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Correspondence to: Margarete Odenthal, PhD, Institute for Pathology, University Hospital Cologne, Kerpener Str. 62, 50924 Cologne, Germany. firstname.lastname@example.org
Telephone: +49-221-4786367 Fax: +49-221-4786360
AIM: To test the occurrence of the Pro12Ala mutation of the peroxisome proliferator-activated receptor-γ (PPARγ)2-gene in patients with non-alcoholic fatty liver disease (NAFLD) or alcoholic fatty liver disease (AFLD).
METHODS: DNA from a total of 622 specimens including 259 blood samples of healthy blood donors and 363 histologically categorized liver biopsies of patients with NAFLD (n = 263) and AFLD (n = 100) were analyzed by Real-time polymerase chain reaction using allele-specific probes.
RESULTS: In the NAFLD and the AFLD collective, 3% of the patients showed homozygous occurrence of the Ala12 PPARγ2-allele, differing from only 1.5% cases in the healthy population. In NAFLD patients, a high incidence of the Ala12 mutant was not associated with the progression of fatty liver disease. However, we observed a significantly higher risk (odds ratio = 2.50, CI: 1.05-5.90, P = 0.028) in AFLD patients carrying the mutated Ala12 allele to develop inflammatory alterations. The linkage of the malfunctioning Ala12-positive PPARγ2 isoform to an increased risk in patients with AFLD to develop severe steatohepatitis and fibrosis indicates a more prominent anti-inflammatory impact of PPARγ2 in progression of AFLD than of NAFLD.
CONCLUSION: In AFLD patients, the Pro12Ala single nuclear polymorphism should be studied more extensively in order to serve as a novel candidate in biomarker screening for improved prognosis.
Fatty liver diseases are becoming a common cause of chronic liver diseases in the Western countries encountering in about 20% of the general adult and child population[1-5]. Excessive accumulation of triglycerides in hepatocytes occurring in etiologically diverse conditions causes hepatic steatosis characterized by more than 5%-10% fat stored either in macrovesicles or in microvesicles of hepatocytes[6,7]. Whereas in the past, regular and excess alcohol consumption was the most common reason for hepatic steatosis[8,9], fatty liver diseases are now most frequently associated with obesity, insulin resistance and type 2 diabetes due to an unbalanced and rich diet in industrial nations[3,5].
The spectrum of fatty liver diseases (FLD) independent of causative agents ranges from simple steatosis to steatohepatitis, which can progress to liver fibrosis ending up in cirrhosis or hepatocellular carcinoma[3,5,10-13]. Thus, 5%-10% of non-alcoholic fatty liver disease (NAFLD) patients with steatosis develop a steatohepatitis accompanied by a high risk of progression to fibrosis[3,4]. Although fatty liver diseases can have various causes, features of steatohepatitis in NAFLD and alcoholic fatty liver disease (AFLD) are difficult to distinguish histologically[6,7]. Both are characterized by foci of liver cell necrosis and lobular inflammatory infiltrates with polymorphonuclear leukocytes. Furthermore, the onset of steatohepatitis is accompanied by ballooned hepatocytes, often harboring Mallory’s hyaline and megamitochondria or undergoing apoptosis. Whereas steatosis seems to be more pronounced in non-alcoholic steatohepatitis (NASH) than in alcoholic steatohepatitis (ASH), features of necroinflammatory and cholestatic activity are more prominent in ASH liver biopsies. Progression of steatohepatitis then results in pericellular fibrosis[6,7,15] involving myofibroblastic activation of sinusoidal hepatic stellate cells responsible for elevated extracellular matrix deposition.
Members of the peroxisome proliferator-activated receptors (PPAR) seem to play a key role in the pathophysiology of FLD by modulating increased glucose uptake and hepatic triglyceride accumulation, but also perform anti-inflammatory signals when steatohepatitis has occurred[17-19]. The PPAR family consists of PPARα, PPARγ, and PPARδ nuclear receptors, functioning as transcription factors, that mediate transcriptional response to insulin resulting in glucose uptake, increased fatty acid oxidation, lipogenesis and lipid storage, respectively. Whereas the PPARα is highly present in hepatocytes, the splice variants PPARγ1 and 2 triggering adipogenesis are mainly expressed in adipose tissues and only to a minor extent in the liver. PPARγ increases the expression of genes that promote fatty acid storage, whereas it represses genes that induce lipolysis in adipocytes. In patients suffering from FLD, hepatic expression of PPARγ is shown to be involved in insulin sensitivity, triglyceride clearance and hepatic steatosis.
Due to its high impact as an insulin-sensitising transcription factor involved in adipogenesis and lipogenesis, the occurrence of single nucleotide polymorphisms (SNP) in the PPARγ gene was recently addressed by numerous reports studying subjects with insulin resistance, type 2 diabetes, arteriosclerosis, and hypertension[21-23]. A prevalent SNP association with impaired lipid homeostasis was observed in terms of the N-terminal proline alanine exchange (Pro12Ala) of the extra domain in the PPARγ2 variant. This PPARγ splice form includes 30 additional amino acids, which are responsible for a 5-6-fold increase of PPARγ’s transcriptional activity. The Pro12Ala exchange in the activating extra region of the PPARγ2 is the result of a cytosine to guanine substitution in the PPARγ gene, as a consequence encoding the Ala-allele form with a heavily reduced function. In several populations, the association of the Pro12Ala polymorphism with insulin-sensitivity, type 2 diabetes, obesity and adipositas have been shown[25-27]. However, the role of the Pro12Ala polymorphism of PPARγ gene in occurrence and progression of fatty liver diseases is not yet defined.
In the present study, we analyzed the frequency of the Pro12Ala polymorphism in the PPARγ gene by a highly sensitive LNA-probe based polymerase chain reaction (PCR) approach in a total of 622 subjects of a Caucasian population, suffering from fatty liver disease (n = 359) or being healthy blood donors (n = 263). In agreement with reports showing a high Ala allele prevalence in patients with impaired lipid metabolism in obese and adipose patients[26,28], in FLD patients the Ala allele also occurs more often than in the healthy control group. Interestingly, the interpretation and linkage of the allele frequency to histological evaluation and clinical data demonstrates a prominent risk in AFLD patients bearing the Ala allele to develop severe steatohepatitis and fibrosis. Furthermore, our data revealed for the first time a higher anti-inflammatory impact of PPARγ in progression of human AFLD than NAFLD.
From a total of 622 cases, 259 blood samples and 363 biopsies were studied for occurrence of the Pro12Ala exchange in the PPARγ gene. Local research ethics guidelines were followed. We collected 363 cases from the files of the Department of Gastroenterology and Hepatology, University Hospital of Essen (GER) and the Institute for Pathology, University Hospital of Cologne (GER) according to their histological criteria of fatty liver disease (Table (Table1).1). 263 tissue specimens from patients were classified as NAFLD according to the clinical information about alcohol consumption (less than 20 g alcohol per day). One hundred specimens of patients who consume more than 20 g alcohol per day met the definition of AFLD as described by Neuschwander-Tetri et al. Clinical data, such as GOT, GPT, and γGT, were compiled along with the state of diabetes. There was no appreciable difference between the mean age of AFLD (53.93 ± 10.63, range 20-81 years) and NAFLD (50.48 ± 15.25, range 16-80 years). All specimens, stained with haematoxylin and eosin (HE) and by the Gomori method for visualization of reticular fibers, were independently classified by three experienced liver pathologists (Hardt A, Drebber U, Dienes HP), according to the histological score described by Kleiner et al (Table (Table1).1). Additionally, 259 DNA extracts from blood samples of healthy blood donors were taken as references for local gene distribution.
Extraction of DNA from 363 formalin fixed and paraffin embedded (FFPE) biopsies was performed from three 7 μm-microtome sections after deparaffinization and proteinase K treatment, as previously described. Then, DNA was purified by means of magnetic bead technology (FormaPure™ Kit of Agentcourt, Beverly MA, USA). All DNA purification steps were carried out by the BioMek FX laboratory automatic workstation (Beckman Coulter, USA) according to the work file and recommendations of Agentcourt.
Furthermore, DNA from 259 blood samples was prepared by the robotic workstation using the Genfind™ Kit of Agentcourt according to the manufacturer’s instructions.
For construction of a reference system with sequences of the Pro12Ala locus (rs1805192) carrying either the mutation or the wild type sequence of PPARγ2 gene we used oligonucleotides comprising the proline or alanine encoding sequences (Figure (Figure1).1). These oligonucleotides were dimerized in 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L Tris pH 7.4, creating EcoRI and SpeI compatible overhangs and inserted into pBluescript SKII (+) (Stratagene, Texas, USA) by the respective restriction sites (Figure (Figure11).
The Pro12Ala exchange of the PPARγ gene (rs1801282) was examined in DNA from normal blood donors and patients by a Taqman probe associated real-time PCR. Genomic DNA was amplified by real-time PCR in a total volume of 10 μL using the Eppendorf MasterMix (Hamburg, Germany) and 0.3 μL of each primer (10 μmol/L). The Plas A and Plas S primer set was used for amplification of reference plasmids; the PPARγ A and PPARγ mis primer set was used for genomic DNA samples (Table (Table22).
Allelic discrimination was achieved by adding 0.4 μL of 2.5 μmol/L LNA probes (TIB Molbiol, Berlin, Germany) recognizing the wild type and the mutant variant of the Pro12Ala locus of the PPARγ2 gene (Table (Table2).2). In parallel to the allelic Pro12Ala discrimination, plasmid reference sequences diluted from 105 to 10 copies in herring’s sperm DNA (1 ng/mL) were applied to all assays as positive controls. Amplification and analyses were accomplished by the following cycling conditions using a MX3000P qPCR System of Stratagene (Texas, USA): initial denaturation at 95°C for 2 min, following 50 cycles 95°C for denaturation, 55°C for annealing, 65°C for extension, each step lasting 20 s.
Pro12Ala distribution was evaluated using the SPSS software 17 of IBM® (Chicago, USA). Significance of cross-classification was calculated by the Fisher’s exact test. Odds ratios were used to describe the risk of disease progression.
Prominent occurence of the PPARγ alanine variant in patients with fatty liver disease
In order to detect the Ala12Pro polymorphism in patients with fatty liver disease we established an assay using locked nucleotide acid (LNA) probes for allelic discrimination. For this purpose, both variants of the Ala12Pro locus were cloned (Figure (Figure1A)1A) and the sequences, encoding either the proline or the alanine variant, were used as reference sequences for efficient allele detection and discrimination. The LNA probe hybridization assay linked to real-time PCR efficiently detected both, the proline wild type and the alanine mutated variant of the PPARγ gene (Figure (Figure1B).1B). Up to 10 copies of each reference sequence were successfully proven. In addition to the high sensitivity, application of the reference sequences attested that the LNA probe based PCR assay was highly specific, enabling the differentiation of the alanine and the proline encoding sequence.
In the 259 healthy blood donors the assay accounted for 1.5% homozygous variants carrying the alanine encoding sequence (Figure (Figure2).2). Analyses of the Pro12Ala distribution in the collective of patients with fatty liver disease (n = 363) revealed an increased incidence of the alanine mutant (3%) compared to the healthy population (1.5%). However, the difference was not statistically significant.
In order to identify the association of enhanced alanine occurrence, clinical data of patients with fatty liver disease were considered, revealing that 263 patients suffered from NAFLD, whereas fatty liver disease was linked to alcohol consumption in 100 cases (Table (Table3).3). In both cohorts, 12%-14% of the subjects were diabetes positive.
Furthermore, we analyzed the transaminase values in the two cohorts, showing a GPT/GOT ratio of 2 in NAFLD patients, but a GPT/GOT ratio of 1 in sera of AFLD patients (Figure (Figure3).3). The high GPT values in NAFLD patients are in accordance with numerous reports, characterizing the progress of steatohepatitis due to non-alcoholic steatosis in comparison to alcoholic steatosis. With respect to the distribution of the Pro12 and the Ala12 alleles in these two cohorts, we found that in both the AFLD and the NAFLD collective the frequency of the alanine genotype was higher (about 3%) compared to the healthy population (Figure (Figure22).
We next addressed the question whether the elevated incidence of the alanine allele in the population of fatty liver diseases is associated with the grade of steatosis, ballooning, steatohepatitis, or liver fibrosis (Table (Table44).
The degree of steatosis is traditionally classified into mild (< 30%), moderate (30%-60%), and severe (> 60%). More than 50% of the patients with NAFLD and AFLD had developed severe steatosis, however, in patients carrying the alanine allele severe steatosis occurred slightly more often. Pronounced ballooning was observed likewise often in NAFLD patients with the mutant or the wild type isoform of the PPARγ2 (Table (Table55).
Histological scoring for inflammatory alterations and fibrosis according to the recommendations described by Kleiner et al (Table (Table1)1) revealed that in most of the patients steatosis was accompanied by moderate, mild or severe steatohepatitis. In particular, in patients suffering from AFLD steatohepatitis has passed over to fibrosis in 71% of the cases.
Whereas in NAFLD patients inflammation was not significantly associated with the allelic incidence, in AFLD patients the frequency of the Ala12 variant of the PPARγ2 gene was significantly increased when prominent inflammation had occurred (P = 0.028). The higher risk of AFLD patients developing several inflammatory processes ending in liver fibrosis was also shown by elevated Odds ratios (Odd inflammation = 2.50, CI: 1.05-5.90 and Odd fibrosis = 2.48, CI: 0.81-7.53) (Table (Table66).
FLD has a high incidence of approximately 20% worldwide and is regarded as a major cause of liver-related morbidity and mortality due to its risk of progression into cirrhosis or hepatocellular carcinoma. Since the transcription factor PPARγ as been shown to be markedly involved in adipogenesis, hepatic lipid storage and metabolism, we first analyzed the frequency of the Pro12Ala polymorphism of the PPARγ gene in a German cohort of patients with FLD compared to German healthy blood donors. A highly sensitive and robust test was established which was certain to distinguish the Ala and the Pro alleles, even though only low copy numbers from some FFPE biopsies might be available. The genotype distribution (77.5% wt, 21.2% heterozygous, and 1.5% homozygous Ala/Ala mutants) in the collective of the healthy blood donors resembles previous data collected on more than 600 Caucasians by Yen et al and Ghoussaini et al. This genotype distribution in Caucasians, however, differs from the Asian or African frequency, in which less Ala alleles of the PPARγ occur[23,32]. In contrast to the data of healthy blood donors, PCR analyses of DNA from subjects with FLD revealed an increased frequency of the homozygous Ala-subtype up to almost 3.5% in both the AFLD and the NAFLD collectives. Recent meta-analyses summarized data of the Pro12Ala polymorphism in patients with diabetes and identified the mutated Ala variant as a protection factor of diabetes type 2[34-36]. The malfunctioning Ala variant was also shown to be associated with coronary heart disease and with obesity indicated by significant higher BMI in homozygous Ala carriers than in subjects expressing the heterozygous or the wild type PPARγ2 form[38,39]. Although in some reports higher insulin sensitivity and BMI could not be confirmed[40,41], a comprehensive study on 1170 British patients with coronary heart disease and a meta-analysis including 19 136 subjects clearly identified the Ala carriers as individuals with significant higher BMI. Additionally, cholesterol, LDL-cholesterol and apolipoprotein B concentrations are elevated in Ala carriers[42,44]. Therefore, this Ala-associated hyperlipidemia is assumed to be a reason for the 2-fold higher incidence of the Ala genotype in patients with FLD compared to healthy blood donors. Since free fatty acids are shown to be involved in upregulation of Fas/CD-95 death receptor, enhanced levels of circulating fatty acids due to impaired PPARγ function in Ala/Ala patients may result in apoptosis and inflammatory processes. In contrast to NAFLD, where no or only a moderate link of inflammatory progression to the Pro12Ala polymorphism was shown, a prominent risk of developing steatohepatitis was observed in AFLD patients carrying the Ala allele. This difference in the associated frequency of the Ala variant encoding the minor active PPARγ2 form argues for a divergent role of the PPARγ2 in mechanisms of AFLD and NAFLD progression. The PPARγ2 isoform is upregulated by phosphatidylinositol 3-kinase activation in response to free fatty acids or by insulin. In post-ischemic liver injury and also in alcohol-induced fibrosis, the PPARγ1 andγ2 variants were shown to be downregulated and to function protectively[47,48]. Therefore, the 2-3-fold higher risk of ALFD patients, but not of NAFLD patients, to develop inflammatory and fibrotic progression if they carry the malfunctioning Ala variant of PPARγ2, emphasizes a more prominent anti-inflammatory impact of the PPARγ2 in AFLD than in NAFLD wt-carriers.
The anti-inflammatory action of PPARγ was also demonstrated by previous studies on hepatic stellate cells[49,50], which take centre stage of sinusoidal liver fibrosis due to their tremendous matrix production and secretion of pro-inflammatory and pro-fibrotic mediators after myofibroblastic transition in chronic liver injury. The authors show that PPARγs repressed in myofibroblastic hepatic stellate cells. Additionally, the inflammatory chemokine expression by hepatic stellate cells is markedly inhibited in response to the activation of PPARγ by the agonistic ligand glitazone[49,50]. Taken into account that the malfunctioning Ala variant is associated with a higher risk of progression into steatohepatitis in AFLD patients these results lead to the suggestion that in particular inflammation and fibrosis of AFLD wt-patients can be attenuated by a treatment with PPARγ thiazolidinedione ligands such as rosiglitazone and pioglitazone. Patients with NASH, however, may benefit from glitazone therapy by other mechanisms like improved insulin sensitivity, decreased hyperlipidemia and impeded steatosis as a result of the therapeutic approach[51,52].
In conclusion, our data of a comprehensive study of the Pro12Ala polymorphism on biopsies with FLD, well classified concerning inflammatory and fibrotic alterations, revealed for the first time an association of the Pro12Ala polymorphism with the risk of developing ASH and suggests a more prominent anti-inflammatory influence of the PPARγ2 on progression of human AFLD than on NAFLD. Therefore, the Pro12Ala polymorphism should be studied on an expanded cohort of AFLD patients, in order to be later integrated in a panel of genetic markers applied for future improved prognosis of disease progression.
Fatty liver diseases are becoming a common cause of chronic liver diseases in the Western countries encountered in about 20% of the general adult and child population. The spectrum of fatty liver diseases (FLD) independently of causative agents ranges from simple steatosis to steatohepatitis, which can progress to liver fibrosis ending up in cirrhosis or hepatocellular carcinoma. Members of the peroxisome proliferator-activated receptors (PPAR) seem to play a key role in the pathophysiology of FLD by modulating increased glucose uptake and hepatic triglyceride accumulation, but also perform anti-inflammatory signals when steatohepatitis has occurred. Since the transcription factor PPARγ has been shown to be markedly involved in adipogenesis, hepatic lipid storage and metabolism, we analyzed the frequency of the Pro12Ala polymorphism of the PPARγ gene in a German cohort of patients with FLD compared to German healthy blood donors.
Due to its high impact as an insulin-sensitising transcription factor involved in adipogenesis and lipogenesis, the occurrence of single nucleotide polymorphisms (SNP) in the PPARγ gene was recently addressed by numerous reports studying subjects with insulin resistance, type 2 diabetes, arteriosclerosis, and hypertension. A prevalent SNP association with impaired lipid homeostasis was observed in terms of the N-terminal proline alanine exchange (Pro12Ala) of the extra domain in the PPARγ2 variant. The authors analyzed the frequency of the Pro12Ala polymorphism in the PPARγ gene by a highly sensitive LNA-probe based polymerase chain reaction approach in a total of 622 subjects of a Caucasian population, suffering from fatty liver disease (n = 359) or healthy blood donors (n = 263). In agreement with reports showing a high Ala allele prevalence in patients with impaired lipid metabolism in obese and adipose patient, the Ala allele also occurs more often in FLD patients than in the healthy control group. Interestingly, the interpretation and linkage of the allele frequency to histological evaluation and clinical data demonstrates a prominent risk in alcoholic fatty liver disease (AFLD) patients bearing the Ala allele to develop severe steatohepatitis and fibrosis.
In order to detect the Ala12Pro polymorphism in patients with fatty liver disease the authors established an assay using locked nucleotide acid probes for allelic discrimination. The authors' data of a comprehensive study of the Pro12Ala polymorphism on biopsies with FLD, well classified concerning inflammatory and fibrotic alterations, revealed in patients with AFLD, but not with non-alcoholic fatty liver disease (NAFLD), an significant association of the Pro12Ala polymorphism with the risk to develop steatohepatitits. Therefore, PPARγ is suggested to exert a higher anti-inflammatory impact in progression of human AFLD than NAFLD.
The association of the Pro12Ala polymorphism with the risk of developing inflammatory progression in patients with AFLD suggests a more prominent influence of PPARγ2 on progression of human AFLD than on NAFLD. Therefore, the Pro12Ala polymorphism should be studied on an expanded cohort of AFLD patients, in order to be later integrated in a panel of genetic markers applied for future improved prognosis of disease progression and therapy planning.
This review article provides an overview of SLE-related gastrointestinal system involvements, and there are only few review article in the international literatures in recent years.
We thank Dr. Kasper HU (Institute for Pathology, Medical Centre Muenster, Germany) for his support in histopathological evaluation of the AFLD biopsies and part of the NAFLD biopsies. Furthermore, we thank Melanie Ruhrlaender and Katharina Wendland for excellent technical assistance.
Supported by A grant of Marga and Walter Boll foundation
Peer reviewer: Teng-Yu Lee, MD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Taichung Veterans General Hospital, 160, Sec. 3, Taichung Harbor Road, Taichung 407, Taiwan, China
S- Editor Wang YR L- Editor O’Neill M E- Editor Zheng XM