遗传性多发性骨软骨瘤EXT1基因一个新的剪接突变及致病机制分析

A novel splice mutation in EXT1 gene of hereditary multiple osteochondroma and analysis of its pathogenic mechanism

目的对遗传性多发性骨软骨瘤致病基因EXT1一个新的剪接突变进行分析，并研究其致病机制。方法2013年4月厦门大学附属福州第二医院骨肿瘤科收治的多发性骨软骨瘤患者即先证者因全身多处关节畸形20余年于门诊收入院，采集先证者及父母外周血并提取基因组DNA，PCR扩增EXT1／EXT2基因编码区及邻近内含子序列并测序；生物信息学分析所得突变；以先证者、母亲及正常人血组织cDNA为模板，扩增EXT1基因编码区并将产物做TA克隆测序，用卡方检验统计分析各组异常转录本数以研究该突变的致病机制。结果基因测序发现先证者及母亲EXT1基因第4内含子5’剪接位点存在杂合性缺失突变（c．1284＋2del）；生物信息学预测该突变可导致外显子4跳跃或异常剪接；TA克隆测序及统计分析表明先证者及母亲含外显子4跳跃的转录本其比例均明显高于正常人（P=0．000，P〈0．01）。结论EXT1基因突变c．1284＋2del导致较大数量的EXT1基因转录本外显子4跳跃，而影响其正常转录翻译。

Objective To analyse a novel splice mutation in EXT1 gene of hereditary multiple osteochondroma, and study its pathogenic mechanism. Methods In April of 2013, the proband was hospitalized from the outpatient department with multiple joint deformity for more than 20 years, peripheral blood of the proband and his parents were collected and genomic DNA was extracted. Coding regions and adjacent intron sequences of EXT1/EXT2 genes in genomic DNA of the family members were amplified and sequenced. Bioinformatics was used to analyze the mutation from sequencing, cDNA from peripheral blood of the proband, the mother and normal control was made respectively as a template for amplifying coding regions of EXT1 gene, and the product was T-A cloned and sequenced. The abnormal transcripts of each group were counted and analyzed using chi square test to study the pathogenic mechanism of the mutation. Results Sequencing results of family members revealed that there was a heterozygous deletion mutation （ c. 1284 ＋ 2del） in the 5＇ splice site of intron 4 in EXT1 gene of the proband and his mother. Bioinformatics predicted that exon 4 of EXTI gene was skipping or spliced aberrantly due to the mutation. T-A clone and sequencing results as well as the statistical analysis suggested that there was a significantly higher proportion of transcripts with skipping exon 4 in the proband and his mother compared with the normal control（ P = 0. 000, P 〈 0. 01 ）. Conclusions c. 1284 ＋ 2del in EXT1 gene is reported for the first time internationally, which results in a considerable number of abnormal transcripts with skipping exon 4 in EXT1 gene, thereby influences the normal transcription and translation of EXT1 gene.