Functional iron deficiency anaemia and its genetics in a black South African population with chronic kidney disease

Nalado, Aishatu Muhammad
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Background: Anaemia of chronic kidney disease (CKD) and iron deficiency anaemia (IDA) has been associated with morbidity and mortality in patients with CKD. Consequences of these changes differ across different races. Diagnosis of IDA is usually straightforward, except when it occurs in the context of inflammatory disorders such as CKD, where the conventional parameters used to diagnose IDA are not definitive. Hence, there is a need for newer and more accurate biomarkers in the diagnosis of IDA in CKD patients. Studies in developed countries have reported the usefulness of newer parameters in IDA diagnosis; however, the role of these newer biomarkers in IDA diagnosis in Africa is unknown. It remains unclear whether genetic factors may increase susceptibility to the development of IDA in CKD patients. Therefore, this study has been conducted to determine the prevalence and racial prevalence of IDA; to determine the utility of newer biomarkers in the diagnosis of IDA and the effect of these biomarkers on disease outcomes; and to provide important insights into the prevalence and genetic susceptibility of IDA in CKD patients in sub-Saharan Africa. Methods: This was a cross-sectional study carried out from May 2016 to December 2018, involving 365 CKD patients from the Renal Outpatient Clinic of the Charlotte Maxeke Johannesburg Academic Hospital, South Africa, and 144 age- and gender-matched healthy controls comprising hospital staff and the patients’ relatives. The study was approved from the Human Research Ethics Committee of the University of the Witwatersrand (clearance certificate number, 150929). The first part of the study described the prevalence of anaemia and IDA in a multi-ethnic CKD population. The second part of the study determined the utility of reticulocyte haemoglobin content (CHr), and % hypochromic red cells (%HYPO), in the diagnosis of IDA, followed by role of novel biomarkers, GDF-15 and hepcidin, as diagnostic markers of IDA. Hepcidin and GDF-15 were measured using mass spectrometry and ELISA, respectively and was followed by the genetic aspect of the study that determined the susceptibility of a genetic variant to IDA. The last part of the study looked at role of GDF-15 and hepcidin in disease progression and mortality using Spearman’s rank correlation and linear and logistic regression analysis. Receiver operator curves (ROCs) were used to evaluate the diagnostic utility of biomarkers. Using Cox proportional hazard models, a composite endpoint of CKD progression was also investigated. Results: The prevalence of anaemia (Hb < 13 g/dl in males and < 12 g/dl in females) among black CKD patients was 46.9 %, with 26.1% of the sample population suffering from IDA, which proved to be three times more frequent among the CKD patients than among the controls. The proportions of functional iron deficiency anaemia (18.6 % vs 1.4 %) and absolute iron deficiency anaemia (16.7 % vs 7.8 %) were higher among the CKD patients than among the controls. However, 32.3 % (95% CI: 27.90 %– 37.10 %) of the study population presented with iron deficiency without anaemia. The mean age of the CKD patients was higher than that of the controls (52.7  14.3 vs 40.4  12.6 years, P < 0.001). The sensitivity and specificity values for diagnosing IDA among CKD participants were 62.6 % and 80.2 % respectively for CHr at a cut-off value lower than 28 pg, and 63.3 % and 79.8 % for the proportion of hypochromic red cells (%HYPO), respectively. The (define here) AUC of CHr (0.81, 95 % CI: 0.76-0.87) was higher than the AUC of %HYPO (0.76, 95 % CI: 0.76-0.87). The predictive value of diagnosing IDA among CKD patients using GDF-15 and hepcidin was high (AUC= 0.723 and 0.714, respectively). The prevalence of rs 855791 C homozygosity was similar for the iron-deficient and non-iron-deficient anaemia groups (86.1 % vs 84.2 %, p= 0.723). When the analysis was confined to participants with or without functional IDA, however, the C homozygote (88.3 % vs 84.4 %, p= 0.425) value was similar for both groups. Patients with high levels of hepcidin were up to six times more likely to be at risk of dying as opposed to patients with normal levels of hepcidin (HR: 6.14; P< 0.001). On the other hand, it was determined that the levels of GDF-15 and IDA were not associated with mortality in CKD patients. Higher plasma levels of hepcidin were strongly associated with CKD progression when compared to normal hepcidin levels (HR: 3.09; P= 0.002). The risk of CKD progression was two times higher in the presence of hypocalcaemia as opposed to normocalcaemia (HR: 2.24; P= 0.021). The death rate increased with declining haemoglobin levels from 0.96 among patients with mild anaemia, to 4.29 per 100-person years, among patients with severe anaemia. Anaemic patients with a glomerular filtration rate (GFR) of less than 30 mls/min were susceptible to a significantly greater risk of death, due mainly to end stage kidney disease (ESKD) and failure to commence haemodialysis because of a lack of funding (HR 11.51: 95% CI 1.62–78.32, P < 0.001). Participants with hyperphosphatemia had about a 2.6-fold increased risk of death (HR, 2.60; 95% CI, 1.09-6.20, P= 0.02). Although the odds of anaemia were 3.8-fold higher (odds ratio = 3.8, P = 0.059) among CKD stage 5 participants as opposed to those with CKD stage I, the relationship between anaemia and the stages of CKD proved to be non-linear. Conclusions: Anaemia and IDA were common in our pre-dialysis CKD patients. Anaemia, elevated hepcidin levels, hypocalcaemia and low estimated glomerular filtration rates (eGFRs), were found to be associated with an increased risk of death among pre-dialysis CKD patients. Our study also highlighted that newer biomarkers (novel) can be useful in the diagnosis of IDA and can perform more effectively than the conventional parameters (transferrin saturation (TSAT) and ferritin) in an African CKD population. In addition, our study showed that newer biomarkers can be used to predict disease progression and mortality in a CKD population. This suggests that health care services for chronic diseases should be prioritise pre-dialysis CKD patients.
A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Doctor of Philosophy. Johannesburg, 2019
Nalado, Aishatu Muhammad, Functional iron deficiency anaemia and its genetics in a Black South African Population with Chronic Kidney Disease, University of the Witwatersrand, Johannesburg, <>