Multiple osteochondromas (MO), also referred to as multiple hereditary exostoses, is an autosomal dominant bone disease with a prevalence of 1/50,000 in the Western population (1). Although MO cases can be sporadic, ~80% of patients with MO have a family history of the disease (2,3). The size and number of osteochondroma tumors continually increase prior to adulthood (4). The disease is characterized by ≥1 overgrown exostoses capped by cartilage, usually occurring at the metaphyses of the long bones and bilateral limbs (1). Although the majority of cases of MO have been reported to involve benign bone tumors and a lack of typical symptoms, osteochondromas can cause deformities, functional limitation, compression of the nerves and blood vessels, and pain by placing pressure on adjacent tissues (5). Malignant transformation into chondrosarcoma or osteosarcoma has been reported as the most severe complication, occurring in 0.5–5% of patients (6).

Although the molecular mechanisms of MO require further investigation, previous studies have reported that mutations in the exostosin glycosyltransferase genes, EXT1 and EXT2, serve an important role in the pathogenesis of MO (7–9). EXT1 and EXT2 are members of the exostosin family and are located on chromosome 8q24.11-q24.13 and 11p12-p11, respectively. It has been reported that EXT genes encode proteins involved in the biosynthesis of heparan sulfate (HS), a key molecule in adjusting chondrocyte proliferation and bone growth (10). In addition, it has been demonstrated that dysfunction of EXT proteins may trigger the occurrence of MO (11,12). The majority of mutations that have been identified in EXT1 and EXT2 are nonsense, frameshift or splice-site (13), which have been reported to cause truncation, premature termination or premature degradation of EXT proteins (9).

In the present study, a genetic analysis of a family with a history of MO was conducted using polymerase chain reaction (PCR) amplification of the entire coding regions of EXT1 and EXT2, followed by DNA sequencing. A novel frameshift mutation, c.660delG (p.L221Cfs*82), was identified in exon 3 of the EXT2 gene.

EXT1 and EXT2 are tumor-suppressor genes that encode endoplasmic reticulum-type II transmembrane glycoproteins. Dysregulation or loss of function of these genes results in multiple exostoses (13,15). EXT1 and EXT2 combine to form a stable hetero-oligomeric complex, catalyzing the polymerization of HS chains in the Golgi apparatus and endoplasmic reticulum (16). HS is universally expressed on the surface of cells and is a major component of glycoproteins in the extracellular matrix. It also functions as a regulator of cell adhesion, the receptor-ligand binding process and signal transduction (17–20). Previous studies have reported that HS is a crucial determinant in the growth and differentiation of chondrocytes, and irregularities in its production may be associated with MO (21,22).

Mutations in EXT1 and EXT2 are associated with MO pathogenesis, although EXT1 mutations have been reported to be more common than EXT2 mutations (13). However, a reverse effect is observed in the Chinese population, in which EXT2 mutations have been indicated to occur at a higher frequency than EXT1 mutations (23). In the present study, a three-generation family with a history of MO was investigated and an analysis of mutations in EXT1 and EXT2 was performed in five of the family members (three affected by MO and two unaffected individuals). In the proband and the other two affected members of the family, a heterozygous deletion of one nucleotide (G) was identified in exon 3 of EXT2 at c.660 (p.L221Cfs*82). The mutation was absent in the unaffected family members, whose EXT genes were sequenced. In addition, according to the distribution of patients with MO in the family pedigree (Fig. 1), the c.660delG mutation indicates an autosomal dominant pattern of inheritance from the proband's grandfather to the proband's father.

The novel mutation (c.660delG) was identified to be located in the exostosin domain of the EXT2 gene, resulting in a frameshift and an introduction of a premature termination codon at position 302. At only 301 amino acids in size, the truncated EXT2 was identified to be shorter compared with the normal EXT2 protein. In addition, as the c.660delG mutation was indicated to be located within the exostosin domain, the mutant EXT2 protein indicated a defective exostosin domain and was absent in the glycosyltransferase family 64 domain. The glycosyltransferase domain serves a crucial role in HS biosynthesis, and therefore its absence may abolish the function of the EXT2 protein. The novel mutation introduced a stretch of 81 amino acids and may result in a novel function of the mutant EXT2 protein. Further investigation is required to examine the functional change in EXT2 protein caused by the c.660delG mutation.

In conclusion, a novel frameshift mutation in the EXT2 gene was identified (c.660delG), and in silico analysis revealed that this mutation may produce a dysfunctional EXT2 protein. The present study extends the existing knowledge of mutations that occur in EXT2 and provides novel insight into the genetic determinants underlying the pathogenesis of MO. Furthermore, the present study offers valuable information for genetic counseling and prenatal diagnosis in this family.