关键词: 营养不良型大疱性表皮松解症;产前诊断;母源性污染

Abstract:

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 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 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.