LEVELS OF MICRORIBONUCLEIC ACID 125b(miRNA125b) AS A MARKER OF SEVERITY AND FREQUENCY OF VASO-OCCLUSIVE CRISIS IN PATIENTS WITH SICKLE CELL ANAEMIA

Abstract

INTRODUCTION: The disease burden of Sickle cell anaemia (SCA) with its associated morbidity and mortality is very high in our environment. Treatment for this disorder has gradually shifted from symptomatic treatment to treating the molecular basis of the disorder. Vaso-occlusive crisis (VOC) is the commonest type of crisis found in sickle cell anaemia and this has been found to occur because of inflammatory changes in the vessel wall mediated by pro-inflammatory cytokines especially tumor necrosis factor alpha (TNF-α). Expression of TNF-α is regulated by microribonucleic acid 125b (miRNA125b) which enhances degradation of TNF-α messenger ribonucleic acid(mRNA) thus reducing the synthesis and plasma level of TNF-α . This study sets to establish any correlation between miRNA125b and frequency of vaso-occlussive crisis which may possibly open up a viable target for molecular treatment of SCA.
OBJECTIVES: This study aims to assess if there is any significant difference in levels of miRNA125b and frequency of crisis in SCA patients and to determine if quantity of miRNA 125b correlate with plasma concentration of TNF-α.
STUDY SETTING: This was a prospective, non-randomized case controlled study of eighty SCA patients recruited by simple consecutive sampling from the sickle cell clinic of University of Nigeria Teaching Hospital, Enugu. The SCA patients were categorized into Group A : those that have three or more vaso-occlusive crisis per year and group B: those that have zero to one vaso-occlusive crisis per year.
METHODOLOGY: Venous blood (10mls) was collected from each SCA patient and put into bottles containing the anticoagulant ethylene diamine tetra –acetic acid (EDTA). The blood was centrifuged to separate the cells from the plasma. The plasma was stored at minus 80 degrees Centigrade. Red blood cells were lysed using ammonium chloride. The leucocytes were processed to obtain a guanidium isothiocyanate (GITC) lysate which contains genomic DNA and RNA. Total ribonucleic acid (RNA) was extracted from the GITC lysate and complementary DNA (cDNA) was synthesized by reverse transcription.



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MiRNA125b and Abelson gene levels were quantified using polymerase chain reaction (PCR). Quantitation of Abelson gene was done to obtain the level of a normal gene in the sample (a reference parameter) used to calculate the normalized quantity of miRNA125b. Plasma level of TNF-α was also quantified using enzyme linked immunosorbent assay (ELISA) while full blood count was done using a Mythic 22 automated blood cell counter.
RESULT: The median normalized miRNA125b was 0.215 (IR- 0.093- 0.365) for SCA patients who have three or more vaso-occlusive crisis per year and 0.145 (IR- 0.085- 0.218) for those with zero to one vaso-occlusive crisis per year. This difference failed to reach significance level. (p= 0.074).The quantity of miRNA125b in SCA patients showed no correlation with plasma concentration of TNF (p=0.703). There was a significant difference between the mean total leucocyte count in patients who have three or more vaso-occlusive crisis per year (11.2 x 109/l ± 3.8 x 109/l) and mean value of leucocyte count (9.2 x 109 /l± 1.9 x 109/l) in those with zero to one vaso- occlusive crisis per year (p=0.005). Also, the mean of the absolute neutrophil count for patients with three or more VOCs per year (6.3 x109/l ±2.6 x 109/l) was significantly higher than the value (4.4 x109/l ± 1.2 x 10/l) for those with 0-1 VOCs per year (0.000)
CONCLUSION: There was no significant difference between the levels of miRNA125b in sickle cell anaemia patients who have three or more vaso-occlusive crisis per year and those with zero to one vaso-occlusive crisis per year. Also the quantity of miRNA125b in SCA patients had no correlation with plasma concentration of TNF-α