一个多发性骨软骨瘤家系的EXT1基因突变分析

Mutational analysis of the EXT1 gene from a pedigree with hereditary multiple osteochondromas

目的对1个多发性骨软骨瘤家系的致病基因EXT1进行突变分析。方法提取先证者外周血基因组DNA,PCR扩增EXT1基因全部外显子序列并进行序列测定;找到突变后,对相应PCR产物进行T克隆,以证实突变。对其他家庭成员的EXT1基因相应外显子也进行序列分析。结果先证者的临床表现符合多发性骨软骨瘤,其父也有类似临床表现,但明显轻于先证者,且11名同胞及其子女均无发病。先证者序列分析表明,其EXT1基因第6外显子存在杂合的缺失突变:1476-1477delTC。先证者父亲的血液组织、精子和口腔粘膜细胞也存在相同突变,但在PCR产物直接测序中突变体的测序信号明显弱于先证者;在PCR产物的T克隆-测序中,来自先证者父亲的血液、精子、口腔黏膜细胞突变体的重组菌分别占总数的10%、2.6%、13.3%。其他11名同胞未见EXT1基因突变。结论先证者父亲在胚胎早期发生了EXT1基因突变,使其体内部分细胞(包括部分软骨细胞)带有EXT1基因突变,导致症状较轻的多发性骨软骨瘤。

Objective To investigate EXT1 gene mutation in a family with hereditary multiple osteochondromas. Methods The 11 exons of EXT1 gene from the genomic DNA of peripheral blood cells from the proband were amplified by PCR and sequenced. The products of PCR were inserted into pGEM-T cloning vector to confirm the mutation. The corresponding exons of EXT1 gene of other family members were also sequenced. Results The proband＇s clinical manifestations were consistent with hereditary multiple osteochondromas. The clinical manifestations of the proband＇s father were much milder than those of the proband. The 11 siblings of the proband＇s father and their offsprings were free from hereditary multiple osteochondromas. Sequence analysis showed that there was a heterozygous mutation （1476-1477delTC） in exon 6 of the EXT1 gene from the proband. Blood cells, sperms and oral mucosal cells of the proband＇s father carried the same mutation, but the sequencing signals were obviously weaker than that of the proband. In T cloning-sequencing of the PCR products, the recombinants carrying EXT1 gene mutation from blood cells, sperms and oral mucosal cells of the proband＇s father accounted for 10%, 2.6% and 13.3% of the total, respectively. No mutation was found in the EXT1 gene of other 11 siblings. Conclusion A de novo deletion mutation of EXT1 gene had occurred in early embryogenesis of the proband＇s father, resulting in the presence of this mutation in part of the body＇s cells （including part of the chondrocytes）, which in turn leads milder hereditary multiple osteochondromas.