Guo Xiaoyan, Chen Wenxu, Lin Mingrui, Shi Tengfei, Huang Dianhua, Wang Zhihong
Department of Laboratory Medicine, Fuzhou Second Hospital Affiliated to Xiamen University, Fuzhou, Fujian 350007, China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2017 Jun 10;34(3):411-415. doi: 10.3760/cma.j.issn.1003-9406.2017.03.022.
To detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism.
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.
DNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to 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 (< 0.05) compared with the controls.
The c.1164+1G to 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.
检测一个多发性骨软骨瘤家系中EXT1基因的潜在突变，并探讨其致病机制。
对EXT1/EXT2基因的编码区及其侧翼序列进行PCR扩增和Sanger测序。通过排除可能的单核苷酸多态性和生物信息学分析来验证可疑突变。采用TA克隆测序法分析先证者中EXT1基因的转录本，并与健康对照者的转录本丰度进行比较。
DNA测序在先证者中发现EXT1基因第3内含子5'剪接位点处有一个新的杂合点突变（c.1164+1G→A）。健康对照者中未发现相同突变。生物信息学分析表明，该突变高度保守，可导致外显子3跳跃或异常剪接。TA克隆测序表明，与对照相比，先证者中外显子3跳跃的转录本数量显著增加（P<0.05）。
c.1164+1G→A突变导致一部分EXT1基因转录本中外显子3跳跃。结果，具有肿瘤抑制功能的转录本数量相对减少，最终导致肿瘤发生。
