应用变性高效液相色谱技术检测汉族人Ⅰ型多囊肾致病基因突变

Mutation detection of ADPKD PKD1 gene in Hans by denaturing high-performance liquid chromatography

目的通过采用变性高效液相色谱(denaturinghigh performanceliquidchromatography,DHPLC)技术检测汉族人常染色体显性多囊肾病(autosomaldominantpolycystickidneydisease,ADPKD)Ⅰ型致病基因PKD1的突变,建立更为快速、敏感的突变筛查系统。方法以来源于19个ADPKD家系的67名成员血样标本的基因组DNA为模板,通过长链PCR和巢式PCR联合扩增的方法扩增PKD1全编码区,然后采用DHPLC方法进行初步筛查,将存在异常色谱图的扩增产物经核苷酸测序,确定突变的具体位点和类型,并与以往采用单链构象多态性(singlestrandconformationpolymorphism,SSCP)方法检测出的突变结果相比较。结果共检测出14个致病突变,包括10个错义突变、1个插入突变、1个缺失突变、2个无义突变,其中12个突变位点与之前SSCP的检测结果相同,另新发现nt32819G→A和nt37137T→C两个突变位点,突变检出率为73.7%。结论DHPLC方法可以作为更为有效筛查汉族人ADPKDPKD1突变位点的检测途径。

Objective To develop a screening system for more rapid and sensitive mutation detection of autesoreal dominant polycystic kidney disease （ADPKD） gene 1 （PKD1） by using denaturing high-performance liquid chromatography （DHPLC） protocol. Methods Using genomic DNA as templates extracted from blood samples of 19 Hart pedigrees with 67 family members, the complete codon areas were amplified by long-range PCR and nested PCR in succession, and then the PCR products were analyzed by DHPLC. The mutations from screened abnormal PCR products were confirmed by DNA sequencing, and then compared with the mutations identified by single strand conformation polymorphism （SSCP） before. Results There were 14 mutations found in this study, including 10 misseuse, 1 insertion, 1 deletion and 2 nonsense mutations. Besides 12 mutations identified before, mutations nt32819G→A and nt37137T→C were the novel mutations found. The mutation detection ratio was 73.7 %. Conclusion This developed system via DHPLC can be used as a more effective approach for mutation detection of ADPKD PKD1 in Hans.