Division of Human Genetics, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town 7935, South Africa.
Redox Laboratories, Cape Town 7935, South Africa.
Neuromuscul Disord. 2018 Jul;28(7):553-563. doi: 10.1016/j.nmd.2018.05.001. Epub 2018 May 10.
The importance of molecular diagnosis and identification of disease-associated variants for Duchenne muscular dystrophy (DMD) is evident in the age of gene-based therapies and personalised medicine. Detection of the causative DMD variant and determination of its effects on dystrophin expression is best achieved by analysis of RNA extracted from muscle biopsy material. However, this is not done routinely, as the procedure can be traumatic, especially to young children, and carries risk of complications related to the use of anaesthetic. As skin biopsies are safer and straightforward to perform than muscle biopsies, we investigated the utility of cultured human epidermal melanocytes and dermal fibroblasts as alternative tools for RNA-based diagnosis of DMD. Shallow skin biopsies from 5 boys with genetically confirmed diagnoses of DMD were used to culture fibroblasts and melanocytes. Biopsies were sampled, and tolerated without complications, using local anaesthetic cream. Dystrophin expression in the cultured cells was assessed using immunocytochemical staining, quantitative real-time PCR and cDNA sequencing methodologies. We observed differential expression of the full-length dystrophin muscle transcript, with significantly more robust expression in melanocytes, compared to that in fibroblasts. Our results suggest that cultured skin melanocytes may present an alternative tool for RNA-based genetic diagnosis of DMD.
分子诊断和疾病相关变异体鉴定对杜氏肌营养不良症（DMD）的重要性在基因治疗和个性化医疗时代显而易见。通过分析从肌肉活检材料中提取的 RNA 来检测致病的 DMD 变异体及其对肌营养不良蛋白表达的影响是最佳的。然而，由于该程序可能对儿童造成创伤，并且存在与使用麻醉剂相关的并发症风险，因此通常不会进行此操作。由于皮肤活检比肌肉活检更安全且操作简单，因此我们研究了培养的人表皮黑素细胞和真皮成纤维细胞作为 DMD 基于 RNA 的诊断替代工具的用途。使用局部麻醉乳膏从 5 名经基因证实患有 DMD 的男孩的浅层皮肤活检中培养成纤维细胞和黑素细胞。采样活检，无并发症，耐受性良好。使用免疫细胞化学染色、定量实时 PCR 和 cDNA 测序方法评估培养细胞中的肌营养不良蛋白表达。我们观察到全长肌营养不良蛋白肌肉转录物的差异表达，与成纤维细胞相比，黑素细胞中的表达明显更强。我们的结果表明，培养的皮肤黑素细胞可能是 DMD 基于 RNA 的遗传诊断的替代工具。
