DHPLC在X-连锁肾上腺脑白质营养不良分子诊断中的应用(附12例报告)

目的探讨DHPLC在X-连锁肾上腺脑白质营养不良(X-ALD)分子诊断中的应用。方法提取12个X-ALD家系及成员的外周血基因组DNA,分15个片段扩增ABCD1基因的10个外显子,应用DHPLC技术对其进行突变筛查,并对出现异常洗脱峰的PCR产物进行DNA序列测定,证实突变位点的存在。结果12个X-ALD家系存在12种不同的ABCD1基因突变,包括8个错义突变、2个移码突变和2个无义突变,即P534R、G343V、R259W、A141T、R401Q、K276E、Y174C、A314P、fs E471、fs A247、S108X和Q177X。结论DHPLC筛查结合DNA序列测定能快速有效检测出ABCD1基因突变。不同的X-ALD家系有不同的ABCD1基因突变位点,突变类型和表型之间无特殊相关关系。

Application of DHPLC in molecular diagnosis of X-linked adrenoleukodystrophy(report of 12 cases)

Objective To investigate the feasibility of utilizing denaturing high performance liquid chromatography(DHPLC) in molecular diagnosis of X-linked adrenoleukodystrophy.Methods The genomic DNA was extracted from peripheral blood of 12 Chinese patients with X-linked adrenoleukodystrophy and their family members(including 11 cases of X-linked adrenoleukodystrophy and 26 cases with X-linked adrenoleukodystrophy indices).Two methods,DHPLC and DNA direct sequencing,were used to analyze the ABCD1 gene from the genomic DNA.DHPLC was utilized to screen the gene mutations after amplifying the 15 fragments of the 10 exons and exon-intron boundaries of the ABCD1 gene.For confirming the mutations,those PCR products,in which the abnormal DHPLC elution peaks were formed,were subjected to DNA direct sequencing.Results Twelve Chinese pedigrees of X-linked adrenoleukodystrophy were analyzed,and abnormal elution patterns were found in PCR products from all patients or patients' mother,which revealed the presence of mutations in the ABCD1 genes.All the mutations were confirmed by DNA direct sequencing,which revealed twelve distinct mutations in the ABCD1 genes of the twelve pedigrees,including ten base substitutions(1987C>G,1414G>T,1161C>T,807G>A,1588G>A,1212A>G,07A>G,326G>C,709C>A and 915C>T),one dinucleotide deletion(1801-02 del AG) and one base insertion(1125 ins GCCATCG),which resulted in eight missense mutations,two nonsense mutations and two frame shift mutations,namely P534R,G343V,R259W,A141T,R401Q,K276E,Y174C,A314P,S108X,Q177X,fs E471 and fs A247.Conclusion The combined DHPLC and sequencing approach may act as a rapid and efficient method for ABCD1 gene mutation analysis in patients and carriers of X-linked adrenoleukodystrophy families.There exist different ABCD1 gene mutations in different pedigrees,and no obvious correlation between the genotype and phenotype has been found.