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为了研究睾丸特异性乳酸脱氢酶,即乳酸脱氢酶C4(LDH-C4)基因突变在男性不育发病中的作用,利用LDH-C4特异性底物对100名不明原因男性不育症患者的精子LDH-C4进行活性显色,用变性高效液相色谱(DHPLC)技术对LDH-C4活性低下的患者进行LDHC基因PCR产物的突变筛查,对DHPLC峰形异常的PCR产物进行序列测定.筛选到一组精子LDH-C4活性明显下降的患者,其中1名患者的LDHC基因PCR产物在DHPLC中呈异常洗脱峰.对这一PCR产物进行序列测定,发现患者LDHC基因第5外显子的115位碱基发生了T→A的杂合改变(GenBank登录号GU479375),该突变使LDHC基因的178位密码子由原来的TTG(编码亮氨酸)变为TAG(终止密码子),形成截短的C亚基.T克隆-测序进一步证实了该无义突变的杂合状态.这是在人类LDHC基因上发现的第一个突变,提示LDHC基因突变可能是男性不育发病的原因之一.
In order to investigate the role of testicular-specific lactate dehydrogenase (LDH-C4) gene mutation in the pathogenesis of male infertility, LDH-C4-specific substrate was used to investigate the effect of LDH-C4 mutation on 100 infertile male infertility patients LDH-C4 was used to perform colorimetric assay. Mutations of LDHC gene PCR products were screened by DHPLC in patients with low LDH-C4 activity, and the PCR products of abnormal DHPLC peaks were sequenced. A group of patients with significantly decreased LDH-C4 activity was screened, in which LDHC gene PCR products of 1 patient showed abnormal elution peaks in DHPLC.The sequence of this PCR product was sequenced, and found that patients with LDHC gene exon 5 (GenBank accession number GU479375) at codon 115 changed the codon 178 of LDHC gene from the original TTG (encoding leucine) to TAG (stop codon) Forming a truncated C subunit.T cloning-sequencing further confirmed the heterozygous state of this nonsense mutation.This is the first mutation found in human LDHC gene, suggesting that LDHC gene mutation may be the cause of male infertility one.