克山病发病与谷胱甘肽过氧化物酶-1基因多态性的关系研究

目的 研究谷胱甘肽过氧化物酶-1(GPx-1)基因多态性与血硒、GPx-1酶活性的关系,为克山病个体的易感性提供分子遗传学基础.方法 采用分光光度法及酶促法测量71例克山病患者和290名对照(其中内对照78名,外对照212名)的全血硒和GPx-1酶活性,采用测序及聚合酶链反应-限制性内切酶长度多态性(PCR-RFLP)方法分析GPx-1基因198位点基因型,采用新生乳鼠心肌细胞转染表达质粒,探讨GPx-1两种基因型的功能.结果 克山病患者的血硒为(0.8±0.2)μmol/L,内对照组为(0.9±0.2)μmol/L,外对照组为(1.2±0.2)μmol/L(F=4.888,P＜0.001).克山病患者的GPx-1酶活性为(73.0±12.6)×10-10U/RBC,内对照组为(80.9±9.2)×10-10U/RBC,外对照组为(115.8±21.1)×10-10U/RBC(F=5.324,P＜0.001).克山病患者较对照组均明显降低.GPx-1基因上筛查出突变198位点多态性Pro198Leu;克山病患者的Pro198Leu多态性频率为21.1%,对照组的多态性频率为10.7%,病例组的多态性频率明显升高(x2=5.588,P=0.018).突变型组(Pro198Leu和Leu198Leu)的血硒为(0.9±0.2)μmol/L,非突变型为(1.1±0.3)μmol/L(t=3.183,P＜0.01);突变型组的酶活性为(86.1±23.0)×10-10U/RBC,非突变型组的酶活性为(101.8±25.9)×10-10U/RBC(t=5.784,P＜0.01).进一步研究证实198位等位基因多态性与低硒之间存在协同倍增交互作用.在大鼠乳鼠心肌细胞上过表达GPx-1(198Leu)会降低30%的GPx-1酶活性,并且对添加硒的反应不敏感.结论 低硒并GPx-1基因198Leu多态性,与GPx-1酶活力降低有关;低硒和GPx-1-198Leu存在协同倍增交互作用,共同增加克山病发病风险.

Polymorphisms in the glutathione peroxidase-1 gene associated with increased risk of Keshan disease

Objective To assess the association of blood selenium and polymorphism of glutathione peroxidase-1 (GPx-1) genes in patients with Keshan Disease (KD) and provide genetic evidence for KD susceptibility.Methods The levels of whole blood selenium and the activity of GPx-1 were measured with spectrophotometric and enzymatic method among 71 KD patients and 290 controls (including 78 internal controls and 212 external controls).The genotype of GPx-1 at 198 site was analyzed by sequencing and PCRRFLP.The functions of two GPx-1 variants were studied by rat neonatal cardiomyocytes transfection and expression pLamid.Results Blood level of selenium in KD patients was (0.8 ±0.2) μmol/L,the internal controls' was (0.9 ±0.2) μmoL/L,and the external controls' was ( 1.2 ±0.2) μmol/L (F=4.888,P＜0.001 ).GPx-1 activity of KD patients was (73.0 ± 12.6) × 10-10 U/RBC, internal controls' was ( 80.9 ±9.2) × 10-10U/RBC,and external controls' was ( 115.8 ±21.1 ) × 10 -10U/RBC ( F =5.324,P ＜0.001 ).Those of KD patients were significantly lower than controls.The polymorphism (Pro198Leu) of GPx-1 were identified; the frequency of Pro198Leu of KD patients was 21.1% , the frequency of controls was 10.7%(χ2 =5.588 ,P =0.018).The level of blood selenium in variant subgroup( Pro198Leu or Leu198Leu) was(0.9 ± 0.2) μmoL/L, and its in non-variant subgroup was ( 1.1 ± 0.3 ) μmol/L ( t = 3.183, P ＜ 0.01 );The GPx-1 activity in variant subgroup was (86.1 ± 23.0 ) × 10-10U/RBC, and its in non-variant subgroup was ( 101.8 ± 25.9 ) × 10-10 U/RBC ( t = 5.784, P＜ 0.01 ) .Further analysis revealed a synergisticmultiplicative interaction between presence of GPx-1 codon198 alleles and low blood selenium level Overexpression of GPx-1 (198Leu) in rat cardiomyocytes caused 30% lower enzyme activity and less response to increasing concentrations of selenium than with over-expression of GPx-1 (198Pro).Conclusion Low blood selenium in carriers with the 198Leu-susceptible genotype of GPx-1 is associated with low GPx-1 activity,synergistic-multiplicative interaction was found between these two factors.And these two factors may increase the risk of KD.