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目的 探讨肾上腺脑白质营养不良 (AL D)的分子诊断方法。方法 在临床诊断的基础上 ,从 2名患者外周血提取白细胞总 RNA ,进行反转录后 ,用 PCR方法分四段扩增 AL D基因编码区。纯化 PCR产物 ,并对其进行序列测定。查出突变位点后 ,对患者家系成员的基因组 DNA进行 PCR-限制性酶切分析。结果 患者 A的 AL D基因第 2 80位密码子发生 CGC→ CTC改变 ,使原来编码的精氨酸被亮氨酸取代 (R2 80 L突变 ) ;患者 B的 AL D基因第 5 0 8位密码子发生了 CCC→ CTC改变 ,使正常的脯氨酸被亮氨酸替换 (P5 0 8L 突变 )。两个突变通过酶切分析和家系调查得到进一步确证。结论 建立了 AL D的分子诊断技术 ,并在中国人 AL D患者发现了两个新的 AL D基因突变。
Objective To investigate the molecular diagnosis of adrenoleukodystrophy (AL D). Methods Based on clinical diagnosis, leukocyte total RNA was extracted from peripheral blood of two patients and reverse transcribed. The coding region of ALD gene was amplified by PCR in four steps. The PCR product was purified and sequenced. After finding the mutation site, PCR-restriction analysis of the genomic DNA of the patient’s pedigree was performed. Results A codon 280 of ALD gene in patient A changed from CGC to CTC, which resulted in the replacement of the original arginine with leucine (R280 L mutation). The codon of the ALD gene in patient B Sub CCC → CTC changes occur, so that the normal proline is replaced by leucine (P5 0 8L mutation). Two mutations were confirmed by restriction analysis and pedigree investigation. Conclusion The molecular diagnosis of ALD was established and two new ALD mutations were found in Chinese ALD patients.