Funkční varianty genů pro metaloproteinázy MMP‑ 1 a MMP‑ 7 nesouvisejí se závažností portální hypertenze u pacientů s cirhózou
Cirhóza je konečné stadium chronického onemocnění jater. Vysoká morbidita a mortalita souvisejí zejména s komplikacemi vyplývajícími z portální hypertenze. Remodelace fibrotické tkáně prostřednictvím matrixových metaloproteináz (MMP) je permanentní proces, který u pacientů s cirhózou vede k cirhotické přestavbě. Cílem studie bylo posoudit význam funkčních variant genů pro metalproteinázy MMP‑1 a MMP‑7 u pacientů s cirhózou a posoudit jejich souvislost s portální hypertenzí.
Pacienti a metody: 179 pacientů s jaterní cirhózou (střední věk 55,2 ± 11,6 let) bylo vyšetřeno na přítomnost funkčních variant genů pro MMP‑1 (- A; rs1799750) a MMP‑7 (G/ A; rs17884789). U všech pacientů bylo provedeno měření gradientu tlaku v jaterních žilách, laboratorní a ultrazvukové vyšetření. Pro porovnání frekvence výskytu alel s pacienty s cirhózou zařazenými do studie byla pro zdravou populaci použita data z odborné literatury.
Výsledky: Frekvence výskytu genotypů rs1799750 (1G‑ 1G 25 %, 1G‑ 2G 56 %, 2G‑ 2G 22 %) u pacientů s cirhózou jater a u zdravé populace se neliší (p > 0,05). V případě genu pro MMP‑7, byl u našich pacientů pro variantu rs17884789 zjištěn jeden genotyp (wild typ GG). Nebyla zjištěna souvislost mezi frekvencí výskytu zkoumaných genotypů a závažností portální hypertenze nebo hodnotou Child‑ Pughova nebo MELD skóre.
Závěr: Funkční varianty MMP‑1 a MMP‑7 nemají souvislost s portální hypertenzí u pacientů s cirhózou jater.
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Doručeno: 11. 2. 2015
Přijato: 16. 3. 2015
R. Brůha 1; M. Jáchymová 2; K. Dvorak 1; J. Petrtýl 1; J. Smalcova 1; P. Urbánek 3; T. Svestka 1; L. Vítek 1,2
Authors place of work:
th Department of Internal Medicine, 1st Faculty of Medicine, Charles University in Prague
14; Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague
2; Department of Internal Medicine, Central Military Hospital, Prague
Published in the journal:
Gastroent Hepatol 2015; 69(2): 116-120
Hepatologie: původní práce
Cirrhosis is a final stage of chronic liver disease. High morbidity and mortality are mostly related to complications of portal hypertension. Remodelling of liver fibrotic tissue by matrix metalloproteinases (MMPs) is a permanent process leading to cirrhotic scar formation. The aim was to study the significance of functional metalloproteinase MMP‑1 and MMP‑7 gene variants in cirrhotic patients and their relationship to portal hypertension.
Patients and Methods: 179 patients with liver cirrhosis (mean age 55.2 ± 11.6 years) were examined for functional variants of MMP‑1 (- A; rs1799750) and MMP‑7 (G/ A; rs17884789) genes. Measurement of hepatic venous pressure gradient, laboratory and ultrasound examination were performed in all patients. Literary data from a healthy population were used for comparison of allele frequencies with our cirrhotic patients.
Results: The frequency of rs1799750 (1G‑ 1G 25%, 1G‑ 2G 56%, 2G‑ 2G 22%) genotypes in cirrhotic patients does not differ from the healthy population (p > 0.05). In MMP‑7 gene, one genotype (wild type GG) was found in our patients uniformly for rs17884789 variant. No relationship was found between the frequency of examined genotypes and either the severity of portal hypertension or Child‑ Pugh or MELD score.
Conclusions: Functional variants of MMP‑1 and MMP‑7 have no relationship to portal hypertension in liver cirrhosis.
Liver cirrhosis is a final stage of different chronic liver diseases, associated with high morbidity and mortality. Major complications of cirrhosis such as bleeding from oesophageal varices, ascites, and hepatic encephalopathy are related to portal hypertension . Fibrosis and scarring of liver parenchyma leading to cirrhosis is a continuous process controlled by many factors. With chronic liver disease the extracellular matrix undergoes a process of remodelling, leading to new collagen formation and deposition. The extracellular matrix in chronic liver disease consists of type I and type III collagen . Tissue remodelling is regulated by molecular mechanisms, involving proteases, inhibitors and growth factors. The major role in extracellular matrix (ECM) degradation is played by matrix metalloproteinases (MMPs), a class of zinc and calcium‑ dependent enzymes, and their tissue inhibitors (TIMPs) . The three most relevant MMPs are gelatinase A (MMP‑ 2), gelatinase B (MMP‑ 9), and stromelysin (MMP‑ 3), involved in the normal liver matrix degradation under physiological condition. In fibrotic liver there is net deposition of fibrillar matrix that is comprised predominantly of the interstitial collagens, types I and III. These triple helical molecules are relatively resistant to protease activity but are cleaved by interstitial collagenase MMP‑1 . Another important collagenase is MMP‑7, which has been attributed to liver fibrosis under various pathological conditions [5,6], including cirrhosis and hepatocellular carcinoma .
The MMPs genes are located on chromosome 11q22 . The gene for MMP‑1 (which degrades fibrillar collagen types I and III) carries a promoter dimorphism at position 21607, where either one (1G allele) or two (2G allele) guanines are detected. The importance of this functional variant in liver cirrhosis is not known. The single nucleotide polymorphism (SNP) in the MMP‑7 gene (Gly‑ 137 3 Asp‑ 137 [G137D]) was described to be susceptible to liver cirrhosis . The relationship to portal hypertension is not known.
The aim was to study the significance of functional metalloproteinase MMP‑1 and MMP‑7 gene variants in cirrhotic patients and their relationship to portal hypertension.
Material and methods
A group of 179 patients with proven liver cirrhosis, indicated for invasive haemodynamic measurement of portal hypertension, was included in the study. Liver cirrhosis was diagnosed on the basis of biochemical tests, clinical and ultrasound findings and/ or liver biopsy, and the presence of portal hypertension diagnosed by hepatic vein catheterisation.
Hepatic vein catheterisation and a hepatic venous pressure gradient (HVPG) measurement were performed in all patients. The clinical data of patients with liver cirrhosis are given in tab. 1. The data for comparison of allele frequencies in our group of cirrhotic patients with healthy controls were obtained from literature [7,9].
The study was approved by the local Ethics Committee, and the protocol conformed to the latest ethical guidelines of the Declaration of Helsinki. Informed consent was obtained from all subjects.
Measurement of HVPG
Measurement of HVPG was performed using the classical wedged technique . Shortly after overnight fasting, patients were transported to the catheterisation room in supine position. Under local anaesthesia, a 7F catheter introducer was placed in the right jugular vein using the Seldinger technique. Under fluoroscopic control, a 7F balloon‑ tipped catheter (B. Braun Melsungen AG, Germany) was advanced into the right hepatic vein in order to measure both the free hepatic venous pressure and the wedged hepatic venous pressure. All measurements were performed in triplicate. HVPG was calculated as the difference between the wedged hepatic venous pressure and free hepatic venous pressure.
Biochemical and haematology examinations were performed by routine techniques on automatic analysers. The severity of liver disease was evaluated by Child‑ Pugh and MELD score.
Genotyping of the MMP
The functional variants of MMP‑1 (– 1607 bp; – A; rs1799750) and MMP‑7 (Gly137Asp; rs17884789) genes were examined by standard PCR technique. Genomic DNA was isolated from peripheral blood leucocytes and the polymorphic sites of MMP‑1/ MMP‑7 genes were detected using both the polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism technique.
The data are expressed as the mean ± ± SD. Allele frequency was evaluated by the Chi‑ square test. Either a two‑-sample t‑test or the Mann‑Whitney rank test for non‑Gaussian distributed variables was used to estimate the intergroup differences. One Way Analysis of Variance or Kruskal‑ Wallis One Way Analysis of Variance on Ranks were used to estimate the differences between more than 2 groups. A p value of ≤ 0.05 was required for statistical significance. The statistics were computed using STATISTICA CZ v. 8 (StatSoft, Prague, Czech Republic).
Characteristics of patients
One hundred and seventy nine patients were included in the study: 126 males and 53 females. No differences in the age, biochemical parameters and/ or the aetiology and stage of cirrhosis were present between both gender groups (data not shown). The clinical and laboratory parameters of all patients are given in tab. 1. Hepatocellular carcinoma developed in eight patients from our group.
Genotyping, comparison to controls and relationship to portal hypertension and liver dysfunction
The frequencies of MMP‑1 gene variations are presented in tab. 2. No statistical differences between the cirrhotic patients and literary controls regarding the frequencies of analysed genotypes were found. Female cirrhotic patients had lower frequency of 1G‑ 1G genotype compared to male patients with liver cirrhosis (16% vs 25%) with borderline statistical significance (p = 0.04). No differences between clinical/ laboratory parameters were found in different genotype in cirrhotic patients (tab. 3); there were no relationships between different genotypes and either parameters of liver dysfunction, or portal hypertension. The distribution of 1G‑ 1G, 1G‑ 2G and 2G‑ 2G did not differ between Child‑ Pugh classes A, B and C. No relationship between above mentioned genotypes and MELD score was found.
In MMP‑7 gene, only one genotype was found in all patients uniformly for rs17884789 variant (wild type GG).
Liver fibrosis is a pathological process involving many cellular and molecular changes and leading to the deposition of excess matrix proteins in the extracellular space. When combined with ineffective regeneration and repair, increased distortion of the normal liver architecture results in cirrhosis. Liver fibrosis is a dynamic and bidirectional process, involving phases of progression and regression. In addition to increased matrix synthesis, the process of cirrhosis formation involves major changes in the regulation of matrix degradation.
In the extracellular space, matrix degradation occurs under physiological conditions as a consequence of the action of a family of enzymes called the matrix metalloproteinases (MMPs). These are secreted from cells into the extracellular space as proenzymes, which are then activated by a number of specific, usually cell surface‑associated cleavage mechanisms. The active enzymes are in turn inhibited by a family of tissue inhibitors of metalloproteinases (TIMP‑ 1 to - 4). By this combination of mechanisms, extracellular matrix degradation is closely regulated, which prevents inadvertent tissue damage . Specific MMPs and TIMPs are expressed on stellate cells, but the source of MMP1, which plays a crucial role in degrading the excess interstitial matrix in advanced liver disease, is still uncertain . The major producers of matrix metalloproteinase‑ 7 in the liver are the bile ductular epithelial cells, Kupffer cells and hepatocytes, as demonstrated by in situ hybridisation . Huang et al. suggested that MMP‑7 is a major metalloprotease associated with the tissue remodelling during the progression of liver fibrosis, particularly in biliary atresia .
There is abundant evidence that fibrosis and even cirrhosis can be reversible under various clinical situations [12,13]. The crucial underlying mechanism of cirrhosis reversibility remains unclear. One factor that may prevent reversibility is reduced expression of enzymes that degrade matrix or sustained elevation of proteins that inhibit the function of these degradative enzymes .
Recently, naturally occurring sequence variation has been detected in the promoter of a number of MMP genes. These functional variants have been shown to have allele‑ specific effects on the transcriptional activities of MMP gene promoters, and to be associated with susceptibility to many diseases like coronary heart disease, aneurysms, chronic pancreatitis and cancers. Significant association of the MMP‑1 - 1607 1G/ 2G (rs1799750) gene promoter polymorphism with chronic pancreatitis has been described, and it has been suggested that it could be considered a biological marker in the aetiology of chronic pancreatitis . These findings indicate that variation in the MMP genes may contribute to inter‑ individual differences in susceptibility to these common, complex diseases, likely through effects on the balance between the synthesis and degradation of extracellular matrix proteins . Regarding liver diseases most important are MMP‑1 and MMP‑7. The MMP‑1 gene carries a promoter dimorphism at position 21607, where either one (1G allele) or two (2G allele) guanines are found. The 2G allele is associated with higher gene expression than the 1G allele .
All these data raises the question, whether different expression of these enzymes caused by specific gene variation could influence the course of liver fibrosis and cirrhosis. Based on our data the answer is clear – there is no association between the gene variants for MMP‑1 and MMP‑7 and liver cirrhosis, liver dysfunction or portal hypertension. This is partly in concordance with literature data, which are controversial. For example, Wiencke et al. studied Norwegian patients with primary sclerosing cholangitis and found no general associations of the MMP‑1 gene to liver disease . Nevertheless, they described that specific alleles were associated to subsets of PSC patients with cholangiocarcinoma.
The relationship of different MMPs gene variants and carcinomas seems to be close, as MMP single‑nucleotide polymorphisms influence breast cancer behaviour and the MMP‑1 2G/ 2G genotype increases the risk of lymph node metastasis and predicts poor prognosis . Based on meta‑analysis it seems that the polymorphism MMP1 – 1607 1G > 2G is significantly associated with a significantly increased risk of cancers like colorectal cancer and lung cancer . In cirrhosis an association between MMP‑7 functional variants and hepatocellular carcinoma has been described . Unfortunately our data do not provide any meaningful conclusion regarding the relationship between hepatocellular carcinoma and MMPs gene variants, as only a small number of patients with carcinoma has been detected (eight patients).
In our study no relationship between liver dysfunction and MMPs gene variants was found. There may be other factors involved, because in the study by Leeming et al.  the severity of portal hypertension and liver dysfunction correlated with extracellular matrix serological biomarkers. The authors even suggested that the combination of ECM serological markers with clinical scores may aid in the detection of the level of portal hypertension and the degree of liver dysfunction.
Hepatic mRNA expression of MMPs was studied also during the course of chronic hepatitis C, showing either steadily increasing mRNA expression with disease progression (MMP‑1, -2, -7 and -14). The MMPs expression closely correlated to the parameters of fibrogenesis . Unfortunately no data regarding the eventual gene variants are given in that paper.
Another issue is the gender difference. In our patients we found borderline differences in allele frequency for MMP‑1 gene variant between male and female. This observation has probably, in our opinion, no clinical relevance.
Limitation of our study is the absence of our own control population. Nevertheless the data from literature used for the comparison of cirrhotic patients and controls are highly relevant, originating from a Caucasian population [7,9].
In conclusion, our study demonstrates that the presence of functional variants in the gene for metalloproteases MMP‑1 (rs1799750) and MMP‑7 (rs17884789) do not carry any increased risk of liver cirrhosis. No relationship between different genotypes and portal hypertension or liver dysfunction was found. These data could probably indicate that the fibrotic process is too complicated to be affected by one single parameter.
Supported by Grant NT 12290/4 given by Internal Grant Agency, Czech Ministry of Health (IGA MZCR, http://iga.mzcr.cz/publicWeb/).
The authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study.
The Editorial Board declares that the manuscript met the ICMJE „uniform requirements" for biomedical papers.
Submitted: 11. 2. 2015
Accepted: 16. 3. 2015
Radan Bruha, MD, PhD
4th Department of Internal Medicine
General University Hospital
U Nemocnice 2, 128 08 Prague 2
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Gastroenterologie a hepatologie