先天性软骨发育不全的产前快速基因诊断

Rapid genetic prenatal diagnosis for achondroplasia

目的探讨先天性软骨发育不全（ACH）的产前快速基因诊断方法。方法选择2007年5—11月南京大学医学院附属鼓楼医院妇产科产前诊断中心收治的3个ACH家系：其中家系1为年龄6个月的ACH患儿；家系2为妊娠18周的孕妇，待采集胎儿羊水细胞进行产前诊断；家系3为孕39周经超声诊断为ACH的胎儿，待采集脐带血进行诊断。同时采用限制性内切酶（Sfc Ⅰ和Msp Ⅰ）酶切、变性高效液相色谱（DHPLC）分析及序列分析3种检测方法，对外周静脉血或脐血中的成纤维细胞生长因子受体3（FGFR3）基因进行突变检测。结果（1）DHPLC分析：家系1患者的色谱分析结果为异源双链杂合峰，家系2患者和胎儿的结果均为异源双峰，家系3胎儿的结果为异源双峰，正常对照的结果为同源单峰。（2）FGFR3基因10号外显子PCR产物酶切前的聚丙烯酰胺凝胶电泳检测结果：家系1患者、家系2患者及胎儿的PCR产物经Sfc Ⅰ酶切后除有247bp条带外，还产生162和85bp两个酶切产物条带，但不能被Msp Ⅰ酶切，且只有247bp 1条产物条带。家系3胎儿PCR产物不能被Sfc Ⅰ酶切，但经Msp Ⅰ酶切后可出现162和85bp两个产物条带。正常对照PCR产物均不能被Sfc Ⅰ和Msp Ⅰ酶切。（3）PCR产物测序结果：家系1患者PCR产物经测序发现FGFR3基因1138 G→A杂合子突变，家系2患者及胎儿也均为FGFR3基因1138 G→A杂合子突变，家系3胎儿为FGFR3基因1138Gc杂合子突变，正常对照在FGFR3基因1138位点为G纯合子。家系2胎儿以上各项检测结果均与家系2患者相同，是遗传母亲致病突变基因的结果。结论DHPLC和限制性内切酶酶切分析均能准确检测致病突变的FGFR3基因，但DHPLC技术操作方便快捷、准确，而且敏感性更高，可在临床上推广用于ACH的产前快速基因诊断。

Objective To explore the genetic prenatal diagnosis method for achondroplasia （ACH）. Methods During May to November 2007, three ACH pedigrees were diagnosed at the Prenatal Diagnosis Center, Department of Obstetrics and Gynecology, Affiliated Drum Tower Hospital of Medical College, Nanjing University. In family 1, there was a 6-month-old male ACH infant. In family 2, the expectant mother, with 18 weeks of pregnancy, was an ACH patient. Amniocentesis was performed for prenatal diagnosis. The fetus of family 3 was diagnosed as ACH by ultrasound examination on the 39th week of gestation. Umbilical cord blood of this fetus was collected for examination. Totally, three methods, restriction enzyme （Sfc I and Msp Ⅰ ） digestion analysis, denaturing high performance liquid chromatography （DHPLC） and sequencing analysis were performed simultaneously to detect the pathogenic mutation of fibroblastic growth factor receptor 3 （FGFR3） for the three ACH families. Results （ 1 ） The DHPLC detection： heteroduplex was detected in the patient of family 1; both the patient and the fetus of family 2 showed heteroduplex results; the result of the fetus of family 3 was also heteroduplex. （2） The enzyme digestion analysis for the PCR products of 10 exon of FGFR3： after Sfc Ⅰ digestion, the PCR products of patients and the fetus of family 1 and 2 showed not only the band of 247 bp, but also bands of 162 bp and 85 bp. But their PCR products could not be digested by Msp Ⅰ, and it only showed the band of 247 bp. For the fetus of family 3, the PCR products could not be digested by Sfc Ⅰ, while after digestion by Msp Ⅰ, bands of 162 bp and 85 bp were shown up. The PCR products of the normal control could be digested by neither Sfc I nor Msp Ⅰ.（3） The sequencing results： the heterozygote mutation of 1138 G→A was confirmed in the patient of family 1. The pregnant woman and her fetus in family 2 showed the same result. The heterozygote mutation of G→C was confirmed in the fetus of family 3. The site of 1138 was G homozygote in the normal control. The three detection results of the fetus in family 2 were the same as that of the mother, which means that the fetus inherited the same pathogenic mutation from his or her mother. Conclusions Both DHPLC and restriction enzyme digestion analysis could detect the mutation of FGFR3 gene, but DHPLC is more rapid, convenient and sensitive. So DHPLC can be applied to genetic diagnosis and prenatal diagnosis for ACH patients.