GDF‐15 is associated with atherosclerosis in adults with transfusion‐dependent beta‐thalassemia

Abstract Objectives: To study serum growth differentiation factor‐15 (GDF‐15) serum level in β‐thalassemia patients and its relation to carotid intima‐media thickness. Background: Thalassemia is a common genetic disease resulting in decreased beta‐chains, leading to manifested anemia. It may be subsequently complicated by iron overload, which induces numerous morbidities and even death. Growth differentiation factor‐15 (GDF‐15) is a strong and independent predictor of mortality and disease progression in patients with atherosclerosis alongside with carotid‐intimal media thickness (CIMT). Patients and methods: This monocentric case‐control study was done on 90 subjects in the period from January 2020 to March 2021. Sixty transfusion‐dependent beta‐thalassemia (TDβT) cases (≥18 years) were selected from the thalassemia clinic of Hematology division at Menoufia University hospitals. We included also 30 sex and age matched healthy as the controls. Routine investigations were done beside. Serum GDF‐15 was measured by ELISA. CIMT was measured by Doppler Ultrasonography. Results: CIMT on both sides was statistically significant higher in cases (median of 0.08 cm) than in the controls (median of 0.04). GDF‐15 was also significantly higher in cases (median of 1839.89 pg/dl) than in controls (median of 256.14 pg/dl). So, we found that GDF‐15 is a predictor of and associated with atherosclerosis in thalassemic adults (OR = 39.198, p value 0.008, 95% CI: 2.576–596.5). Conclusion: GDF‐ 15 is increased in TDβT. CIMT (as a marker of subclinical atherosclerosis) is increased in these patients alongside with GDF‐15, is a predictor, and associated with atherosclerosis in thalassemic adults.


INTRODUCTION
Thalassemias are a group of inherited blood disorders caused by decrease or absence of beta-globin chain synthesis which is reflected with decrease in hemoglobin level; it is mainly inherited as recessive autosomal disorder [1]. Its prevalence is more in Mediterranean countries, the Middle East, Central Asia, the north coast of Africa, and South America. α-Thalassemia is prevalent in people of Western African and South Asian descent [2]. The beta-globin chains are encoded by a single gene on chromosome 11 [3].
Insufficient amount of normal structure of globin chains causes imbalance between α and β chains leading to clinical features of the disease [4]. Complications from thalassemia are mainly related to blood transfusion and iron overload that include involvement of the heart, liver, and endocrine glands. Splenomegaly, viral hepatitis infection or human immunodeficiency virus (HIV) infection, venous thrombosis, and osteoporosis may occur also [5]. The relationship between chronic hemolysis with subsequent iron overload, inflammation, and premature atherosclerosis has been documented in hemolytic anemia, especially β-thalassemia [6]. Carotid intima-media thickness (CIMT) is a noninvasive method to detect early subclinical atherosclerosis, and it well correlates with vascular injury and severity of coronary artery disease. So, it is a good tool for detecting early atherosclerosis in such patients [7]. Thalassemia intermedia patients usually live longer and, thus, are more prone to complications of atherosclerosis. There is evidence of an increased risk of central ischemia rather than peripheral ischemia in these patients [8]. The growth differentiation factor-15 (GDF-15) is a multifactorial cytokine and a member of the transforming growth factor superfamily. Expression of the GDF-15 gene in cardiomyocytes, vascular smooth muscle cells, and endothelial cells is strongly upregulated in response to oxidative stress, inflammation and tissue injury, while high levels of serum GDF-15 associate with ineffective erythropoiesis and may reflect a certain type of bone marrow stress or erythroblast apoptosis [9]. In many cardiovascular diseases (such as hypertrophy, heart failure, atherosclerosis, endothelial dysfunction), obesity, insulin resistance, diabetes, and chronic kidney diseases are associated with increased GDF-15 and linked with the progression and prognosis of the disease condition [10].

PATIENTS AND METHODS
This is a monocentric case-control study. We included 90 subjects in the period from January 2020 to May 2021. Cases group was 60 transfusion beta-thalassemia adults (≥18 years); we had selected them from the outpatient thalassemia clinic of Hematology division at Menoufia University hospitals and control group formed of 30 sex and agematched healthy subjects.

Ethical approval
Approval of local ethical committee at Menoufia faculty of medicine was obtained under number 121/2021.
Informed consents from all participants were obtained according to local ethical committee guidance.

METHODS
The diagnosis of beta-thalassemic adults was based on clinical symptoms and signs, complete blood count (CBC), and hemoglobin electrophoresis.
Following patients were excluded from the study: patients less than We also did pelviabdominal ultrasound and echocardiography to the thalassemic patients participated in the study.
We measured carotid intimal media thickness (CIMT) by Doppler ultrasonography (GE logic E10 United states) and measured at the

Statistical analysis
After finishing data collection, we tabulated and statistically analyzed it by an IBM compatible personal computer with SPSS Statistical Package Version 22. Two types of statistics were used: descriptive statistics, for example, number and percent for qualitative data, mean, and standard deviation (SD) for quantitative data and analytic statistics, for example, chi-squared test (χ 2 ) was used to study association between two quali-F I G U R E 1 Receiver operating characteristic curve (ROC) analysis of the optimal cutoff of CIMT and GDF15 levels. Blood GDF-15 levels classified into three categories: normal (<1200 pg/ml), moderately elevated (1200-1800 pg/ml), and highly elevated (>1800 pg/ml) [35] tative variables.

RESULTS
Results tabulated in Table 1 show that there was no a statistically significant difference between the cases and controls regarding age and sex (p value > 0.05). There was a statistically significant difference between them in BMI which was higher in controls by mean of 24.36 ± 2.6 Kg/m 2 (p < 0.05) and highly significant difference in height which  was a highly statistically significant value that was higher in cases by median of 1839.89 pg/ml than in controls by median of 256.14 pg/ml (p < 0.001). Echocardiography showed a highly statistically significant difference between cases and controls in diastolic dysfunction that was higher in cases by percentage of 56.7% while 3.3% of cases had systolic dysfunction. Pulmonary hypertension was more in cases by mean of 30.12 ± 9.7 mmHg than in controls by mean of 15.132 ± 2.9 mmHg of highly statistically significance as shown in Table 1.
From Table 2

Correlation between GDF-15 and other variables in beta-
thalassemic patients is shown in Table 3 Receiver operating characteristic curve analysis of the optimal cutoff of GDF-15 for prediction of atherosclerosis was done in thalassemic patients as in Table 4. The serum GDF-15 to CIMT ratio as a predictor of atherosclerosis with cut off value of GDF-15 (≥1446.01 pg/dl) is highly statistically significant (p < 0.001) and area under curve (AUC) is 0.962 of specificity 90% and sensitivity 88% as shown in Figure 1.

DISCUSSION
High-resolution ultrasound is a method for detecting structural and functional atherosclerotic changes in the arterial wall. Intima-medial thickness (IMT) is a measure of the combined thickness of intima and media layers of carotid artery that is assessed by B-mode ultrasound.
Increased CIMT is a structural marker representative of subclinical and asymptomatic atherosclerotic vascular diseases [19]. The aim of our study is to evaluate GDF-15 serum levels and CIMT (as a biomarker for subclinical atherosclerosis) in a cohort of transfusion-dependent betathalassemic adults to explore their possible correlations with clinical, hematological, and laboratory variables and to reveal the association between risk factors and atherosclerosis. The study showed that age varies with a mean of 27.17 ± 5.75 years. There was female predominance by percentage of 61.6%. There was a statistically significant difference between cases and controls in BMI that was higher in controls (p < 0.05) and a highly significant difference in height that was more in controls by mean of 167.7 ± 6.5 cm than in cases by mean of 159.02 ± 8.6.7 cm (p < 0.001), and this is similar to studies of Abd Elsamei et al. [20] and Ghazala et al. [21]. There was a highly statistically significant difference regarding Hb levels that were lower in cases (mean 7.87 ± 1.01 g/dl) than in controls (mean 12 ± 1.41 g/dl) and platelets that were higher in cases (mean 565.47 ± 231.5×10 3 /mm 3

CONCLUSION
Subclinical atherosclerosis was documented among Egyptian transfusion-dependent beta-thalassemic adults. This is evaluated by measurement of CIMT (as a biomarker of subclinical atherosclerosis) by ultrasonography and was positively correlated with dyslipidemia and elevated serum GDF-15, which is detected by ELISA. So, GDF-15 is associated with atherosclerosis in adults with transfusion dependable beta-thalassemia.

CONFLICT OF INTEREST
The authors disclose no conflict of interest.

FUNDING
None.

AUTHOR CONTRIBUTIONS
Alaa Efat and Rana Wahb wrote the manuscript and analyzed the data.
Aly Elkholy and Mohamed Abdelhafez performed data collection and manuscript preparation. Ashraf Dawod performed laboratory studies and analysis. Sabry Shoeib and Essam Abdelmohsen were responsible for selection and follow-up of patients. All authors revised the study and reviewed the article.

ETHICS STATEMENT
Approval of local ethical committee at Menoufia faculty of medicine was obtained under number 121/2021. Informed consents from all participants were obtained according to local ethical committee guidance.