目标基因测序技术检测后纵韧带骨化致病基因突变

Identification of mutations of disease-causing genes in ossification of the posterior longitudinal ligament based on targeted next generation sequencing technology

[目的]建立目标基因测序技术，对后纵韧带骨化（ossification of the posterior longitud inalligament，OPLL）患者的11个已知致病基因进行突变筛查，探讨OPLL与致病基因突变的关系。[方法]提取6例OPLL患者外周血全基因组DNA，利用GenCap目标基因捕获技术（北京迈基诺公司），设计骨形态发生蛋白-2（bone morphogenetic pro—tein2，BMP2）、骨形态发生蛋白-4（bone morphogenetic protein4，BMP4）、骨形态发生蛋白-9（bone morphogenetic protein 9，BMP9）、Ⅺ型胶原蛋白以（collagen type XI alpha 2，COLllA2）、Ⅵ型胶原蛋白理1（collagen type VIM pha1，COL6A1）、核苷酸焦磷酸酶（ectonucleotide pyrophosphatase／phosphodiesterase 1，ENPPl）、成纤维细胞生长因子2（fibroblast growth factor 2，FGF2）、成纤维细胞生长因子受体1（fibroblast growth factor receptor 1，FGFRl）、成纤维细胞生长因子受体2（fibroblast growth factor receptor2，FGFR2）、转化生长因子一β3（transforming growth factor beta3，TGFB3）和转化生长因子一8受体2（transforming growth factor beta receptorII，TGFBR2）基因外显子区域特异性捕获探针，与基因组DNA文库进行杂交，将目标基因组区域的DNA片段进行富集后，再利用illumina hiseq2000第二代测序仪进行测序，通过数据分析，确定突变位点，对选定的突变位点用Sanger测序法进行验证。[结果]设计合成的目标基因特异性捕获探针可有效地捕捉并富集基因组DNA的目标靶片段。目标区域平均测序深度为426．85～976．15，99．30％-100％目标区域覆盖度。在1例患者中发现COL6A1基因的1个错义突变，此位点检测结果与Sanger测序结果完全一致。[结论]目标基因测序技术成功发现了OPLL患者的致病基因突变。该方法快速而有效，对深入研究OPLL的分子病因学有重要意义。

[ Objective] To screen the 11 known genes in the pathogenesis of ossification of the posterior longitudinal liga- ment （OPLL） by using targeted next generation sequencing technique, as so to determine the association of genetic defects and OPLL. [ Methods] Peripheral blood was collected and genomie DNA was isolated from 60PLL patients. A total of 11 known genes of OPLL were selected for deep exome resequencing. GenCap targeted gene capture technique was used to design bone mor- phogenetic protein 2 （ BMP2）, bone morphogenetic protein 4 （ BMP4）, bone morphogenetic protein 9 （ BMP9 ）, collagen type XI alpha 2 （ COLllA2）, collagen type VI alpha 1 （ COL6A1 ）, ectonueleotide pyrophosphatase/phosphodiesterase 1 （ENPP1）, fibroblast growth factor 2 （ FGF2）, fibroblast growth factor receptor 1 （ FGFR1 ）, fibroblast growth factor receptor 2 （ FGFR2）, transforming growth factor beta 3 （ TGFB3 ） and transforming growth factor beta receptor II （ TGFBR2 ） . The enrichment libraries were sequenced on Illumina HiSeq 2000 sequencer to determine the mutation in these genes. Selected variants were further vali- dated by Sanger sequencing. [ Results ] For the samples subjected to targeted gene capture and the second generation sequen- cing, the average sequencing depths on the targeted regions were yielded from 426. 85 to 976. 15. Meanwhile, coverage of targe- ted exons was ranged from 99. 30% to 100%. A missense mutation in COL6A1 was identified. [ Conclusion] Disease - causing gene for OPLL has been determined by targeted next generation sequencing precisely and rapidly in this study. This method provides a reliable strategy for routine gene diagnosis of OPLL.