血友病基因诊断的研究  

The study of the hemophilia gene diagnose

作　　者：赖东波 毛碧涛 李茹 孙新 LAI Dongbo MAO Bitao LI Ru SUN Xin(Guangzhou Women and Children Medical Center, Guangzhou 510000, China)

机构地区：[1]广州市妇女儿童医疗中心,510000

出　　处：《中国小儿血液与肿瘤杂志》2016年第5期237-243,共7页Journal of China Pediatric Blood and Cancer

摘　　要：目的对20名血友病甲患者和4例血友病乙患者进行基因诊断。方法 1血友病甲患者FⅧ基因检测:(1)内含子22倒位检测:首先采用长距离PCR(LD-PCR)对患者FⅧ基因内含子22倒位进行检测;(2)内含子1倒位检测:对内含子22倒位突变阴性患者,采用序列特异性PCR进行内含子1倒位突变检测;(3)点突变检测:对内含子22和1倒位突变阴性患者,针对FⅧ基因26个外显子和外显子-内含子交界区设计引物,采用PCR直接测序法检测点突变;(4)大片段缺失检测:对前三步检测阴性的患者,进一步采用多重连接探针扩增技术(MLPA)检测大片段缺失。2血友病乙患者FⅨ基因检测:(1)点突变检测:针对FⅨ基因的启动子、8个外显子和外显子-内含子交界区设计引物,采用PCR直接测序法检测点突变;(2)大片段缺失检测:对点突变检测阴性者,进一步采用MLPA检测大片段缺失。结果 20例血友病甲患者,共检出内含子22倒位8例,内含子1倒位1例,错义突变5例,插入突变1例,大片段缺失1例,剪切突变4例。4例血友病乙患者检测出错义突变2例,缺失伴插入突变1例,大片段缺失突变1例。其中新发现FⅧ基因突变3例(c.1009+2 C>T,c.670+1 G>C,c.4798delA),F9基因突变1例(c.938-939indelT)。结论本研究对血友病甲和血友病乙患者的基因诊断阳性率为100%。Objective To make genetic diagnosis in hemophilia patients.Methods 1.Genetic testing of F8 gene was performed in emophilia A patients：① Intron 22 inversion detection：intron22 inversion was detected by use of long-distance PCR（LD-PCR） firstly;② Intron 1 inversion：intron1 inversion was detected by use of sequence specific PCR in intron 22 inversion negative patients;③ point mutation detection;primers were designed to amplify the 26 exons and exon-intron boundaries,PCR amplification and direct sequencing were then performed to detect point mutation in inversion mutation negative patients;④ Large deletion detection;large deletion was detected by multiplex ligation-dependent probe amplification（MLPA） in the remaining patients.2.Genetic testing of F9 gene was performed in the hemophilia B patients;① point mutation;primers were designed to amplify the 8 exons and exon-intron boundaries,PCR amplification and direct sequencing were then performed to detect point mutation firstly;② Large deletion detection;large deletion was detected by MLPA in the point mutation negative patients.Results In 20 hemophilia A patients,there were 8 patients with intron 22 inversion,1 with intron 1 inversion,5 with missense,1 with insertion mutation,4 with splicing mutation and 1 with large deletion.In 4 hemophilia B patients,there were 2 patients with missense mutation,1 with indel mutation and 1 with large deletion.Three novel mutations（c.1009 ＋2 C T,c.670 ＋ 1 G C,c.4798delA） were identified in F8 gene,and 1 novel mutation（c.938_939indelT） were identified in F9 gene.Conclusions In this study,genetic tests were 100%for both hemophilia A and B.

关 键 词：血友病甲 血友病乙 基因突变 测序 多重连接探针 

分 类 号：R440[医药卫生—诊断学] R725.5[医药卫生—临床医学]