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首页> 外文期刊>Journal of dermatological science >Single cell PCR amplification of microsatellites flanking the COL7A1 gene and suitability for preimplantation genetic diagnosis of Hallopeau-Siemens recessive dystrophic epidermolysis bullosa.
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Single cell PCR amplification of microsatellites flanking the COL7A1 gene and suitability for preimplantation genetic diagnosis of Hallopeau-Siemens recessive dystrophic epidermolysis bullosa.

机译：微卫星的单细胞PCR扩增侧翼COL7A1基因，适用于Hallopeau-Siemens隐性营养不良表皮表皮细胞分解Blowsa的适用性。

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摘要

BACKGROUND: Hallopeau-Siemens recessive dystrophic epidermolysis bullosa (HS-RDEB) is a severe inherited blistering skin disorder caused by mutations in the anchoring fibril type VII collagen gene, COL7A1. There is currently no effective treatment but DNA-based prenatal testing in families at risk of recurrence is possible, mostly involving chorionic villus sampling at 10-11 weeks' gestation. OBJECTIVES: An alternative method, for avoiding recurrence of HS-RDEB, is preimplantation genetic diagnosis (PGD). This involves DNA analysis of single blastomeres extracted from late cleavage stage embryos following in vitro fertilisation. METHODS: To establish PGD for HS-RDEB, we designed and optimised a sensitive single cell semi-duplex polymerase chain reaction (PCR) assay for two highly polymorphic dinucleotide repeat microsatellite markers, D3S1581 (telomeric) and D3S1289 (centromeric), close to the COL7A1 gene. RESULTS: We demonstrated high PCR efficiency, low allele drop out rates and no contamination in testing this assay on 50 single buccal cells of known heterozygous genotype and 13 research blastomeres from donated embryos. CONCLUSIONS: This semi-duplex PCR method provides robust, reproducible and informative amplification results for single cells. Moreover, this test has now been approved for clinical application by the UK Human Fertilisation and Embryology Authority (HFEA). As such, the development of PGD for HS-RDEB broadens the range of prenatal testing options and personal choice for couples at reproductive risk of this severe genetic skin disease.
机译：背景：Hallopeau-Siemens隐性营养不良表皮细胞分解Bullosa（HS-RDEB）是由锚固原纤维型VII胶原基因，COL7A1中的突变引起的严重遗传的起泡皮肤病。目前没有有效的治疗方法，但在有效的家庭中基于DNA的产前测试可能是可能的，大多涉及10-11周的妊娠绒毛绒毛绒毛。目的：一种替代方法，用于避免HS-RDEB的复发，是预体遗传诊断（PGD）。这涉及在体外施肥后从晚期切割阶段胚胎中提取的单卵囊的DNA分析。方法：为了建立HS-RDEB的PGD，我们设计并优化了两个高度多态性二核苷酸重复微卫星标记，D3S1581（端粒）和D3S1289（Centromeric）的敏感单细胞半双工聚合酶链反应（PCR）测定，接近COL7A1基因。结果：我们证明了高的PCR效率，低等位基因脱落率，并且在捐赠胚胎的已知杂合基因型的50个颊细胞和13个研究卵泡中测试该测定中没有污染。结论：该半双工PCR方法为单细胞提供鲁棒，可重复和信息的放大结果。此外，该测试现已批准由英国人类施肥和胚胎局（HFEA）批准临床应用。因此，HS-RDEB的PGD的发展扩大了这种严重遗传皮肤病的生殖风险的夫妻产前测试选择和个人选择的范围。

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