Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by “dual-fluorescence labeled PCR-restriction fragment length analysis”
Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by “dual-fluorescence labeled PCR-restriction fragment length analysis”
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DOI：
10.1007/s10038-007-0242-2
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发表时间：
2008-02
影响因子：
3.5
通讯作者：
Jiang X Lin;K. Ishikawa;Masaki Sakamoto;T. Tsunemi;T. Ishiguro;T. Amino;S. Toru;I. Kondo;H. Mizusawa
中科院分区：
文献类型：
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作者：
Jiang X Lin;K. Ishikawa;Masaki Sakamoto;T. Tsunemi;T. Ishiguro;T. Amino;S. Toru;I. Kondo;H. Mizusawa
脊髓小脑性共济失调1型是一种常染色体显性遗传性小脑性共济失调，其病因是ATXN1基因编码多谷氨酰胺的CAG重复序列的扩增。ATXN1蛋白中多聚谷氨酰胺的长度是该病发病机制的关键决定因素。SCA1的分子诊断通常是通过使用跨越该构型的引物来评估CAG重复构型的长度来进行的。然而，这种传统的方法可能会导致在评估多谷氨酰胺编码的CAG重复长度时的误诊，因为CAT中断可能存在于CAG重复构型中，不仅在正常对照中，而且在有神经症状的受试者中也是如此。我们开发了一种新的方法来评估不被CAT序列中断的实际CAG重复数。用一对标记了两种不同荧光的引物进行聚合酶链式反应，然后用识别GCATC(N)5序列的SfaNI限制性内切酶酶切，直接检测到编码聚谷氨酰胺的实际CAG重复序列的长度。我们将该方法命名为“双荧光标记聚合酶链式反应-限制性片段长度分析”。我们发现，编码聚谷氨酰胺的实际CAG重复数在我们的正常染色体队列(n=385)和SCA1染色体(n=5)之间并不重叠。结论：本方法是SCA1分子诊断的一种有效方法。
Spinocerebellar ataxia type 1 (SCA1; OMIM:# 164400) is an autosomal dominant cerebellar ataxia caused by an expansion of CAG repeat, which encodes polyglutamine, in the ataxin-1 (ATXN1) gene. Length of polyglutamine in the ATXN1 protein is the critical determinant of pathogenesis of this disease. Molecular diagnosis of SCA1 is usually undertaken by assessing the length of CAG repeat configuration using primers spanning this configuration. However, this conventional method may potentially lead to misdiagnosis in assessing polyglutamine-encoding CAG repeat length, since CAT interruptions may be present within the CAG repeat configuration, not only in normal controls but also in neurologically symptomatic subjects. We developed a new method for assessing actual CAG repeat numbers not interrupted by CAT sequences. Polymerase chain reaction using a primer pair labeled with two different fluorescences followed by restriction enzyme digestion with SfaNI which recognizes the sequence “GCATC (N) 5”, lengths of actual CAG repeats that encode polyglutamine were directly detected. We named this method “dual fluorescence labeled PCR-restriction fragment length analysis”. We found that numbers of actual CAG repeat encoding polyglutamine do not overlap between our cohorts of normal chromosomes (n= 385) and SCA1 chromosomes (n= 5). We conclude that the present method is a useful way for molecular diagnosis of SCA1.