Panagopoulos I, Lassen C, Kristoffersson U, Aman P
Department of Clinical Genetics, University Hospital, Lund, Sweden.
Hum Mutat. 1999;13(3):232-6. doi: 10.1002/(SICI)1098-1004(1999)13:3<232::AID-HUMU7>3.0.CO;2-N.
Huntington disease (HD) is an autosomal dominant neurodegenerative disorder associated with expansions of an unstable CAG trinucleotide repeat in exon 1 of the IT15 gene. In normal individuals, IT15 contains up to 35 CAG repeats, while in affected the repeat length is >36. Polymerase chain reaction (PCR) is used to estimate the number of CAG repeats but may be inefficient in long repeats because of the high C+G content of the HD locus. We present a novel PCR approach for the diagnosis of HD, which permits direct visualization of the amplified products on agarose gel, using ethidium bromide. It is based on the methylation-sensitive conversion of C residues to U by bisulfite treatment of single-stranded DNA and subsequent amplification of the sense strand with specific primers. The bisulfite treatment dramatically reduces the C + G content of the region; thus, the high Tm and stable secondary structures are no longer obstacles to PCR. In both normal and affected individuals, UAG repeats (5'- CAG-3', before bisulfite treatment) in the sense strand can easily be amplified and visualized on a gel by ethidium bromide staining. The method has considerable advantages compared with other described PCR-based diagnostic tests for HD.
亨廷顿舞蹈症（HD）是一种常染色体显性神经退行性疾病，与IT15基因外显子1中不稳定的CAG三核苷酸重复序列扩增有关。在正常个体中，IT15含有多达35个CAG重复序列，而在患病个体中，重复长度大于36个。聚合酶链反应（PCR）用于估计CAG重复序列的数量，但由于HD基因座的C+G含量高，对于长重复序列可能效率不高。我们提出了一种用于诊断HD的新型PCR方法，该方法允许使用溴化乙锭在琼脂糖凝胶上直接观察扩增产物。它基于通过对单链DNA进行亚硫酸氢盐处理将C残基甲基化敏感地转化为U，随后用特异性引物扩增有义链。亚硫酸氢盐处理显著降低了该区域的C+G含量；因此，高解链温度和稳定的二级结构不再是PCR的障碍。在正常个体和患病个体中，有义链中的UAG重复序列（亚硫酸氢盐处理前为5'-CAG-3'）都可以很容易地扩增，并通过溴化乙锭染色在凝胶上观察到。与其他已描述的基于PCR的HD诊断测试相比，该方法具有相当大的优势。
