Journal of DNA and RNA Research
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Research Article | Open Access
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  • | Provisional

    Molecular Study of Hepcidin HAMP (-582A/G) Gene Polymorphisms and Measurement of Serum Hepcidin Level among Sudanese Patients with Anemia of Chronic Kidney Disease

    Amged Hussen Abdelrhman 1       Khalid Mohammed Khalid 2     Enaam Abdelrhman Abdelgadir 1    

    1Assis Professor, Department of Hematology and Immunohematology, Omdurman Islamic university / Sudan

    2Assoc Professor, Head Department of Pathology, Faculty of Medicine, alneealin university / Sudan

    Abstract

    Background

    Anemia of chronic disease is anemia found in certain chronic disease states, is typically marked by the disturbance of iron homeostasis or hypoferremia. Chronic renal failure is currently known as Chronic Kidney Disease (CKD) or Chronic Renal Insufficiency (CRI) implies long-standing, progressive and irreversible renal parenchyma disease resulting in diminished renal function up to 40 to 60%. Often, chronic kidney disease is diagnosed as a result of screening of people known to be at risk of kidney problems, such as those with high blood pressure or diabetes and those with a blood relative with chronic kidney disease. This disease may also be identified when it leads to one of its recognized complications such as cardiovascular disease, anemia, or pericarditis.                            

    Methods

    Sysmex kx21 used to CBC and the Cobase411 used to iron profile. Enzyme-Linked immunoassay (ELISA) was used to determine the level of serum hepcidin. 

    Sample preparation and PCR detection of HAMP DNA Polymorphisms: Restriction digestion of PCR products was done using Fast Digest. (Figure 1).                                                                                        

    Results

    Serum hepcidin levels higher in patients with anemia of chronic kidney disease compared with healthy controls mean. The polymorphisms of the hepcidin gene promoter in Sudanese patients with ACKD showed that the hepcidin HAMP AA genotype 70, AG 23, and GG 7 in 100 patients dialysis-dependent and AA 83, AG 17 and GG 0, and the allele A are more frequent in patients affected by ACKD. Significant statistical association observed between the hepcidin level and end-stage kidney disease.

    Conclusion

    This study evaluates for the first time the association between anemia of chronic kidney disease and hepcidin genes promoter polymorphisms and show that the hepcidin HAMP AA genotype and the allele A are more frequent in patients affected by ACKD, further investigation is needed, our data support the hypothesis and hepcidin HAMP are important in the pathophysiology of ACKD.

    Received 28 Apr 2020; Accepted 24 May 2020; Published 28 May 2020;

    Academic Editor:Wentao Xu, Food safety and molecular biology, China.

    Checked for plagiarism: Yes

    Review by:Single-blind

    Copyright©  2020 Amged Hussen Abdelrhman, et al.

    License
    Creative Commons License    This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

    Competing interests

    The authors have declared that no competing interests exist.

    Citation:

    Amged Hussen Abdelrhman, Khalid Mohammed Khalid, Enaam Abdelrhman Abdelgadir (2020) Molecular Study of Hepcidin HAMP (-582A/G) Gene Polymorphisms and Measurement of Serum Hepcidin Level among Sudanese Patients with Anemia of Chronic Kidney Disease . Journal of DNA And RNA Research - 1(2):23-30.
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    DOI10.14302/issn.2575-7881.jdrr-20-3343

    Introduction

    Anemia of chronic disease is anemia found in certain chronic disease states, is typically marked by the disturbance of iron homeostasis or hypoferremia. This condition leads to a shortage of iron for hemoglobin synthesis but the iron storage in bone morrow is left undisturbed. Anemia of chronic disease is the second most prevalent anemia after iron deficiency anemia, it can be triggered by the wide range of inflammatory disorders such as infection, autoimmune disease, chronic diseases aging process and malignancy, also known as anemia of inflammation1,2. Kidney failure is a medical condition in which the kidneys fail to adequately filter waste products from the blood. Studies of hepcidin, an antimicrobial peptide that have a role as a modulator of iron homeostasis has given a new insight for the management of the anemia of chronic disease. Hepcidin, a peptide composed of 25 amino acid, is synthesized by hepatocyte, it inhibits iron release from macrophages, intestinal epithelial cells and placental syncytiotrophoblasts by its interaction with the transmembrane iron exporter ferroportin, accelerating degradation of ferroportin mRNA increased production of hepcidin is induced by inflammatory via interleukin 6(IL-6), hepcidin synthesis and secretion are controlled by proteins, HFE hemojuvelin and transferrin receptor.

    Materials and Methods                                  

    Study Participants

    This study included 100 Sudanese patients diagnosed as anemia of chronic kidney disease,50 CKD end-stage dialysis-dependent, and 50 CKD on dialysis who were attended Bahri dialysis center. Besides 50 healthy individuals were recruited from the same center as co-patients and included as a healthy control group. Sysmex kx21 used to RBCs profile, Cobas e411 used to determine the iron profile and ELISA used to determine the level of hepcidin.

    Sample Preparation and PCR Detection of HAMP DNA Polymorphisms

    The concentration of DNA was measured on a nanodrop 1000 spectrophotometer (Thermo-scientific –USA ) at 260 and 280nm. The mean concentration of the purified genomic DNA was 20.56ng/ul. After extraction, using published primer set: {5-GTACTCATCGGACTGTAGATGATTTAGC(forward),5GTGACAGTCGCTTTTATGGGGCCTGC-3 (reverse ) HAMP gene promoter region also amplified. Restriction digestion of PCR products was done using Fast Digest. As regards the detection of HAMP (-582A/G) gene polymorphism, the Fast Digest HindIII restriction enzyme (Fermentas-Thermo-USA) was used. (Figure 1), the digestion products were subjected to 2.5% agarose gel electrophoresis and showed that allele A which did not contain the HindIII restriction enzyme site was digested to 200 bp fragments, whereas allele G yields 190 bp and 90 bp fragments (Figure 1).     

    Figure 1. Gel picture of PCR products digest by HindIII restriction enzyme (HAMP). 100 b.p DNA Ladder (100–1000 b.p). Lane 1, 2, 3, 4, 6, 7 : GG homozygote alleles (bands at 90 bp and190 bp). Lane 8, 9, 10, 11: AG heterozygote alleles (bands at 90 b.p, 190 b.p, and 200 b.p). Lane 5: AA homozygote alleles (band at 200 bp).
    Figure 1.

    Statistical Analysis              

    Statistical assessment was carried out with a statistical package for social sciences (SPSS), Genotypes and allele frequencies between groups was analyzed by Chi-Squared test.

    Results

    The current study show , mean of the RBCs profile (RBCs count , Hb , PCV ) (3.353±0.88cell/l, 10.62±2.4g/dl , 32.59±6.82% ) in patients with anemia of chronic kidney disease Vs (4.048±0.47cell/l, 12.52±1.57g/dl, 37.92±4.79%) in control groups P.value (0.00 , 0.00 , and 0.00) respectively Table 1. The mean value of the iron profile, serum iron, serum ferritin and transferring saturation percentage (61.353±29,8ug/dl, 195.3.62±19.4ng/ml, 21.59±12.82% ) in patients with anemia of chronic kidney disease Vs (82.048±0.47ug/dl, 80.52±1.57ng/ml, 28.92±4.79%) in control groups P.value (0.00 , 0.00 , and 0.00). (Table 2). Serum hepcidin levels higher in patients with anemia of chronic kidney disease compared with healthy controls, mean (161.55±29.8ng/ml,195.322±19.224ng/l. P.value (0.000) (Table 3). The polymorphisms (SNP) of the hepcidin (HAMP) gene promoter in Sudanese patients with anemia of chronic kidney disease showed that the hepcidin HAMP (-582A/G) (SNP) AA genotype 70 (35%), AG 23(11.5) and GG 7 (3.5%) in 100 patients dialysis-dependent and AA 83 (41.5%), AG 17 (8.5%) and GG 0 (0%), and the allele A are more frequent in patients affected by anemia of chronic kidney disease (Table 4). Significant statistical association observed between the hepcidin level and end-stage kidney disease (Table 5). When the measured variables compared with different polymorphisms of the HAMP gene, an insignificant relation observed (Table 6).

    Table 1. Mean of Some Red Blood Cell Profile in 100 Patients with Anemia of Chronic Kidney Disease and 100 Control Group
    RBCs profile  Mean Case  Mean Control  P. value 
    RBCs count cell/l 3.353±0.88 4.048±0.47 0.000S
    HB g/dl 10.62±2.4 12.52±1.57 0.000S
    PCV % 32.59±6.82 37.92±4.79 0.000S
    MCV fl 93.09±10.12 93.04±4.28 0.974NS
     MCH pg 34.95±33.55 30.59±1.42 0.068NS
    MCHC g/l 316.34±37.58 368.10±397.37 0.070NS

    Table 2. Mean of Serum Iron Profile in 100 Patients and 100 Control Group
     Iron profile  Mean Case  Mean Control  P. value 
    Iron µg/dl 61.55±29.8 82.05±13.4 0.000S
    Ferritin ng/ml 195.322±192.24 80.89±60.94 0.000S
    TIBC µg/dl 253.97±77.87 260.32±52.49 0.586NS
    TS % 21.33±12.72 28.17±4.70 0.000S

    Table 3. Mean of Hepcidin Levels in 100 Patients and 100 Control Group
     Variables  Mean n Case  Mean Control  P. value
    Hepcidin ng/ml 61.55±29.8 82.05±13.4 0.000S

    Table 4. Frequencies of Genotype to the HAMP (-582A/G) gene Polymorphisms to 100 Patients with ACKD (Dialysis Dependent) and 100 Control Group
     Genotype Participants   Total P value
    Case Control
     AA  70(35%)  83 (41.5%)  153(76.5%)   0.076NS
     AG  23(11.5%)  17(8.5%)  40(20%)
     GG  7(3.5%)  0 (0%)  7(3.5%)
     Total  100(50%)  100 (50%)  200(100%)

    Table 5. Comparison of the Hepcidin and all CKD stages (Five stages)
    Variables CKD stages (I) CKD stages (J) Mean P value
    Hepcidin ng/ml  Stages Stage1 -8.908 0.007S
    Stage2 -3.092 0.342NS
    Stage3 -0.800 0.805NS
    Stage4 -18.751 0.000S
    Stage 5 20.777 0.000S

    Table 6. Comparison study of the Measured Variables and Different Genotypes of HAMP (-582A/G) Polymorphisms
    Variables Genotypes Frequencies P.value
    RBCs Count AA 0.997 0.373
      AG    
      GG    
    Hb AA 1.169 0.315
      AG    
      GG    
    PCV AA 0.848 0.431
      AG    
      GG    
    MCV AA 0.827 0.440
      AG    
      GG    
    MCH AA 0.177 0.838
      AG    
      GG    
    MCHC AA 0.450 0.639
      AG    
      GG    
    S.iron AA 2.071 0.132
      AG    
      GG    
    S.ferritin AA 0.622 0.539
      AG    
      GG    
    TIBC AA 0.139 0.870
      AG    
      GG    
    TS% AA 0.339 0.713
      AG    
      GG    
    S.hepcidin AA 1.237 0.295
      AG    
      GG    

    Discussion

    This current study focuses on the possible association between hepcidin HAMP (-582A/G) gene polymorphisms and anemia of chronic kidney disease in Sudanese patients. No studies have been performed to test whether the A/G HAMP gene polymorphisms associated with CKD. The current study, also focused on measurements of the serum hepcidin levels using the ELISA technique. In this study, there is a significant association between CKD and RBCs profile (RBCs count, Hb, PCV ) , confirming that the patients with CKD are anemic, and the type of anemia found is dimorphic normocytic normochromic and microcytic hypochromic anemia. Hepcidin levels were significantly higher in patients with ACKD compared with healthy controls, comparable results were also reported in other studies 3, who found that the hepcidin levels were higher in patients with ACKD in Indian patients. It has also been indicated that hepcidin levels were approximately two to three-folded higher in patients with ACKD than in the controls 4. Hepcidin is expected to be elevated in patients with ACKD due to limited hepcidin excretion in urine, tissue iron overload and inflammation5. Also agreed with the results of 6,who reported that the hepcidin levels are likely to be higher in ACKD patients due to inflammation, and agreed with the results of 7, who found who reported that the hepcidin levels are likely to be higher in Egyptian patients with ACKD. Among this study group patients, the decreased levels of serum iron, TIBC, and TS% were found, However, serum ferritin levels were found to be elevated in this group. Findings consistent to this results in agreement with a study on patients with CKD 8, who reported that the serum ferritin levels are likely to be higher in ACKD patients due to inflammation The situation in which the serum iron is low and the serum ferritin is high is frequently seen among ACKD patients, High ferritin levels may be observed in this disease because of functional iron deficiency or reticuloendothelial blockade. The present study shows the comparison between the study variables (RBCs profile, Iron profile, hepcidin levels) in the all stages of CKD in Sudanese patients, ( five stages according to GFR), showed statistically significant differences in the RBCs count, Hb, PCV, S.iron, S.ferritin, TIBC. TS %, hepcidin level in the end stage of CKD (Dialysis dependent), and no statistically significant differences seen in the MCV, MCH, and MCHC, concluding that the severity of CKD can increase the severity of anemia, influencing in the iron status and increase the levels of hepcidin. The present study show in the first time that the genotype distribution and allele frequency for HAMP (-582A/G) in Sudanese were compared in all subjects, no significant difference was observed among studied groups. Besides, no significant difference was observed in the frequency of HAMP (-582A/G) alleles among studied groups P.value =(0.076). When male and female patients analyzed, a similar distribution of HAMP A/G genotype and allele frequency is found P.value 0.238, demonstrating that the HAMP A/G polymorphism is not involved in the pathophysiology of ACKD in both men and women. No significant association between the -582A/G genotype and serum iron, serum ferritin, transferrin saturation, or ferritin levels were found, which might reflect no differences in liver iron concentration. Also, no significant relation was found between HAMP(-582A/G) variants and the different others studied parameters. These results are in agreement with 9, who found no association between the -582A/G genotype and serum iron, serum ferritin, and transferrin saturation, and the present study also disagreement with 10, who found that the -582A/G in the human HAMP promoter has no effect on the hepcidin transcription in normal situations but have some effect in pathophysiological situations where more hepcidin was needed. However, from the findings of the present work, the hepcidin HAMP (-582A/G) genetic variations are unlikely to play an important role in the genetic predisposition to ACKD, conflicting results may be due to various reasons such as demographic features of subject and different lifestyle, also sample size plays a crucial role. This situation encourages more and more attempts to be made to further assess the associations of these polymorphisms with the disease. 

    Conclusion                        

    This study evaluates for the first time the association between anemia of chronic kidney disease (ACKD) and hepcidin (HAMP) genes promoter polymorphisms and show that the hepcidin HAMP (-582A/G) AA genotype and the allele A are more frequent in patients affected by ACKD, further investigation is needed, our data support the hypothesis and hepcidin HAMP (A/G) are important in the pathophysiology of ACKD.

    Acknowledgment

    By the grace of Almighty Alla and his help I complete thia study , all praise to him, my gratitude goes to Dr Enaam A, Rhman and Dr Khalid M, Khalid my supervisors whose guides me to complete this work , finally special thanks to patients who were so cooperative and despite their pain.

    Abbreviations

    ACKD - Anemia of Chronic Kidney Disease

    A - Adenine,

    CRP - C-Reactive Protein

    C - Cytosine

    DNA - Deoxyribonucleic Acid

    G - Guanine

    HAMP - Human Antimicrobial Peptide

    HFE - High Iron Fe

    IL-6 - Interleukin-6 IL-1 - Interleukin-1

    SPSS - Social Packages Statistical

    TNF - Tissue Necrosis Factor

    mRNA - Messenger Ribonucleic Acid

    NS - Non Significant

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