The Kruppel-like factor 6 genotype is associated with fibrosis in nonalcoholic fatty liver disease

Abstract

Background & aims: Although nonalcoholic fatty liver disease (NAFLD) is increasingly common, only a minority of affected individuals develop fibrotic liver disease. Based on its role in liver growth and repair, we explored whether Kruppel-like factor 6 (KLF6) plays a role in NAFLD progression.

Methods: KLF6 expression in 31 fibrosis scored NAFLD liver biopsy specimens was assessed by real-time polymerase chain reaction. Transfected minigene constructs were used to study the effect of a polymorphism, KLF6-IVS1-27G>A, that promotes KLF6 alternative splicing in vitro. We genotyped KLF6-IVS1-27G>A in 3 groups of patients (UK group 1, n = 306; Italian group 2, n = 109; trio group 3, n = 61 children and parents).

Results: KLF6 expression was increased in association with increased steatosis, inflammation, and fibrosis in NAFLD livers. KLF6-IVS1-27G>A promoted alternative splicing of KLF6 and abrogated the up-regulation of both alpha-smooth muscle actin and collagen 1 in LX-2 cells. Group 1 genotyping identified KLF6-IVS1-27G>A in 44 of 306 (14.4%) patients. Notably, KLF6-IVS1-27G>A was associated significantly with milder NAFLD, with only 25% having more advanced fibrosis compared with 45% of wild-type (wt) individuals. This trend was confirmed in group 2. A linear regression analysis including all 415 patients, adjusted for age, sex, body mass index, and blood glucose level, confirmed that presence of the wt KLF6 allele was an independent predictor of fibrotic NAFLD. Furthermore, we have shown preferential transmission of the wt allele to children with fibrotic NAFLD.

Conclusions: We report a functional polymorphism in the KLF6 gene associated with advanced NAFLD and believe further study of KLF6 may enhance our understanding of this disease process.

Figure 1

Increased wt KLF6 expression with advanced stages of NAFLD. Either Total KLF6 mRNA isoforms (represented by black bars) or the wt isoform only (grey bars) have been quantified by semiquantitative TaqMan RT-PCR. The individual Brunt et al histology scores and KLF6 RQ values are shown in Table 1, whereas the mean expression levels (RQ log) ± standard error within histologically defined grades of NAFLD are shown in this Figure (n = 31; P values are shown). (A) Stepwise increase in both Total KLF6 isoforms and wt KLF6 increased stepwise in association with increasing levels of inflammation (A). Although wt KLF6 also was increased significantly in association with advanced steatosis (B) and fibrosis (C), Total KLF6 isoforms were not. (A) ■, Total, P = .001; , wt, P = .010; (B) ■, P = .049; , P = .001; (C) ■, NS; , P = .03.

Figure 2

KLF6 IVS1-27G>A promotes alternative splicing of KLF6 and abrogates the up-regulation of αSMA and collagen 1. Cells were transfected with either transfection reagent alone (CON), a control minigene expressing LacZ (LacZ), a KLF6 minigene with the IVS1-27G>A SNP (G>A), or a KLF6 wt minigene construct (wt). (A) Agarose gel electrophoresis of cDNA products amplified using 5′ and 3′ flanking primers detecting all KLF6 isoforms identified is shown. KLF6 in the wt minigene transfected cells was predominantly the full-length wt isoform, whereas in the cells transfected with the KLF6 IVS1-27G>A minigene there were additional smaller bands representing alternative splice forms. Relative quantities of these isoforms were confirmed by semiquantitative RT-PCR (n = 3, data not shown). (B) Semiquantitative RT-PCR data from RNA extracted from stably transfected LX-2 cells harvested at 1 week. Total KLF6 was increased in cells transfected with either of the KLF6 minigene constructs, whereas only a relatively modest amount of this total KLF6 was the wt isoform in the G>A–transfected cells. (C) The marked increase in wt KLF6 in the LX-2 cells transfected with the wt KLF6 minigene was associated with significant increases in both αSMA and collagen I mRNA, which was not present in those cells transfected with the G>A SNP containing minigene-expressing dominant-negative KLF6 isoforms in addition to the wt isoform.