Hayashi Saori, Ohsawa Yutaka, Takahashi Toshiaki, Suzuki Naoki, Okada Tadashi, Rikimaru Mitsue, Murakami Tatsufumi, Aoki Masashi, Sunada Yoshihide
Department of Neurology, Kawasaki Medical School, Kurashiki.
Intern Med. 2010;49(24):2693-6. doi: 10.2169/internalmedicine.49.3771. Epub 2010 Dec 15.
Mutations in the dysferlin gene cause limb-girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy (MM), which are collectively named dysferlinopathy. Dysferlinopathy is the most frequent type of LGMD in the Japanese population. Molecular genetic analysis is essential for the diagnosis of dysferlinopathy because of its variable immunohistochemical patterns of biopsied muscles, including patterns similar to normal controls. The analysis of the entire dysferlin gene however, is time-consuming and laborious; therefore a simple and rapid screening method to detect hot spot mutations in the dysferlin gene is essential for the diagnosis of dysferlinopathy.
We previously showed that 4 mutations, c.937+1G>A, c.1566C>G, c.2997G>T and c.3373delG account for 50% of all the mutations identified in Japanese dysferlinopathy patients. We performed a one-tube multiplex PCR, followed by extension of primers for each mutation with a fluorescence-labeled dideoxynucleotide to screen the 4 hot spot mutations.
The multiplex primer-extension reaction was developed on samples of known mutations. The extension products were represented as 4 different peaks that corresponded to a mutated nucleotide on electropherogram. Using the developed screening method, we were able to detect mutations in these hot spots in 3 samples out of 8 clinically suspected LGMD2B/MM patients in only approximately 8 hours. These 3 cases were definitely diagnosed as LGMD2B/MM by exonic sequencing.
We have developed a simple and rapid screening method which could facilitate the definitive diagnosis of dysferlinopathy, contributing to an understanding of the genotype-phenotype correlations for dysferlinopathy.
dysferlin基因的突变会导致肢带型肌营养不良症（LGMD）2B和宫下肌病（MM），这两种疾病统称为dysferlin病。在日本人群中，dysferlin病是LGMD最常见的类型。由于活检肌肉的免疫组化模式存在差异，包括与正常对照相似的模式，因此分子遗传学分析对于dysferlin病的诊断至关重要。然而，对整个dysferlin基因进行分析既耗时又费力；因此，一种简单快速的检测dysferlin基因热点突变的筛查方法对于dysferlin病的诊断至关重要。
我们之前发现，4种突变，即c.937+1G>A、c.1566C>G、c.2997G>T和c.3373delG，占日本dysferlin病患者中所有已鉴定突变的50%。我们进行了单管多重PCR，随后用荧光标记的双脱氧核苷酸对每个突变的引物进行延伸，以筛查这4个热点突变。
在已知突变的样本上开展了多重引物延伸反应。延伸产物在电泳图上表现为4个不同的峰，分别对应一个突变的核苷酸。使用开发的筛查方法，我们仅用了大约8小时就从8例临床疑似LGMD2B/MM患者的样本中检测出了3例热点突变。通过外显子测序，这3例病例被明确诊断为LGMD2B/MM。
我们开发了一种简单快速的筛查方法，该方法有助于dysferlin病的明确诊断，有助于了解dysferlin病的基因型与表型的相关性。
