St John's Institute of Dermatology, King's College London (Guy's Campus), 9th Floor Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, UK.
Br J Dermatol. 2010 Jun;162(6):1330-6. doi: 10.1111/j.1365-2133.2010.09701.x. Epub 2010 Feb 15.
Herlitz junctional epidermolysis bullosa (HJEB) is a severe, life-threatening, autosomal recessive blistering skin disease for which no cure is currently available. Prenatal diagnosis for couples at risk is feasible through fetal skin biopsy or analysis of DNA extracted from chorionic villi, but these methods can be applied only after pregnancy has been established. An alternative approach, which involves the analysis of single cells from embryos prior to establishment of pregnancy, is preimplantation genetic diagnosis (PGD). Until now, its clinical uptake has been hindered by lengthy delays in establishing mutation-specific protocols, and by the small amount of template DNA that can be obtained from a single cell. A new method that addresses these problems, preimplantation genetic haplotyping (PGH), relies on whole genome amplification followed by haplotyping of multiple polymorphic markers using standard DNA-based polymerase chain reaction (PCR) assays.
To design and validate a generic PGH assay for HJEB and to transfer this into clinical practice.
We established a multiplex PCR-based PGH assay involving 16 markers within and flanking the LAMB3 gene (the most frequently mutated gene in HJEB). The assay was then validated in 10 families with at least one previously affected offspring. After licensing by the Human Fertilisation and Embryology Authority (HFEA), the new test was used for PGD in a couple at risk of HJEB.
The chromosome 1 LAMB3 markers within the assay were shown to be of sufficient heterogeneity to have widespread application for preimplantation testing of HJEB. In one couple that were heterozygous carriers of nonsense mutations in LAMB3, we used the new assay to identify unaffected embryos in a series of PGD cycles. Pregnancy was established in the third PGD cycle and a healthy, unaffected child was born. DNA analysis of cord blood confirmed the predicted single-cell mutation status of wild-type LAMB3 alleles.
PGH represents a major step forward in widening the scope and availability of preimplantation testing for serious mapped single-gene disorders. We have established a generic test that is suitable for the majority of couples at risk of HJEB.
遗传性交界性大疱性表皮松解症（HJEB）是一种严重的、危及生命的常染色体隐性水疱性皮肤病，目前尚无治愈方法。对于有风险的夫妇，可以通过胎儿皮肤活检或从绒毛膜绒毛中提取的 DNA 分析进行产前诊断，但这些方法只能在怀孕确定后才能应用。另一种方法是在怀孕前对胚胎进行单细胞分析，这就是植入前遗传学诊断（PGD）。到目前为止，由于建立特定突变协议的过程冗长，以及从单个细胞中获得的模板 DNA 量较少，其临床应用受到了阻碍。一种新的方法——植入前遗传单体型分析（PGH），解决了这些问题，该方法依赖于全基因组扩增，然后使用标准的基于 DNA 的聚合酶链反应（PCR）检测对多个多态性标记进行单体型分析。
设计和验证用于 HJEB 的通用 PGH 检测，并将其应用于临床实践。
我们建立了一种基于多重 PCR 的 PGH 检测方法，该方法涉及 LAMB3 基因（HJEB 最常见的突变基因）内和周围的 16 个标记。然后，在至少有一个先前受影响的后代的 10 个家庭中对该检测方法进行了验证。在人类受精和胚胎管理局（HFEA）授权后，新的检测方法被用于一对有 HJEB 风险的夫妇的 PGD。
检测中的染色体 1 LAMB3 标记显示出足够的异质性，可广泛应用于 HJEB 的植入前检测。在一对 LAMB3 基因上存在无义突变的杂合携带者夫妇中，我们使用新的检测方法在一系列 PGD 周期中识别出未受影响的胚胎。在第三个 PGD 周期中成功妊娠，并产下一名健康的、未受影响的孩子。脐带血的 DNA 分析证实了预测的野生型 LAMB3 等位基因的单细胞突变状态。
PGH 代表了在扩大严重的单基因疾病的植入前检测范围和可用性方面向前迈出的重要一步。我们已经建立了一种适用于大多数有 HJEB 风险的夫妇的通用检测方法。
