三例眼皮肤白化病患者TYR和P基因的突变分析

Mutation screening of the TYR and P gene in three patients with oculocutaneous albinism

目的分析眼皮肤白化病（oculocutaneous albinism，OCA）患者酪氨酸酶（tyrosinase，TYR）基因和P基因的基因突变。方法应用聚合酶链反应（polymerase chain reaction，PCR）和变性高效液相色谱（denaturing high-performance liquid chromatography，DHPLC）技术对3例患者的眼皮肤白化病Ⅰ、Ⅱ型相关基因（TYR和P基因）的外显子进行突变检测，并对DHPLC检出的突变样本进行测序和限制性内切酶分析以验证该突变。针对未见报道的新突变，筛查100名表型正常的无关个体，排除多态的可能。结果在3例患者中检测出两种P基因突变，未检测到TYR基因突变。其中，患者1的P基因第13外显子发生杂合突变T450M；患者2的P基因发生两个杂合突变，分别是第13外显子T450M和第23外显子G775R；患者3的P基因第23外显子发生杂合突变G775R。P基因第13外显子限制性内切酶分析显示，患者1、2均出现杂合突变T450M导致的0在I酶切位点部分消失，100名表型正常的无关个体未检出该突变；经检索，T450M为一未见报道的新突变。结论联合应用PER、DHPLC、DNA测序和限制性内切酶分析的方法可有效的对白化病进行基因诊断。

Objective To identify the mutations of the tyrosinase gene （TYR） and P gene in patients with oculocutaneous albinism（OCA）. Methods Polymerase chain reaction （PCR） and denaturing high-performance liquid chromatography （DHPLC） were applied to detect the mutations in all exons of TYR gene and P gene. Then DNA sequencing and restriction endonuclease analysis were used to confirm the mutations detected by DHPLC. Novel mutations were screened in 100 unrelated persons with normal phenotypes to exclude the possibility of polymorphism. Results Two mutations were detected in the P gene of the three patients and none in TYR gene. Heterozygous mutation of T450M in exon 13 of the P gene was detected in patient 1. Patient 2 had a heterozygous mutation of T450M in exon 13 and a heterozy, gous mutation of G775R in exon 23 of the P gene. Patient 3 had a heterozygous mutation of G775R as well. Restriction endonuclease analysis of the P gene exon 13 showed that the Oli I site had partly disappeared resulting from the heterozygous mutation T450M in patient 1 and patient 2, but not in 100 unrelated individuals. The heterozygous mutation Td50M is a novel mutation. Conclusion Gene diagnosis of OCA can be carried out effectively by combining PCR, DHPLC, DNA sequencing and restriction endonuclease analysis.