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目的 对 3个肾上腺脑白质营养不良 ( adrenoleukodystrophy,AL D) ( MIM# 30 0 10 0 )家系进行基因突变分析。方法 从 3个 AL D患儿及其主要家系成员的外周血白细胞 ,提取总 RNA和基因组 DNA。应用逆转录聚合酶链反应技术 ,对 3个家系的 ABCD1基因编码区 ,分 4个片段进行 PCR扩增并对 PCR产物直接测序。同时应用 PCR-限制性酶切或扩增阻滞突变系统分析相应的基因组 DNA,进一步确证ABCD1基因的突变位点。结果 3名患儿的 ABCD1基因上均存在错义突变 ,其中患儿 1的 ABCD1基因第 5 34位密码子发生 CCC→ CGC改变 ,使脯氨酸被精氨酸取代 ( P5 34R) ;患儿 2的 ABCD1基因第 2 6 6位密码子发生 GGG→AGG改变 ,使甘氨酸被精氨酸取代 ( G2 6 6 R) ;患儿 3母亲的 ABCD1上一个等位基因第 6 17位密码子发生 CGC→ GGC改变 ,使精氨酸被甘氨酸取代 ( R6 17G) ,另一个等位基因未发生突变。结论 在中国人 AL D患者中发现 1个新的 ABCD1基因突变 ( P5 34R) ,并首次在中国人 AL D患者中检测到G2 6 6 R、R6 17G突变
Objective To analyze the gene mutation in three adrenoleukodystrophy (ALD) (MIM # 30 0 10 0) pedigrees. Methods Total RNA and genomic DNA were extracted from peripheral blood leukocytes from 3 children with ALD and their major familial members. The ABCD1 gene coding region of three pedigrees was amplified by PCR and sequenced directly by PCR using reverse transcription polymerase chain reaction (PCR). At the same time, the corresponding genomic DNA was analyzed by PCR-restriction analysis or amplification and blockade mutation system to further confirm the mutation site of ABCD1 gene. Results There was a missense mutation in ABCD1 gene in all three children, in which CCC → CGC was changed in codon 34 of ABCD1 in children 1, and proline was replaced by arginine (P5 34R) 2 ABCD1 gene codon 266 GGG → AGG changes in the glycine by arginine (G2 6 6 R); 3 children with a mother ABCD1 allele at the 6th codon CGC → GGC changes, leaving arginine substituted for glycine (R6 17G) and no mutation of the other allele. Conclusions A novel ABCD1 gene mutation (P5 34R) was found in Chinese ALD patients and the G2 6 6 R and R6 17G mutations were first detected in Chinese ALD patients