Kozuka Naoki, Tachi Nobutada, Ohya Kazuhiro, Uchida Eiji, Kikuchi Shin, Sengoku Yasuhito
School of Health Sciences, Sapporo Medical University, Sapporo, Hokkaido.
No To Hattatsu. 2003 Sep;35(5):380-7.
Myotonic dystrophy (MyD) is a hereditary neuromuscular disorder of an autosomal dominant trait. MyD is caused by an expansion of unstable CTG trinucleotide repeat in the 3' untranslated region of mRNA coding myotonin protein kinase (MT-PK). We analyzed CTG repeat expansion in 10 patients with congenital MyD and their relatives using the non-radioactive PCR Southern method. The region containing the CTG repeat was amplified by PCR using specific primers. The PCR products were electrophoresed on a 1% agarose gel and transferred to a nylon membrane. The CTG repeat expansion was shown using a fluorescein-labelled (CTG) 10 probe. To estimate the number of CTG repeats, we compared the smears obtained on Southern blotting with a picture of PCR products and a DNA size marker (100 bp). We compared our results of radioactive Southern blotting for genomic DNA digested by Eco RI or Bgl I and for PCR products. In congenital MyD patients, heterogeneous smears (3.89-10.22 kb:about 1252-3362 CTG repeat) were observed, whereas in the adult type MyD had heterogeneous smears (0.92-1.82 kb:about 262-562 CTG repeat). In asymptomatic MyD patients, there were heterogeneous smears (0.35-1.16 kb:about 72-342 CTG repeat). These results demonstrated anticipation. We conclude that the non-radioactive PCR Southern method is useful and convenient for the DNA diagnosis of MyD.
强直性肌营养不良（MyD）是一种常染色体显性遗传的神经肌肉疾病。MyD是由编码肌强直性蛋白激酶（MT-PK）的mRNA的3'非翻译区中不稳定的CTG三核苷酸重复序列扩增引起的。我们使用非放射性PCR Southern方法分析了10例先天性MyD患者及其亲属的CTG重复序列扩增情况。使用特异性引物通过PCR扩增包含CTG重复序列的区域。将PCR产物在1%琼脂糖凝胶上进行电泳，然后转移到尼龙膜上。使用荧光素标记的（CTG）10探针显示CTG重复序列扩增。为了估计CTG重复序列的数量，我们将Southern印迹上获得的涂片与PCR产物图片和DNA大小标记（100 bp）进行比较。我们比较了用Eco RI或Bgl I消化的基因组DNA以及PCR产物的放射性Southern印迹结果。在先天性MyD患者中，观察到异质性涂片（3.89 - 10.22 kb：约1252 - 3362个CTG重复），而成年型MyD有异质性涂片（0.92 - 1.82 kb：约262 - 562个CTG重复）。在无症状的MyD患者中，存在异质性涂片（0.35 - 1.16 kb：约72 - 342个CTG重复）。这些结果证明了遗传早现现象。我们得出结论，非放射性PCR Southern方法对于MyD的DNA诊断是有用且方便的。
