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

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.

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).

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. 

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.

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