|Year : 2017 | Volume
| Issue : 1 | Page : 133-138
Growth differentiation factor 15 as a marker of ineffective erythropoiesis in patients with chronic C virus infection
Omaima M Abbas1, Mohamed A Helwa MD 2, Ashraf Y El Fert3, Iman S Osheba1
1 Department of Clinical Pathology, National Liver Institute, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Medical Biochemistry, National Liver Institute, Menoufia, Egypt
|Date of Submission||26-Dec-2015|
|Date of Acceptance||07-Mar-2016|
|Date of Web Publication||25-Jul-2017|
Mohamed A Helwa
Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebein El kom, Menoufia, 32511
Source of Support: None, Conflict of Interest: None
The aim of this study was to determine the expression of growth differentiation factor 15 (GDF-15) in patients with chronic hepatitis C virus and its association with iron-loading anemia in these patients.
Significant shortened red cell survival has been reported in patients with liver diseases even in the absence of anemia. The mechanism for the observed decreased red cell lifespan is not fully understood, and it is most probably multifactorial. GDF-15, a member of the transforming growth factor-β super family, was first cloned from human monocytoid cell line and was found to inhibit tumor necrotizing factor production by macrophages and negatively regulate hepcidin. Recent studies suggest that GDF-15 levels increase in conditions of cell stress and apoptosis. In bone marrow (BM), GDF-15 increases in case of ineffective erythropoiesis.
Patients and methods
This study was carried out at Clinical Pathology Department, Faculty of Medicine and National Liver Institute, Menoufia University, in the period from May 2013 to May 2015. The study included 70 individuals: 50 patients with compensated chronic hepatitis C with unexplained anemia and 10 patients with idiopathic thrombocytopenic purpura who had normal red blood cell counts and no signs of erythroid dysplasia, as a control group for BM expression of GDF-15. In addition, 10 unrelated healthy age-matched and sex-matched adult individuals were included as a normal control group. Serum GDF-15 was measured by ELISA, and GDF-15 mRNA was measured by real-time RT-PCR.
There were significant differences between the different studied groups regarding iron profile, GDF-15 serum level, and BM expression of GDF-15 mRNA, being higher in patients with chronic hepatitis C than other groups. GDF-15 was positively correlated with serum iron indices and negatively correlated with hemoglobin concentration.
Ineffective erythropoiesis contributes to iron overload and anemia in patients with chronic hepatitis C.
Keywords: anemia of liver disease, chronic hepatitis C, growth differentiation factor 15
|How to cite this article:|
Abbas OM, Helwa MA, El Fert AY, Osheba IS. Growth differentiation factor 15 as a marker of ineffective erythropoiesis in patients with chronic C virus infection. Menoufia Med J 2017;30:133-8
|How to cite this URL:|
Abbas OM, Helwa MA, El Fert AY, Osheba IS. Growth differentiation factor 15 as a marker of ineffective erythropoiesis in patients with chronic C virus infection. Menoufia Med J [serial online] 2017 [cited 2019 Apr 19];30:133-8. Available from: http://www.mmj.eg.net/text.asp?2017/30/1/133/211514
| Introduction|| |
Anemia develops in ∼75% of patients with chronic liver disease. Significantly shortened red cell survival has been reported in patients with alcoholic liver disease, biliary cirrhosis, obstructive jaundice, and infectious hepatitis, even in the absence of anemia. The mechanism for the observed decrease in red cell lifespan is not fully understood and is most probably multifactorial .
In the last few years, several published reports have demonstrated an important relationship between chronic hepatitis C virus (HCV) infection and iron overload .
Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily and is expressed in nearly all tissues, suggesting its general importance in essential cellular functions . Under normal physiological conditions, GDF-15 expression is largely restricted to the placenta, but in disease states such as acute injury, inflammation, or cancer, its expression can be dramatically increased . GDF-15 expression in the serum may, therefore, be eventually used as a clinical marker of cell stress or apoptosis, and its measurements is helpful for predicting ineffective erythropoiesis . GDF-15 suppresses hepcidin leading to iron overload ,. The aim of this study was to study the association of GDF-15 with ineffective erythropoiesis and iron overload in patients with chronic HCV.
| Patients and Methods|| |
This case–control study was conducted on 50 treatment-naïve patients with compensated chronic HCV with unexplained anemia. Patients with any obvious cause of anemia such as bleeding, nutritional deficiency, vitamin B12 or folic acid deficiency, portal hypertension, alcoholic use, or treatment of chronic hepatitis C by interferon and/or ribavirin were excluded. Pregnant women and patients with hepatocellular carcinoma or chronic inflammatory diseases were also excluded from the study.
They were compared with 10 patients with idiopathic thrombocytopenic purpura (ITP) with normal peripheral red blood cell count and normal bone marrow (BM) erythroid cells as control group for all parameters. In addition, 10 apparently healthy individuals of comparable age and sex were included as a normal control group for all parameters other than BM examination and GDF-15 expression. A written consent was taken from all the participants, and the study was approved from the ethics committee, Faculty of Medicine, Menoufia University.
All individuals were subjected to the following:
- Clinical assessment including full history and clinical examination
- Abdominal ultrasonography, computed tomography scanning, or liver biopsy when possible
- Laboratory investigations: morning fasting venous blood samples were drawn under aseptic conditions and were collected into vacutainer tubes to perform the following:
- Complete blood count was done by Sysmex Counter K-1000 (catalogue number: A 5873; Wakinohama-kaigandori, Chuo-ku, Japan)
- Liver function tests were performed using Integra 400 Auto analyzer (catalogue number: M 87432; Germany)
- Iron profile: iron and Total iron binding capacity (TIBC) were measured using Integra 400 Auto analyzer. Ferritin levels were measured by electrochemiluminescenceimmunoassay, which is intended for use on Elecsys (Roche Diagnostics, USA) immunoassay analyzers
- Serum GDF-15 was evaluated using a Duo-Set ELISA (R and D Systems, Minneapolis, Minnesota, USA)
- GDF-15 expression in BM was evaluated by real-time RT-PCR. Total RNA was isolated using a commercially available kit (QIAamp RNA Blood Mini Kit; QIAGEN GmbH, Hilden, Germany). Quality, purity, and quantity of extracted RNA were verified before the downstream steps. Reverse transcription was performed by the Thermo Scientific Versoc DNA Synthesis Kit (Thermo Scientific, Carlsbad, California, USA) in one cycle at 42°C for 30 min followed by inactivation of residual RNA at 92°C for 2 min. Complementary DNA (cDNA) samples were stored at −20°C. cDNA of GDF-15 was amplified using the ABI 7500 real-time PCR (Applied Biosystems, Foster City, California, USA). For each sample, GDF-15 mRNA expression level was normalized to the level of GAPDH mRNA as housekeeping genes. The expression ratio was analyzed using the delta comparative cycle threshold method.The GDF-15 reaction mix consisted of 4-μl cDNA, 10-μl 2 × SYBR green master mix (Applied Biosystems), 4-μl nuclease-free distilled water, and 1.0-μl of each 20 × primer, including forward: 5′-CGGTGA ATGGCTCTCAGATG-3′ and reverse: 5′-CAGGTCCTCGTAGCGTTTCC-3′ (Metabion International AG, Martinsried, Germany) . Initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and elongation at 72°C for 1 min was done. For amplification of cDNA of GAPDH, we used primers, including forward: 5′-AGCCACATCGCTCAGACA C-3′ and reverse: 5′-GCCCAATACGAC CAA ATC C-3′ (Metabion International AG).
The data collected were tabulated and analyzed by SPSS (SPSS Inc., Chicago, Il, USA) statistical package version 11 on IBM compatible computer.
Quantitative data were expressed as mean and SD (X + SD) and analyzed by applying Mann–Whitney U-test to compare two groups with non-normally distributed variables. Kruskal–Wallis test was used to compare more than two groups of non-normally distributed variables. One-way analysis of variance test was used to compare more than two groups with normally distributed variables; in this case, post-hoc test was applied to indicate which groups were significantly different from which others.
Pearson's correlation (r) was used to study association between quantitative variables.
Results obtained from these tests were considered to be statistically different at P less than 0.05.
| Results|| |
Patients with HCV group had significantly higher aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, direct bilirubin, and γ-glutamyl transferase levels and lower albumin levels when compared with healthy and ITP groups [Table 1].
|Table 1 Comparison between the studied groups regarding liver profile tests|
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Significantly higher levels of serum iron [Figure 1], transferrin saturation [Figure 2], and ferritin were shown in patients with HCV group than healthy and ITP groups (P< 0.001). No statistically differences between the studied groups were found regarding TIBC (P > 0.05) [Table 2].
Regarding GDF-15 levels, significantly higher levels of serum GDF-15 were detected in patients with HCV group than healthy and ITP groups (P< 0.001) [Figure 3] and [Table 3] and significant higher levels of GDF-15 expression in BM were detected in patients with HCV group than patients with ITP group (P< 0.05) [Table 4].
|Figure 3: Serum growth differentiation factor 15 levels in different studied groups.|
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|Table 3 Comparison between the studied groups regarding serum growth differentiation factor 15|
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|Table 4 Comparison between patients with chronic hepatitis C and idiopathic thrombocytopenic purpura control regarding growth differentiation factor 15 expression in BM|
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Serum GDF-15 levels showed a negative correlation with hemoglobin levels and a positive correlation with serum iron [Figure 4], transferrin saturation [Figure 5], and ferritin [Table 5].
|Figure 4: Correlation between serum growth differentiation factor 15 level and serum iron level in patients with chronic hepatitis C virus.|
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|Figure 5: Correlation between serum growth differentiation factor 15 level and transferrin saturation in patients with chronic hepatitis C virus.|
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|Table 5 Pearson's correlation between serum growth differentiation factor 15 and other parameters in studies group|
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| Discussion|| |
GDF-15 is a negative regulator of hepcidin leading to iron overload ,. Its expression increases in conditions of cell stress and apoptosis. In BM, increased GDF-15 expression is a feature of increased apoptosis of erythroid precursors but not in the case of normal or increased erythropoiesis associated with erythroid recovery, and so it can be used for predicting ineffective erythropoiesis . High GDF-15 levels were seen in anemias characterized by ineffective erythropoiesis such as thalassemia  and refractory anemia with ringed sideroblasts . This study aimed to determine the role of GDF-15 in unexplained anemia and iron overload in patients with compensated chronic hepatitis C.
In the present study, regarding liver function tests, there were highly significant differences in AST, ALT, ALP, albumin and bilirubin levels between patients with chronic HCV and other groups. This is in agreement with Parsian et al.  who showed that the serum levels of AST and ALT in patient groups were significantly higher than the control group (P< 0.001). Moreover, these results agree with Alboraie et al. , who showed that there was a highly significant positive correlation between stage of fibrosis and bilirubin, AST, ALP, and γ-glutamyl transferase. Similarly, Demir et al.  found a positive correlation between fibrosis score and AST and ALT owing to presence of hepatocellular disorders in patients with liver fibrosis.
The present study revealed higher level of serum ferritin, iron, and transferrin saturation in patients with chronic HCV than control and ITP groups.
These results agree with Galal et al. , who reported significantly elevated serum iron indices in patients with chronic hepatitis C.
This was also in agreement with Harrison-Findik , who reported increased levels of serum iron, transferrin saturation, and ferritin in 20–35% of patients with chronic HCV associated with mild to moderate increase of hepatic iron load, predominantly in sinusoidal location.
Iron excess in patients with chronic hepatitis C is multifactorial. It may be because of associated hereditary hemochromatosis, hematological diseases, multiple transfusions, porphyria cutanea tarda, and chronic alcohol abuse .
In the present study, the mean level of serum GDF-15 concentration was significantly higher in patients with chronic hepatitis C than in healthy and ITP controls. This is in agreement with Si et al.  and Liu et al. . It was demonstrated that the expression of HCV structural proteins (core, E1 or E2) significantly induced GDF-15 expression . Hsiao et al.  showed upregulation of GDF-15 following multiple types of liver injury, so they hypothesized that GDF-15 may be involved in the regulation of the acute injury response in hepatocytes.
In this work, a positive correlation was present between serum GDF-15 levels and serum iron indices whereas a negative correlation was present between serum GDF-15 and hemoglobin concentration. Higher levels of GDF-15 expression were found in BM of patients with chronic HCV than ITP control group. This reflects a higher level of ineffective erythropoiesis in patients with chronic HCV because BM GDF-15 expression increases in patients with ineffective erythropoiesis but not in diseases associated with increased erythropoiesis without increased intramedullary apoptosis (e.g. hemolytic anemia) . Ineffective erythropoiesis and suppressed hepcidin secondary to high GDF-15 are involved in iron overload of patients with chronic HCV.
| Conclusion|| |
These results suggest that HCV viral infection increases the GDF-15 levels secondary to increased cell stress and apoptosis with subsequent iron overload. The high GDF-15 expression in BM of patients with chronic HCV suggests that ineffective erythropoiesis contributes to the unexplained anemia and iron overload in patients with compensated chronic hepatitis C.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Means RT. Jr. Anemias secondary to chronic disease and systemic disorders. In: Greer JP, Foerster J, Rodgers GM, et al
. editors. Wintrobe's clinical hematology
. Baltimore: Lippincott Williams & Wilkins; 2009. pp. 1221–1238.
Souza RM, Freitas LA, Lyra AC, Moraes CF, Braga EL, Lyra LG. Effect of iron overload on the severity of liver histologic alterations and on the response to interferon and ribavirin therapy of patients with hepatitis C infection. Braz J Med Biol Res 2006; 39:79–83.
Yatsuga S, Fujita Y, Ishii A, et al.
Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders. Ann Neurol 2015; 78:814–823
Tanno T, Noel P, Millera JL. Growth differentiation factor 15 in erythroid health and disease. Curr Opin Hematol 2010; 17:184–190
Tanno T, Bhanu NV, Oneal PA, et al.
High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med 2007; 13:1096–1101.
Casanovas G, Swinkels DW, Altamura S, et al.
Growth differentiation factor 15 in patients with congenital dyserythropoietic anaemia (CDA) type II. J Mol Med (Berl) 2011; 89
Osada K, Shiotani T, Tockary TA, Kobayashi D, Oshima H, Ikeda S, et al.
Enhanced gene expression promoted by the quantized folding of pDNA within polyplex micelles. Biomaterials 2012; 33:325–32.
Ramirez JM, Schaad O, Durual S, et al.
Growth differentiation factor 15 production is necessary for normal erythroid differentiation and is increased in refractory anaemia with ring-sideroblasts. Br J Haematol 2009; 144:
Parsian H, Mohammad N, Ali R, Mohammad H, Durdi Q. Comparison of five liver fibrosis indexes with serum levels of laminin and N
terminal peptide of procollagen type III in chronic hepatitis patients. J Gastrointestin Liver Dis 2011; 22:344–357.
Alboraie M, Afifi ME, Elghamry FG, Helmy A, et al.
Egy-Score predicts severe hepatic fibrosis and cirrhosis in Egyptians with chronic liver diseases: a pilot study. Hepat Mon 2013; 13:e10810.
Demir N, Servet K, Serap O, Gokhan G, Sua S, Lutfi S, et al.
Evaluation of the relation between hepatic fibrosis and basic laboratory parameters in patients with chronic hepatitis B fibrosis and basic laboratory parameters. Hepat Mon 2014; 14:16975–16980.
Galal G, Muhammad E, Salah Eldeen F, et al.
Serum prohepcidin, iron and hepatic iron status in chronic hepatitis C in Egyptian patients. JASMR 2011; 6:91–101.
Harrison-Findik D. Gender-related variations in iron metabolism and liver diseases. World J Hepatol 2010; 2:302–31.
Sumida Y, Yoshikawa T, Okanoue T. Role of hepatic iron in non-alcoholic steatohepatitis. Hepatol Res 2009; 39:213-22.
Si Y, Liu X, Cheng M, Wang M, et al.
Growth differentiation factor 15 is induced by hepatitis c virus infection and regulates hepatocellular carcinoma-related genes. PLoS One 2011; 6
Liu X, Chi X, Gong Q, Gao L, et al.
Association of serum level of growth differentiation factor 15 with liver cirrhosis and hepatocellular carcinoma. PLoS One 2015; 10:e0127518.
Hsiao EC, Koniaris LG, Zimmers-Koniaris T, et al
. Characterization of growth differentiation factor 15, a transforming growth factor beta superfamily member induced following liver injury. Mol Cell Biol 2000; 20:3742–51.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]