摘要
目的应用DNA测序和高分辨熔解曲线（high-resolution melting，HRM）分析技术对3个原发性肥大性骨关节病（primaryhypertrophicosteoarthropathy，PH0）家系进行HPGD基因的突变分析，了解中国PHO患者可能的热点突变。方法PCR扩增HPGD基因的全部外显子及外显子／内含子衔接区，并对PCR产物进行测序；针对先证者候选突变位点，通过HRM完成家系验证，并进一步通过基因测序技术对HRM方法的准确性进行鉴定。结果3例PHO先证者均存在HPGD基因第3外显子c．310_31ldelCT（P．L104Afs＊3）突变，其中2例为纯合子，1例为杂合子。对先证者和家系成员及正常对照样本的HRM分析结果显示3种不同的曲线型，经测序验证表明3种曲线分别为野生型与突变C．310-311delCT的杂合子和纯合子。结论L310-311del CT（P．L104Afs＊3）很可能是中国PHO患者的基因突变热点，HRM分析检测HPGD基因突变具有方便快捷、灵敏特异的优点，可作为PH0患者及携带者突变筛查HPGD基因突变的优选方法。
Objective To detect mutation of HPGD gene among three pedigrees affected with primary hypertrophic osteoarthropathy （PHO） by DNA sequencing and high-resolution melting （HRM） analysis. Methods Genomic DNA was extracted from peripheral blood samples collected from the pedigrees. PCR and direct sequencing were carried out to identify potential mutations of the HPGD gene. Amplicons containing the mutation spot were generated by nested PCR. The products were then subjected to HRM analysis using the HR-1 instrument. Direct sequencing was carried out in family members and healthy individuals to confirm the result of HRM analysis. Results A homozygous mutation c. 310_311delCT was detected in 2 affected probands, while a heterozygous mutation c. 310 311delCT was detected in the third proband. HRM analysis of the fragments encompassing HPGD exon 3 showed 3 curve patterns representing three different genotypes, i.e. , the wild type, the c. 310_311delCT homozygote, and the c. 310 311delCT heterozygote. Result of DNA sequencing was consistent with that of the HRM analysis and phenotype of the subjects. Conclusion The c. 310_311delCT mutation may be the most prevalent mutation among Chinese population. HRM analysis has provided an optimized method for genetic testing of HPGD mutation for its simplicity, rapid turnover and high sensitivity.
作者
张婉莹
王韬
黄帅武
赵秀丽
Zhang Wanying, Wang Tao, Huang Shuaiwu, Zhao Xiuli(Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, Chin)
出处
《中华医学遗传学杂志》
CAS
CSCD
2018年第2期156-159,共4页
Chinese Journal of Medical Genetics
基金
中国医学科学院医学与健康科技创新工程协同创新团队项目（2016-12M-3-003）
北京协和医学院大学生创新训练计划项目（2013zlgc0648）
