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ORIGINAL ARTICLE  
Year : 2023  |  Volume : 66  |  Issue : 1  |  Page : 81-84
Study of growth differentiation factor-15 in polytransfused children with β-thalassemia


1 Department of Pathology, Lady Hardinge Medical College and Associated SSK and KSC Hospitals, New Delhi, India
2 Department of Paediatrics, KSCH and Lady Hardinge Medical College and Associated Hospitals, New Delhi, India

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Date of Submission08-Aug-2021
Date of Acceptance05-Nov-2021
Date of Web Publication18-Jan-2023
 

   Abstract 


Background: Ineffective erythropoiesis is a predominant feature in β-thalassemia major (β-TM), causing marked erythroid expansion leading to highly raised levels of growth differentiation factor-15 (GDF-15), which, in turn, suppresses hepcidin production in liver resulting in increased iron absorption from gut. We aim to study the serum GDF-15 in polytransfused β-TM patients and its correlation with serum ferritin and serum hepcidin. Method: Thirty-nine polytransfused β-TM children aged between 5 and 17 years and 33 age- and gender-matched healthy controls were enrolled in the study. Complete blood count, serum GDF-15, serum ferritin, and serum hepcidin were performed. Results: The mean serum GDF-15, serum hepcidin, and serum ferritin levels were 638.65 ± 306.96 pg/ml, 108.21 ± 191.30 ng/ml, and 2274.60 ± 1216.08 ng/ml, respectively, which were significantly higher than control group (P < 0.001, P = 0.003, P < 0.001, respectively). There was significant positive correlation of GDF-15 with blood transfusions (r = 0.415, P = 0.009), positive correlation with serum ferritin (r = 0.653, P = 0), and significant negative correlation with serum hepcidin (r = −0.508, P = 0.001). Conclusion: The findings of the present study suggest that GDF-15 is an important regulator of hepcidin in β-TM patients. GDF-15 and serum hepcidin together can be used to monitor iron overload and its related complications in such patients.

Keywords: β-Thalassemia, ferritin, GDF-15, hepcidin

How to cite this article:
Meena S, Sharma K, Sharma S, Chandra J. Study of growth differentiation factor-15 in polytransfused children with β-thalassemia. Indian J Pathol Microbiol 2023;66:81-4

How to cite this URL:
Meena S, Sharma K, Sharma S, Chandra J. Study of growth differentiation factor-15 in polytransfused children with β-thalassemia. Indian J Pathol Microbiol [serial online] 2023 [cited 2023 Feb 2];66:81-4. Available from: https://www.ijpmonline.org/text.asp?2023/66/1/81/367989





   Introduction Top


Thalassemia is the most common genetic disorder worldwide. It occurs at a high frequency in a broad belt extending from Mediterranean basin through Middle-East, Indian subcontinent, Burma, and South-East Asia.[1] These are heterogeneous groups of inherited anemias resulting from reduced or absent synthesis of α- or β-globin chains of hemoglobin, leading to an imbalance between α- and β-chains. Anemia in thalassemia patients is due to ineffective erythropoiesis and hemolysis which results in lifelong tissue hypoxia that drives the extreme overexpansion of erythropoiesis. Iron regulation is mediated through erythroblast secretion of growth differentiation factor-15 (GDF-15), which is increased in circulation in these individuals. GDF-15 circulates to the liver and suppresses hepcidin expression; as a result, increased amounts of dietary iron are absorbed, and secondary hemochromatosis develops over the lifetime of the patient. In recent times, GDF-15 has emerged as an important factor contributing to iron overload in thalassemia patients apart from repeated blood transfusions and increased iron absorption from gut due to paradoxically low hepcidin. Since iron overload is the major cause of morbidity and mortality in β-thalassemia, understanding the pathogenesis is of utmost importance. The present study was carried out to analyze serum GDF-15 levels in polytransfused β-TM patients and its correlation with serum ferritin and serum hepcidin levels. Most of the reports available in literature are of Western population and included both children and adult in their study groups. The present study is done on GDF-15 levels in Indian pediatric thalassemia patients.


   Materials and Methods Top


The present work is a prospective study based on pretransfusion drawn blood samples of thalassemia patients registered in Thalassemia Day Care Centre at Kalawati Saran Children's Hospital. The samples were received in the Department of Pathology, Lady Hardinge Medical College over a 2-year period (2015–2017). Out of 250 cases registered in Thalassemia Day Care Centre, 39 children of β-thalassemia major (β-TM) between 5 and 17 years of age were randomly taken. The control group comprised 33 age- and sex-matched individuals. Those with clinical evidence of acute infection, undergone splenectomy, and seropositivity were excluded from this study. Detailed clinical history was documented and physical examination of all patients was performed.

The subjects evaluated were classified into cases and controls.

Cases

The cases composed of 39 cases of thalassemia major, all of whom had received more than 20 transfusions ranging from 57 to 204 with mean of 121.44 ± 41.66. The diagnosis was based on both clinical and laboratory findings. All the patients were on regular chelation therapy.

Control

Composed of 33 age- and sex-matched individuals, none had received any blood transfusion.

Complete blood count was determined using hematology auto-analyzer, Sysmex KX-21. Serum GDF15 was assessed using ELISA (human GDF15 PicoKineTM ELISA Kit), serum ferritin by Beckman Coulter Unicel Dxl 600 access immunoassay system using Beckman Coulter access ferritin (33020), and serum hepcidin by Hepcidin ELISA (DRG, EIA-5258, Germany).

Data analysis was done using the statistical package for social sciences version 16.0. The results were expressed as mean ± SD. Comparison of values between two groups was done using the Student t-test. Pearson correlation test was used to see the relationship between the two variables. P value (significance value) <0.05 was considered significant.


   Results Top


The age of the patients in the case group ranged from 5 to 17 years with mean age of 10.46 ± 3.69 years. Most of the patients (27/39, 69.23%) were in the age group of 5–11 years with 26 males and 13 females (male:female ratio of 2:1).

The age of the patients in the control group ranged from 5 to 17 years with mean age of 9.96 ± 3.54 years. Most of the patients (23/30, 69.6%) were in the age group of 5–11 years with 14 males and 19 females (male:female ratio of 0.7:1).

The serum ferritin in cases was markedly raised with a mean value of 2274.60 ± 1216.08 ng/ml ranging from 1000 to 6717 ng/ml which was significantly higher than controls (P ≤ 0.001) [Table 1].
Table 1: Comparison of serum ferritin, hepcidin, and GDF-15 in cases and controls

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The mean serum hepcidin value in cases ranged from 8.72 to 680.6 ng/ml with a mean of 108.21 ± 191.30 ng/ml, which was significantly higher than controls (P = 0.003) [Table 1].

The mean GDF-15 concentration in cases was 638.65 ± 306.96 pg/ml, ranging from 190.25 to 1217.31 pg/ml which was significantly higher than controls (P ≤ 0.001) [Table 1].

GDF-15 and its correlation with other parameters

There was significant positive correlation of serum GDF-15 with number of transfusions in cases (r = 0.415, P = 0.009) [Figure 1]. The GDF-15 levels in cases negatively correlated with the hemoglobin concentration but the degree of correlation was not statistically significant (P = 0.482).
Figure 1: Correlation of GDF-15 and number of transfusions in cases

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Serum GDF-15 showed positive correlation with serum ferritin; however, it was not statistically significant in cases (r = 0.653, P = 0) [Figure 2].
Figure 3: Correlation of GDF-15 and serum hepcidin in cases

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There was a significant negative correlation between serum hepcidin and GDF-15 levels in Group A (r = −0.508, P = 0.001) [Figure 3].{Figure 3}


   Discussion Top


Thalassemia syndromes are a heterogeneous group of inherited anemias resulting from reduced or absent synthesis of α- or β-globin chains of hemoglobin leading to an imbalance between α- and β-chains. In β-thalassemia, there is deficient production of β-chains and its clinical severity is related to the extent of imbalance between the α-globin and non-α-globin chains.[2]

Patients with symptomatic thalassemia syndromes and iron regulation are mediated by erythroblast secretion of GDF-15. Ineffective erythropoiesis and hemolysis causing anemia result in chronic tissue hypoxia that drives the extreme overexpansion of erythropoiesis in patients with these syndromes.[3] GDF-15 and possibly other proteins are increased in the circulation in these individuals. GDF-15 produced by erythroblasts circulates to the liver and suppresses hepcidin expression causing increased amounts of dietary iron absorption which results in development of secondary hemochromatosis. In thalassemia major patients, GDF-15, which was initially identified as macrophage inhibitory cytokine-1, is a recently identified protein which suppresses hepcidin production in liver.[4] GDF-15 is not erythroid specific, its expression has also been found to be increased in stress (anoxia, cancer, tissue injury), and in placenta during pregnancy in the second and third trimesters. It causes growth inhibition and induction of apoptosis in epithelial and other tumor cell lines. Studies have shown its role in p53 pathway activation, as it can serve as tumor burden marker and used to assess therapies directed at p53 pathway activation.[5],[6] Recently, role of GDF-15 in immune modulation in various pathological conditions has been described.[7]

Hepcidin is a negative regulator of iron transport into plasma and is thought to be regulated by the erythroid regulators like GDF-15, TGFβ-1, and TWSG1, out of which GDF-15 is the most potent regulator.[8],[9] GDF-15 is a member of TGFβ superfamily, secreted by the proliferating erythroblasts, so any cause of marrow expansion, hypoxia, and erythroblast apoptosis (ineffective erythropoiesis) will lead to increased secretion of GDF-15.[10] In β-TM, ineffective erythropoiesis is a predominant feature, causing marked erythroid expansion leading to highly raised levels of GDF-15, which, in turn, suppresses hepcidin production in liver resulting in increased iron absorption from gut.

Initially the GDF-15 was measured in the form of messenger RNA in early and late erythroblasts in healthy individuals by Tanno et al.[11] They found 64-fold increase of GDF-15 mRNA in late erythroblast as compared to early forms. Further, they measured GDF-15 in thalassemia patients and found markedly raised levels of GDF-15 in cases as compared to controls (P = 0.00010). Musallam et al.[12] measured GDF-15 concentration in adult patients of thalassemia intermedia and they found considerably higher levels of GDF-15, however lower than β-TM with a mean of 25.197.8 ± 16.208.9 pg/ml. Their findings suggest a role of GDF-15 in assessing the clinical severity of ineffective erythropoiesis.

A significant increase in serum GDF-15 level in β-thalassemia patients (median of 5504 pg/ml) as compared to controls (279 pg/ml) was also documented by Porter et al.,[13] although the levels were not as high as in previous studies.

Eissa et al.[14] determined levels of GDF-15 in sera of the thalassemic children of >12 years and <12 years of age. They found that the GDF-15 was higher in both the groups as compared to controls (<0.001) but was comparable between two subgroups. They found significantly raised levels of GDF-15 in patients with low BMI. Similarly, Athiyarth et al.[15] conducted a study on 134 β-TM patients and found that the mean GDF-15 level was significantly high as compared to controls (P < 0.0001).

In the present study, the levels were not so high as reported by other authors, which could be due to younger age of the cases and regular transfusions, reducing hypoxia and suppressing the erythroid proliferation.

Various authors documented a significant positive correlation between GDF-15 and number of transfusions (r = 0.562, P < 0.0001; r = 0.767, P < 0.001 respectively).[15],[16] The current study showed a significant positive correlation of serum GDF-15 with number of transfusions in cases (r = 0.415, P = 0.009).

GDF-15 showed a negative correlation with hemoglobin in various studies as observed by Tanno et al.[11] (r = −0.80, P < 0.01), Musallam et al. (r = −0.312, P = 0.048), and Tantawy et al.[16] (r = −0.408, P = 0.015).[15]

In the present study, GDF-15 level in cases negatively correlated with the hemoglobin concentration, although the degree of correlation was not statistically significant (r = −0.014, P = 0.907). With increasing degree of anemia, the hypoxia increases, causing erythroid expansion which is much more marked in cases with ineffective erythropoiesis as observed in β-TM. Contrary to above, Eissa et al.[14] observed a positive correlation of GDF-15 with hemoglobin but it was not statistically significant (r = 0.053, P = 0.77).

Previously, a significant positive correlation between GDF-15 and serum ferritin levels (P < 0.01) has been described in thalassemia patients.[11],[17] The current study also showed a positive correlation of GDF-15 with serum ferritin (r = 0.653, P = 0); however, it was not statistically significant in cases. A similar finding was observed by Athiyarath et al.[15] in their study on Indian patients comprising both TI and TM.

Pasricha et al.[18] found a significant negative correlation between GDF-15 levels and hepcidin levels (r = −0.32, P = 0.001) and showed that the high levels of GDF-15 in the sera of the patients suppress hepcidin, thus enhancing intestinal iron absorption leading to further iron overload. Other studies made similar observations, recording significant inverse correlation between serum GDF-15 and serum hepcidin (r = −0.422, P = 0.002).[19]

In accordance with the above studies, the present study also showed a significant negative correlation between serum hepcidin and GDF-15 levels in thalassemia cases (r = −0.508, P = 0.001) [Figure 3].

In β-TM, the GDF-15 concentration reflects competing influences of anemia (ineffective erythropoiesis) and iron overload. It has been shown that transfusion suppresses erythropoiesis and thus GDF-15 is lower in patients on regular repeated multiple transfusions. In the present study, GDF-15 levels were lower than recorded in other studies possibly because of repeated transfusions which suppress the erythroid drive.[11],[12],[13],[14],[15],[16] On the contrary, elevated levels of GDF-15 may indicate that even adequately transfused patients with β-thalassemia, despite stable overall erythroid suppression, have ongoing ineffective erythropoiesis with apoptosis.


   Conclusion Top


We can conclude that GDF-15 may serve as a biomarker for evaluating erythropoiesis which can assist in estimating the frequency of transfusion in β-TM patients. In the present study, we could establish a negative correlation between GDF-15 and hepcidin. Thus, combined assessment of these parameters may provide a better overview of the current state of erythropoiesis in the patient. Further studies can be directed to understand their role which may have important implications in the diagnosis and treatment of these patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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2.
Lee YK, Kim HJ, Lee K, Park SH, Song SH, Seong MW, et al. Recent progress in laboratory diagnosis of thalassemia and hemoglobinopathy: A study by the Korean Red Blood Cell Disorder Working Party of the Korean Society of Hemtology. Blood Res 2019;54:17-22.  Back to cited text no. 2
    
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Lawton LN, Bonaldo MF, Jelenc PC, Qiu L, Baumes SA, Marcelino RA, et al. Identification of a novel member of the TGF-β superfamily highly expressed in human placenta. Gene 1997;203:17-26.  Back to cited text no. 5
    
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Wischhusen J, Melero I, Fridman WH. Growth/differentiation factor-15 (GDF-15): From biomarker to novel targetable immune checkpoint. Front Immunol 2020;11:951.  Back to cited text no. 7
    
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Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont S. Hepcidin, a new iron regulatory peptide. Blood Cell Mol Dis 2002;29:327-35.  Back to cited text no. 8
    
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Tanno T, Bhanu NV, Oneal PA, Goh SH, Staker P, Lee YT, et al. High levels of gdf-15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med 2007;13:1096-101.  Back to cited text no. 11
    
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Musallam KM, Taher AT, Duca L, Cesaretti C, Halawi R, Cappellini MD. Levels of growth differentiation factor-15 are high and correlate with clinical severity in transfusion- independent patients with β thalassemia intermedia. Blood Cells Mol Dis 2011;47:232-4.  Back to cited text no. 12
    
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Porter JB, Walter PB, Neumayr LD, Evans P, Weyhmiller MG, Harmatz P, et al. Iron trafficking and distribution in transfusional overload: Insights from comparing diamond blackfan anemia with sickle cell disease and thalassemia. Blood 2012;120:995.  Back to cited text no. 13
    
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  [Full text]  
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Athiyarath R, George B, Mathews V, Srivastava A, Edison ES. Association of growth differentiation factor 15 (GDF15) polymorphisms with serum GDF15 and ferritin levels in β-thalassemia. Ann Hematol 2014;93:2093-5.  Back to cited text no. 15
    
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Pasricha SR, Frazer DM, Bowden DK, Anderson GJ. Transfusion suppresses erythropoiesis and increases hepcidin in adult patients with β-thalassemia major: A longitudinal study. Blood 2013;122:124-33.  Back to cited text no. 18
    
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Huang Y, Lei Y, Liu R, Liu J, Yang G, Xiang Z, et al. Imbalance of erythropoiesis and iron metabolism in patients with thalassemia. Int J Med Sci 2019;16:302-10.  Back to cited text no. 19
    

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Correspondence Address:
Kusha Sharma
C-2/2001, Vasant Kunj, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_803_21

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