摘要
目的:探讨X-连锁肾上腺脑白质营养不良分子诊断中排除假基因干扰的新方法。方法:应用长链RT-PCR技术扩增3个X-连锁肾上腺脑白质营养不良患者的ABCD1基因编码区全长,再分4个片段进行二次PCR,并对PCR产物直接测序;应用巢式PCR对ABCD1基因相应区域进行扩增,其中第一轮PCR产物覆盖ABCD1基因从外显子6至3′非编码区的一个大片段,并对PCR产物进行测序,分析患者的基因组DNA,进一步确证其ABCD1基因突变。结果:在3个X-ALD患者的ABCD1基因上,存在3个不同的碱基改变(2235C>T,2065C>T和2190A>T),分别造成2个错义突变(R617C和P560L)和1个无义突变(K602X)。结论:应用巢式PCR能快速、有效地排除X-ALD分子诊断中ABCD1假基因的干扰。
Objective：To introduce a new method which can avoid the interference of ABCD1 pseudogenes in the molecular diagnosis of X-linked adrenoleukodystrophy. Methods：The coding regions of ABCD1 gene of 3 unrelated Chinese patients with X-linked adrenoleukodystrophy were amplified from the total RNA of peripheral blood by long distance RT-PCR; the product was further amplified in 4 segments in a second round PCR; and the PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA from peripheral blood cells of the patients was analyzed by direct sequencing after amplification of the ABCD1 genes by nested PCR,in which the product of the first round PCR covered the fragment starting from exon 6 and ending at 3＇UTR of the ABCD1 gene. Results：The 3 Chinese patients with X-linked adrenoleukodystrophy had 3 different base substitutions（2235C〉T,2065C〉T and 2190A〉T）in the ABCD1 genes of the 3 probands and their mothers, which resulted in 2 missense mutations （R617C and P560L） and one nonsense mutation （K602X）. Conclusion：Nested PCR can rapidly and efficiently avoid the interference of ABCD1 pseudogenes in the molecular diagnosis of X-ALD.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2009年第2期217-219,共3页
Academic Journal of Second Military Medical University
基金
南京军区医药卫生"十一五"科研基金(06 MA136)~~
