Biomarkers and genetic Polymorphisms associated with Stroke Risk in Children with Sickle Cell Anaemia in Lagos, Nigeria

Author: Ojewunmi, Oyesola Oyewole

Supervisors: Osuntoki A. A. and Imaga N. O. A.

Stroke, initiated by an increase in cerebral blood flow velocity, is one of the neurological complications associated with sickle cell anaemia, contributing significantly to childhood morbidity and mortality. Stroke risk detection using Trans-cranial Doppler ultrasound (TCD) scan is a useful screening tool for the prevention of first stroke occurrence in children with sickle cell anaemia (SCA) thus averting stroke complications. TCD measures elevated blood flow velocity through the time-averaged mean of maximum velocities (TAMMVs) in the internal carotid arteries and middle cerebral arteries. This study investigated the relationship between TCD velocity and clinical/laboratory markers of oxidative stress and inflammation, and polymorphisms of some associated genes that could predict stroke in children with sickle cell anaemia in Lagos, Nigeria. This study consisted of one hundred and forty-seven children with sickle cell anaemia (mean age, 7.16±3.84 years) and seventy three age-matched apparently healthy non-sickle cell anaemia subjects (mean age, 7.04±3.37 years). TCD was performed on subjects with SCA following standard protocol. Subjects with normal TCD measurements (TAMMV <170cm/s) were classified as negative TCD (NTCD) while subjects with conditional and abnormal risk were classified as having positive TCD (PTCD) measurements (TAMMV ≥170cm/s). Haemoglobin oxygen saturation (SpO2) and haematological profile including haemoglobin fractions were determined. Nitric oxide metabolites and lactate dehydrogenase activity (LDH) as markers of intravascular haemolysis were also evaluated. Genomic DNA was extracted from subjects’ blood samples to determine the genetic polymorphisms in the genes of glutathione S-transferase (GST: T1, M1, and P1), Platelet glycoprotein Ia and IIIa (GP Ia and GP IIIa), C667T and A1298C of Methylene tetrahydrofolate reductase (MTHFR), TNF-alpha (308 G>A), Factor V Leiden (G1691A) and Prothrombin (G20210A) using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and restriction fragment length polymorphism (RFLP). Results showed that increased risk of positive TCD was associated with lower SpO2 (<96%; p=0.046; OR: 7.48; 95% CI: 2.10 - 26.65), higher Leucocytes count (>13.1 x 109/L; p=0.011; OR: 2.44; 95%CI: 1.22 – 4.91), and higher LDH (>663.5 IU/L; p= 0.001; OR: 4.04; 95%CI: 1.71- 9.56) compared to the negative TCD. Both GSTT1 and GSTM1 deletions did not show any association with stroke risk in subjects with SCA (p>0.05). GSTP1 genotyping showed that the frequency of the combined Val variant genotype (Ile/Val + Val/Val) was significantly lower in non-SCA subjects than the SCA subjects (37.9% vs. 69.9%, p<0.05; OR: 3.91, 95%CI: 2.10-7.29) but the frequency of subjects in PTCD group was similar to those with NTCD. The frequency of GpIa T allele was higher in SCA subjects compared to non-SCA subjects (28.0% vs. 10.0%) with a significant association of GpIa TT genotype in SCA subjects (OR= 16.8, 95% CI= 0.97-289.9; p<0.01) compared to non-SCA subjects. T allele of C667T MTHFR gene in PTCD group was significantly lower (p<0.05) compared to NTCD group. This study revealed that low haemoglobin oxygen saturation, high Leucocyte count, and high lactate dehydrogenase are independent risk factors for abnormal cerebral blood flow velocity. These are suggestive of chronic hypoxia and severe anaemia which are risk factors for stroke in subjects with SCA. The presence of T allele of Methylene tetrahydrofolate reductase C667T gene confers protection against stroke risk while polymorphisms of Factor Leiden and Prothrombin genes known to contribute to stroke development are not present in our population.