CAJ | 학술논문

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.
목적 대일례이생육과Hallopeau-Siemens형은성영양불량형대포성표피송해증환인차돌변위점명학적잉부진행DNA위기출적산전진단,병탐색진단과정중모체세포오염적배제방법.방법 우잉16주행양막강천자술,추제양수세포중태인기인조DNA.PCR확증、DNA직접측서법명학태인시부대유치병돌변.양수세포첩벽배양기술장양수중태인세포여모체혈세포분리,거제모체세포오염.핵형분석법이기증실양수중유부원성신식.미위성표기련쇄분석기술진일보증실태인기인형.결과 B 초하시태반위우복전벽,고천자침무법피개태반,수경복벽、태반후입양막강추취양수,리심후시양수세포중유육안가견혈세포오염.직접측서현시,잉부이생육적녀인(7세,이학진위Hallopeau-Siemens형은성영양불량형대포성표피송해증)12호외현자상유모원성돌변R525X,105호외현자상유부원성돌변R2610X,태인12호외현자상존재화모친상동적돌변R525X,105호외현자정상.위배제해결과위천자과정중모친혈세포오염양수소치,장양수세포첩벽배양후,재차진행직접측서급가족단배형련쇄분석,현시량차직접측서화련쇄분석결과일치,배제모체오염,증실태인위대유여모친상동돌변적림상표형정상적휴대자.잉부우잉40주산하일표형정상녀영.결론 용직접측서、양수세포첩벽배양、핵형분석、련쇄분석등다충기술연합,가제고산전진단준학성.
Objective To perform a DNA-based prenatal diagnosis in a family with recessive dys-trophic epidermolysis bullosa, and to develop a strategy to eliminate matemal cell contamination in arnniotic 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 gone. Karyotype analysis was used to confirm paternal information in amniotic fluid. Linkage analysis between micro-satellite markers was performed to confirm the fetal genotype. Resulta Centrifugation showed visible contamination of aminotic cells by blood cells. Direct sequencing revealed that the proband was a carrier of both maternal 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 hapiotype analysis after elimination of mater-nal blood cells by amniotic fluid culture confirmed that the fetus was a carrier of maternal mutation with nor-real 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.