conrad EU3rd, Raskind WH ( 1994) The natural history of hereditary multiple exostoses. J Bone Joint Surg Am 76:986–992.,.

Tian C, Yan R, Wen S, et al. ( 2014) A splice mutation and mRNA decay of EXT2 provoke hereditary multiple exostoses. PLoS One 9:e94848.,. Wu Y, Xing X, Xu S, et al. ( 2013) Novel and recurrent mutations in the EXT1 and EXT2 genes in Chinese kindreds with multiple osteochondromas. J Orthop Res 31:1492–1499.,.

Wu YQ, Heutink P, de Vries BB, et al. ( 1994) Assignment of a second locus for multiple exostoses to the pericentromeric region of chromosome 11. Hum Mol Genet 3:167–171.,. Wuyts W, Van Hul W, Wauters J, et al. ( 1996) Positional cloning of a gene involved in hereditary multiple exostoses. Hum Mol Genet 5:1547–1557.

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