Use of RNA fluorescence in situ hybridization in the prenatal molecular diagnosis of myotonic dystrophy type I
Use of RNA fluorescence in situ hybridization in the prenatal molecular diagnosis of myotonic dystrophy type I
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DOI：
10.1373/clinchem.2005.056283
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发表时间：
2006-02-01
影响因子：
9.3
通讯作者：
Novelli, G
中科院分区：
文献类型：
--
作者：
Bonifazi, E;Gulotta, F;Novelli, G
背景资料：强直性肌营养不良1型（DM 1; OMIM #160900）是一种常染色体显性遗传性疾病，具有与染色体19q13.3上DMPK基因3'非翻译区CTG扩增相关的多系统临床特征。一个长的PCR协议来检测DM 1扩增是快速，灵敏，准确，但解释的局限性时，可能会发生扩增的大小超过PCR扩增范围和体细胞mosaicity.Methods的情况下：为了克服这些问题，我们使用RNA荧光原位杂交（RNA-FISH）研究培养的细胞来自绒毛膜绒毛样品（CVS）与DM 1突变。RNA-FISH方法设计用于检测独特的DM 1细胞表型，其特征在于存在具有含有DMPK扩增转录物的局灶性核糖核包涵体（病灶）的细胞核。结果：在4例CTG扩增> 200 CTG的DM 1预测胎儿中，所有细胞中均清晰可见不同数量的核糖核包涵体。1例DMPK突变体细胞嵌合现象显示15%的细胞无核灶。结论：当CTG扩增> 200 copies时，利用RNA-FISH技术可以在滋养层细胞间期核上有效地检测到核灶，从而检测到由核灶引起的DM 1突变。(c)2006年美国临床化学协会
Background: Myotonic dystrophy type 1 (DM1; OMIM #160900) is an autosomal-dominant genetic disorder with multisystemic clinical features associated with a CTG expansion in the 3' untranslated region of the DMPK gene on chromosome 19q13.3. A long-PCR protocol to detect the DM1 expansion is rapid, sensitive, and accurate, but interpretative limitations can occur when the expansion size exceeds the PCR amplification range and in cases of somatic mosaicism.Methods: To overcome these problems, we used RNA fluorescence in situ hybridization (RNA-FISH) to study cultured cells derived from chorionic villus samples (CVS) with the DM1 mutation. The RNA-FISH method is designed to detect the distinctive DM1 cellular phenotype, characterized by the presence of nuclei with focal ribonuclear inclusions (foci) containing the DMPK expanded transcripts. We analyzed 6 CVS from DM1-predicted pregnancies and 6 CVS from DM1-negative pregnancies.Results: In 4 DM1-predicted fetuses with a CTG expansion > 200 CTG, varying numbers of ribonuclear inclusions were clearly visible in all cells. One case with a somatic mosaicism for the DMPK mutation showed 15% of cells with no nuclear foci. No nuclear signals were detected in all controls examined (n = 6) and in 1 DM1-positive sample with a CTG expansion < 100 copies.Conclusion: Nuclear foci, and therefore the DM1 mutation they are caused by, can be detected efficiently on interphase nuclei of trophoblast cells with RNA-FISH when the CTG expansion is > 200 copies. (c) 2006 American Association for Clinical Chemistry