A novel splice mutation induces exon skipping of the EXT1 gene in patients with hereditary multiple exostoses

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three-generation family with HME. Bioinformatics and TA clone-sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) revealed that the expression levels of wild-type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1-DEL) and full-length EXT1 mRNA (EXT1-FL) were overexpressed in 293-T cells and Cos-7 cells using lentivirus infection. RT-qPCR demonstrated that the expression level of EXT1-DEL was significantly increased, compared with EXT1-FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1-DEL was detected by western blot analysis, and the level was increased, compared with EXT1-FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1-DEL was located in the cytoplasm of Cos-7 cells, which was consistent with the localization of the EXT1-FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1-DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.