成功实施一例去除母体细胞污染的大疱性表皮松解症的产前诊断  

Suecessful prenatal diagnosis following elimination of maternal cell contamination in a family with recessive dystrophic epidermolysis bullosa

作　　者：梁键莹[1] 陶炯[2] 张定国[1] 祁怀山[1] 杨丽萍[1] 杨祖菁[3] 虞荷莲 姚志荣[1] 

机构地区：[1]上海交通大学医学院附属新华医院皮肤科,200092 [2]上海市儿科医学研究所 [3]上海市儿科医学研究所妇产科

出　　处：《中华皮肤科杂志》2009年第7期448-450,共3页Chinese Journal of Dermatology

基　　金：上海市卫生局科研课题计划项目（2006042）

摘　　要：目的对一例已生育过Hallopeau—Siemens型隐性营养不良型大疱性表皮松解症患儿且突变位点明确的孕妇进行DNA为基础的产前诊断，并探索诊断过程中母体细胞污染的排除方法。方法于孕16周行羊膜腔穿刺术，抽提羊水细胞中胎儿基因组DNA。PCR扩增、DNA直接测序法明确胎儿是否带有致病突变。羊水细胞贴壁培养技术将羊水中胎儿细胞与母体血细胞分离，去除母体细胞污染。核型分析法以期证实羊水中有父源性信息。微卫星标记连锁分析技术进一步证实胎儿基因型。结果B超下示胎盘位于腹前壁，故穿刺针无法避开胎盘，须经腹壁、胎盘后入羊膜腔抽取羊水，离心后示羊水细胞中有肉眼可见血细胞污染。直接测序显示，孕妇已生育的女儿（7岁，已确诊为Hallopeau—Siemens型隐性营养不良型大疱性表皮松解症）12号外显子上有母源性突变R525X，105号外显子上有父源性突变R2610X，胎儿12号外显子上存在和母亲相同的突变R525X，105号外显子正常。为排除该结果为穿刺过程中母亲血细胞污染羊水所致，将羊水细胞贴壁培养后，再次进行直接测序及家族单倍型连锁分析，显示两次直接测序和连锁分析结果一致，排除母体污染，证实胎儿为带有与母亲相同突变的临床表型正常的携带者。孕妇于孕40周产下一表型正常女婴。结论用直接测序、羊水细胞贴壁培养、核型分析、连锁分析等多种技术联合，可提高产前诊断准确性。Objective To perform a DNA-based prenatal diagnosis in a family with recessive dystrophic epidermolysis bullosa, and to develop a strategy to eliminate maternal cell contamination in amniotic fluid samples. Methods Amniocentesis was carried out at gestation week 16, amniotic fluid culture was used to separate fetal cells from maternal blood cells. Peripheral blood was obtained from the proband, and her parents. Genomic DNA was extracted from peripheral blood and aminotic cells. Subsequently, PCR and direct sequencing were performed to detect pathogenic mutations in the COL7A1 gene. Karyotype analysis was used to confirm paternal information in amniotic fluid. Linkage analysis between micro-satellite markers was performed to confirm the fetal genotype. Results Centrifugation showed visible contamination of aminotic cells by blood cells. Direct sequencing revealed that the proband was a carrier of both matemal mutation, R525X in exon 12, and paternal mutation, R2610X in exon 105, while the fetus only carried the maternal mutation, R525X. The second direct sequencing and haplotype analysis after elimination of maternal blood cells by amniotic fluid culture confirmed that the fetus was a carrier of maternal mutation with normal phenotype. The pregnancy continued and a clinically unaffected girl was born at gestation week 40. Conclusion The accuracy of DNA-based prenatal diagnosis could be improved by the combination of direct sequencing, amniotic fluid culture, karyotype analysis and linkage analysis, etc.

关 键 词：表皮松解 大疱性 营养不良性 产前诊断 

分 类 号：R714.5[医药卫生—妇产科学]