Association of GDF15 levels with body mass index and endocrine status in β‐thalassaemia

Abstract Objective GDF15 has emerged as a stress‐induced hormone, acting on the brain to reduce food intake and body weight while affecting neuroendocrine function. Very high GDF15 levels are found in thalassaemia, where growth, energy balance and neuroendocrine function are impaired. We examined the relationships between GDF15 and anthropometric measures and endocrine status in β‐thalassaemia. Design Cross sectional study. Patients All β‐thalassaemia patients attending the thalassaemia unit of Colombo North Teaching Hospital for blood transfusions. Measurements Anthropometric data, appetite scores, circulating GDF15, IGF, thyroid and reproductive hormone levels in 103 β‐thalassaemia patients were obtained. Results GDF15 levels were markedly elevated in thalassaemia patients (24.2‐fold with β‐thalassaemia major compared with healthy controls). Among patients with β‐thalassaemia major, the relationship between GDF15 and body mass index (BMI) was curvilinear with all individuals with GDF15 levels above 24,000 pg/mL having a BMI below 20 kg/m2. After adjustment for BMI, age and Tanner stage, serum IGF1 concentrations correlated negatively with GDF15 in all thalassaemia patients (β = −.027, p = .02). We found a significant positive relationship between GDF15 and gonadotropin (in both sexes) and testosterone (in males). Conclusions GDF15 levels were markedly elevated in patients with β‐thalassaemia and its association with BMI is consistent with the known effect of GDF15 to reduce body weight. The inverse association between GDF15 with IGF1 levels may reflect a neuroendocrine impact of GDF15 or an indirect effect via impaired nutritional state. The positive association with testosterone in males and gonadotropins in both sexes, was surprising and should prompt further GDF15 studies on the hypothalamic pituitary gonadal axis.


| INTRODUCTION
GDF15 is an endocrine signal of cellular stress. 1 Its levels are elevated in a wide range of chronic human diseases where they frequently correlate with disease severity and adverse outcomes. [2][3][4] GDF15 acts on receptors in the brain to elicit a range of illness behaviours and a neuroendocrine stress response. 5 Patients with thalassaemia have previously been reported to have extremely high levels of GDF15, which is believed to be derived from their expanded mass of abnormal red cell precursors. [6][7][8] GDF15 expression is increased in cells subject to an unfolded protein response 9 which is a prominent feature of thalassaemia erythroblasts. 6 Other studies have suggested a role for intracellular iron in the control of GDF15 expression. 10 Despite regular transfusion and the use of iron chelating agents, patients with thalassaemia still frequently suffer from problems of growth and development, endocrine dysfunction and poor quality of life. [11][12][13] We have undertaken studies in patients with thalassaemia to examine the relationship between levels of GDF15, anthropometric variables and endocrine parameters.

| Study participants
A cross sectional study was conducted at the Adolescent and Adult Thalassaemia Centre of the Colombo North Teaching Hospital, Ragama, Sri Lanka from January to March 2021. All patients with β-thalassaemia attending for blood transfusions during the study period were recruited into the study after obtaining informed written consent. A total of 103 patients with β-thalassaemia [78 with β-thalassaemia major, 18 with haemoglobin E (HbE) β-thalassaemia and 7 with β-thalassaemia intermedia] were recruited. The diagnosis of β-thalassaemia had been confirmed by haemoglobin subtype quantification by high performance liquid chromatography. We also recruited 5 carriers of β-thalassaemia trait (randomly selected from parents of patients with thalassaemia) and 5 non-thalassaemia controls (randomly selected from unaffected family members of patients) for the study. The ethical approval was obtained from the Ethics Review Committee of the Faculty of Medicine, University of Kelaniya, Sri Lanka (Ref. P/228/11/20l9) and performed in accordance to the declaration of Helsinki.

| Data collection procedure
After recruitment, basic sociodemographic and clinical data were gathered using a data collection form by interviewing patients and perusing clinical records. Height and weight were measured using calibrated instruments and pubertal stage was assessed using Tanner staging. Nonspecific symptoms related to food intake and lifestyle were gathered using questions that were answered 'yes' or 'no'.
Pretransfusion venous blood samples were collected during morning hours (at 9.00 AM where possible) and freshly separated plasma were stored and shipped frozen to Cambridge, United Kingdom for the measurement of GDF15, IGF1, oestradiol and testosterone. Haematological and other measurements were done in Sri Lanka. All participants were afebrile and free from signs of infection or inflammation at the time of blood sampling.

| Analytical methods
Samples were analysed for GDF-15 using the Roche Elecsys ® e411 GDF-15 Cobas electrochemiluminescent immunoassay (Roche Diagnostics). The assay range is 400−20,000 pg/mL, with samples that were initially above the measuring range being diluted in Roche 'Multiassay Diluent'. Assays were calibrated and quality controlled using the manufacturer's reagents. Over two levels of controls, the coefficient of variation (CoV) of both controls was <5%.
IGF-1 levels were measured using the DiaSorin Liaison ® XL IGF-1 one-step sandwich chemiluminescence immunoassay after prior separation of IGF-1 from binding proteins (DiaSorin). The assay range is Full blood counts were performed using Beckman Coulter LH 500 automated full blood count analyser. Serum TSH, free thyroxine, LH, FSH and cortisol were measured using Tosoh Bioscience ® AIA-360 automated chemiluminescence immunoassay analyser in a clinically accredited laboratory.

| Comparative statistical analyses
Continuous variables were analysed using linear regression (transforming data before analysis where necessary so that the residuals were normally distributed). Confounders [particularly sex, age and body mass index (BMI)] were added to statistical models as appropriate. After visual inspection of data using scatter diagrams, in certain cases quadratic associations were tested by adding squared independent variables (e.g., BMI) into linear regression models.
Missing data were treated by pairwise deletion. Data are shown as geometric means (95% confidence intervals) unless stated otherwise.

| Background characteristics of study participants
Of the 103 patients with thalassaemia, 59 (57.3%) were males. The mean age of β-thalassaemia patients was 24.6 (±9.5) years and that of βthalassaemia carriers and non-thalassaemia controls were 40.4 (±1.5) and 34

| Association between GDF15 and BMI
Next, we analysed for the associations of GDF15 concentration with anthropometric, appetitive and endocrine markers, both for the All Thalassaemias group [Thal major, E-Thal and Thal intermedia (n = 103)] and for the Thalassaemia Major alone (n = 78). In adult participants (age >17 years), after adjustment for age and sex, there was no association between GDF15 and height (Figure 2A

| DISCUSSION
In this study we confirmed previous reports that the β-thalassaemias are associated with a substantial elevation in levels of circulating GDF15 and that, in patients with β-thalassaemia major, pretransfusion haemoglobin levels are inversely associated with circulating GDF15. The reason for the higher levels of GDF15 in males than females is unclear.
GDF15 is known to be expressed at high levels in erythroblasts 6 and its expression is highly responsive to a range of cellular stresses 5 several of which are likely to be active in the thalassaemic erythroblast undergoing ineffective erythropoiesis. It has been assumed that the extremely high levels of circulating GDF15 in thalassaemia major are derived largely from the haematopoietic compartment but this has not been formally established. All cells can increase GDF15 secretion when stressed and it is possible that non haematopoietic compartments make some contribution to the high GDF15 levels in thalassaemia, which to date has been reported to be a marker of ineffective erythropoiesis and an indicator of disease severity. 14 The suggestion that it might play a more direct role in the regulation of iron homoeostasis through actions in the liver 6 are difficult to reconcile with the fact that the only known receptor for GDF15 is exclusively expressed in the brain.
Our study population consisted of patients with transfusion dependent β-thalassaemia major, HbE β-thalassaemia and βthalassaemia intermedia. Although, HbE β-thalassaemia is considered a less severe phenotype, our results showed that these patients have the highest GDF15 levels. This could be because patients with HbE βthalassaemia are frequently suboptimally transfused due to the lack of uniform guidelines to guide transfusion therapy in these patients. 15 We recently reported that patients with HbE βthalassaemia are chronically under-transfused and have lower pretransfusion haemoglobin levels and higher prevalence of hepatosplenomegaly compared to patients with β-thalassaemia major. 16 The β-thalassaemias are frequently accompanied by a range of systemic disorders 17,18 not all of which are necessarily directly related to a low oxygen carrying capacity. 19 These include failure to thrive, impaired linear growth, 20  thalassaemia. Similarly, a study done among a cohort of gastric cancer patients did not demonstrate any association between blood transfusion and circulating GDF15 levels. 24 It has long been established that GDF15 suppresses food intake and body weight in many different species including primates. [25][26][27] In various disease states, the development of cachexia is strongly associated with the elevation of levels of GDF15. 2 It is therefore notable that we did see a relationship between GDF15 and BMI in the context of thalassaemia that would be consistent with the known effects of GDF15 to reduce energy stores. The correlations we found were nonlinear, suggesting that there may be some threshold of GDF15 at which effects on energy balance begin to become more marked. Certainly, none of the patients who had a BMI above 21 kg/ m 2 had a GDF15 level that was above 21,000 pg/mL.
There is only limited data on the food intake behaviour and appetite in thalassaemia. There is evidence that patients with thalassaemia have micronutrient deficiencies related to inadequate intake which could be related to poor appetite. 28 In addition to its effects on appetite, GDF15 powerfully stimulates the hypothalamic pituitary adrenal axis. 29 No significant correlation was seen with cortisol levels in this study, but the dynamic and circadian nature of cortisol secretion is very poorly captured by random plasma samples, so it is perhaps unsurprising that no correlation was discernible.
Central hypogonadism is one of the commonest endocrine complications of β-thalassaemia. 30 We found no differences in GDF15 concentration between patients receiving replacement therapy with gonadal steroids and those not receiving such therapy.
This might be due to the fact that hormonal treatment in thalassaemia is rather complex due to the severity of iron overload associated with further endocrine complications 31 such as accumulation in different endocrine glands. 32 We noted a surprising, positive relationship between GDF15 levels and LH, FSH and testosterone in males (but not oestradiol in females) each of which remained statistically significant after correction for age, BMI (and sex in the case of LH and FSH). The effects of GDF15 on the hypothalamic pituitary gonadal axis have not been systematically studied in any detail and these findings should prompt further studies.
Disruption of thyroid function is a common feature of Thalassaemia. 33 Although GDF15 levels were somewhat higher in patients treated with thyroxin than those who were not, this did not reach formal statistical significance. There was no correlation between GDF15 and free T4 or TSH in patients not treated with thyroxine.

DATA AVAILABILITY STATEMENT
The data sets generated and/or analysed during the current study are not publicly available, since they are subject to national data protection laws and restrictions imposed by the ethics committee to ensure data privacy of the study participants. However, they can be applied for through an individual project agreement with the principal investigators.