Lindblad K, Lunkes A, Maciel P, Stevanin G, Zander C, Klockgether T, Ratzlaff T, Brice A, Rouleau G A, Hudson T, Auburger G, Schalling M
Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden.
Mol Med. 1996 Jan;2(1):77-85.
Several neurological disorders have recently been explained through the discovery of expanded DNA repeat sequences. Among these is Machado-Joseph disease, one of the most common spinocerebellar ataxias (MJD/SCA3), caused by a CAG repeat expansion on chromosome 14. A useful way of detecting repeat sequence mutations is offered by the repeat expansion detection method (RED), in which a thermostable ligase is used to detect repeat expansions directly from genomic DNA. We have used RED to detect CAG expansions in families with either MJD/SCA3 or with previously uncharacterized spinocerebellar ataxia (SCA).
Five MJD/SCA3 families and one SCA family where linkage to SCA1-5 had been excluded were analyzed by RED and polymerase chain reaction (PCR).
An expansion represented by RED products of 180-270 bp segregated with MJD/SCA3 (p < 0.00001) in five families (n = 60) and PCR products corresponding to 66-80 repeat copies were observed in all affected individuals. We also detected a 210-bp RED product segregating with disease (p < 0.01) in a non-SCA1-5 family (n = 16), suggesting involvement of a CAG expansion in the pathophysiology. PCR analysis subsequently revealed an elongated MJD/SCA3 allele in all affected family members.
RED products detected in Machado-Joseph disease families correlated with elongated PCR products at the MJD/SCA3 locus. We demonstrate the added usefulness of RED in detecting repeat expansions in disorders where linkage is complicated by phenotyping problems in gradually developing adult-onset disorders, as in the non-SCA1-5 family examined. The RED method is informative without any knowledge of flanking sequences. This is particularly useful when studying diseases where the mutated gene is unknown. We conclude that RED is a reliable method for analyzing expanded repeat sequences in the genome.
最近，通过发现扩展的DNA重复序列，几种神经系统疾病得到了解释。其中包括Machado-Joseph病，它是最常见的脊髓小脑共济失调之一（MJD/SCA3），由14号染色体上的CAG重复序列扩展引起。重复序列扩展检测方法（RED）提供了一种检测重复序列突变的有效方法，该方法使用热稳定连接酶直接从基因组DNA中检测重复序列扩展。我们已使用RED检测患有MJD/SCA3或先前未明确特征的脊髓小脑共济失调（SCA）的家族中的CAG扩展。
通过RED和聚合酶链反应（PCR）分析了5个MJD/SCA3家族和1个已排除与SCA1 - 5连锁的SCA家族。
在5个家族（n = 60）中，180 - 270 bp的RED产物所代表的扩展与MJD/SCA3共分离（p < 0.00001），并且在所有受影响个体中观察到对应于66 - 80个重复拷贝的PCR产物。我们还在一个非SCA1 - 5家族（n = 16）中检测到一个与疾病共分离的210 bp的RED产物（p < 0.01），提示CAG扩展参与了病理生理过程。随后的PCR分析在所有受影响的家族成员中揭示了一个延长的MJD/SCA3等位基因。
在Machado-Joseph病家族中检测到的RED产物与MJD/SCA3基因座处延长的PCR产物相关。我们证明了RED在检测重复序列扩展方面的额外有用性，这些疾病的连锁分析因成年发病的渐进性疾病中的表型问题而变得复杂，如在所检测的非SCA1 - 5家族中。RED方法无需任何侧翼序列的知识即可提供信息。这在研究突变基因未知的疾病时特别有用。我们得出结论，RED是分析基因组中扩展重复序列的可靠方法。
