家族性玻璃体淀粉样变性甲状腺激素结合蛋白基因突变及表达研究

Transthyretin gene mutation and expression in patients with familial vitreous amyloidosis

目的 观察家族性玻璃体淀粉样变性（FTA）一家系甲状腺激素结合蛋白（TTR）基因突变位点及其mRNA、蛋白表达变化.方法 来自同一家系的家族性玻璃体淀粉样变性患者7例（发病组）、家系中未发病成员19名（未发病组）以及与该家系来自同一地区的正常健康者9名（对照组）纳入研究.采集3组受检者外周静脉血,提取DNA,采用逆转录聚合酶链反应（PCR）扩增TTR基因外显子1～4,进行测序.实时荧光定量PCR检测受检者血细胞中TTR mRNA的表达.蛋白免疫印迹法检测受检者血清中TTR蛋白表达.结果 发病组7例、未发病组3名受检者TTR外显子3的107位碱基出现杂合突变,由G变为C,出现套峰;即第83氨基酸的密码子发生碱基突变,由GGC突变成CGC,甘氨酸突变为精氨酸（Gly83Arg）,其氨基酸的极性由中性转变为碱性.其他受检者未发现该位点基因突变.发病组患者TTRmRNA表达均较未发病组、对照组低,差异有统计学意义（P=0.032、0.001）;未发病组受检者血细胞中TTR mRNA表达较对照组低,差异有统计学意义（P=0.001）.发病组患者血清中TTR蛋白表达均较未发病组、对照组低,差异有统计学意义（P=0.035、0.038）;未发病组与对照组受检者血清中TTR蛋白表达比较,差异无统计学意义（P=0.491）.结论 该家系7例患者、3名未发病家系成员TTR基因第3外显子发生杂合突变,即Gly83Arg.发病患者TTR mRNA及蛋白表达较未发病家系成员及正常者降低.

Objective To observe the transthyretin （TTR） gene mutation,protein and mRNA expression in patients with familial vitreous amyloidosis.Methods Subjects were divided into three groups：（1） illness group：seven patients with familial vitreous amyloidosis.（2） No-illness group：9 unaffected family members.（3） Control group：9 healthy individuals in same area.Subjects＇ peripheral venous blood were collected and DNA were extracted,4 exons of TTR gene were amplified by reverse transcription polymerase chain reaction（RT-PCR）,the gene fragments were sequencing by the fluorescence labelling method.Serum TTR protein expression was detected by Western blot,and TTR mRNA in leukocyte was assayed by RT-PCR.Results 4 exons of TTR gene of all samples were amplified,and DNA sequencing data showed that 7 patients and 3 subjects DNA from unaffected family members had mutated in the 3rd exon of 107th base,changing from G to C.Heterozygous mutation occurred in codon of the 83th amino acid in exon 3,namely,Gly83Arg,resulted in the change of GGC to CGC.The protein and mRNA expression of TTR was lower in illness group than no-illness group and control groups（P〈0.05）.Compared with control group,TTR mRNA expression in unaffected family members groups was significant decreased（P〈0.05）.Conclusion Heterozygous mutation occurred in codon of the 83th amino acid in exon 3,namely Gly83Arg,and suggested that Gly83Arg is connected with the change of TTR mRNA and protein expression.