Abstract
Objective To develop a novel method for genotyping rs738409 (C>G) single nucleotide polymorphism(SNP), and explore the association between the rs738409 genotypes and non-alcoholic fatty liver disease (NAFLD). Methods Method establishment and analysis of genetic susceptibility. The principle of amplification refractory mutation system (ARMS) and combined a TaqMan fluorogenic probe as signal report were used, by monitoring the difference of cycle threshold (ΔCt=C allele-special primer Ct values minus G allele-special primer Ct values) between the two PCR reactions in a real-time PCR, the method for rs738409 genotyping was established (ARMS-TaqMan). 618 subjects (men: 401; women: 217), in an annual health check-up program from January 2011 to December 2014 in Aerospace Center Hospital, were performed for rs738409 genotyping by the ARMS-TaqMan assay. Fatty liver was diagnosed based on abdominal ultrasonography. The chi-square test and multiple logistic analyses were used to analyze the relationship of rs738409 genotypes and NAFLD. Results The ΔCt by ARMS-TaqMan for rs738409 genotyping were -13.1±1.4 of CC alleles (243 cases), 0.01±0.45 of CG alleles (282 cases), and 12.7±1.9 of GG alleles (93 cases), respectively. The GG alleles frequency of rs738409 were significantly higher in patients with NAFLD compared with subjects without NAFLD (21.5% vs 12.3%, χ2=8.677, P=0.003). In comparison to subjects with CC alleles, the OR (95% confidence interval) adjusted for age, gender, total cholesterol, triglyceride, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol, fasting blood glucose and body mass index was 1.35 (0.91-2.00) in subjects with CG alleles and was 2.21 (1.32-3.71) in subjects with GG alleles (P=0.013). Variant rs738409 genotypes were associated with significant increased trend in alanine aminotransferase (ALT) level from CC alleles, CG alleles to GG alleles (F=8.980, P<0.001), and in aspartate aminotansferase (AST) between GG alleles and CC alleles (F=6.491, P<0.001). Conclusions The novel ARMS-TaqMan assay had the features of accuracy, one step and high-throughput for rs738409 genotyping. The G allele of rs738409 was a risk factor of NAFLD susceptibility and associated with higher level serum ALT and AST.(Chin J Lab Med, 2016, 39: 45-49) Key words: Polymorphism, Genetic; Non-alcoholic fatty liver disease; Real-time polymerase chain reaction
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