摘要目的 对1个多发性骨软骨瘤家系EXT1基因进行突变分析,探索其致病机制.方法 PCR-测序分析家系成员外周血DNAEXT1/EXT2基因编码区及邻近序列;计算机及生物信息学分析预测变异;单核苷酸多态性筛查、TA克隆-测序分析先证者血组织的EXT1基因转录本,卡方检验统计分析比较其异常转录本数与正常对照的差异以探讨变异的致病机制.结果 基因测序发现先证者EXT1基因第3内含子5'剪接位点存在一新的杂合性点突变(c.1164+1G＞A),而正常个体未发现该突变;计算机及生物信息学预测突变位于保守区并可导致第3外显子跳跃或异常剪接;TA克隆测序及统计分析表明,与正常对照相比,先证者含第3外显子跳跃的转录本数出现增加(P＜0.05).结论 c.1164-+ 1G＞A导致较多拷贝的EXT1基因转录本第3外显子跳跃,导致具有肿瘤抑制功能的拷贝数减少,从而引起多发性骨软骨瘤的发生.

abstractsObjective To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism.Methods The coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing.Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis.Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing,with its abundance compared with that of healthy controls.Results DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G＞A) at the 5'splice sites of intron 3 of the EXT1 gene.The same mutation was not found in the healthy controls.Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing.TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (P ＜ 0.05) compared with the controls.Conclusion The c.1164+1G＞A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts.As the result,the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.