Abstract

Several genetic factors modulate the clinical course of sickle cell anemia (SCA), such as the βs-haplotypes, 3.7кb α-globin gene deletions (-α3.7), glucose-6-phosphate dehydrogenase deficiency (G6PD), UGT1A1 polymorphism and influencers of fetal hemoglobin production. While many studies on these modifiers have been conducted among patients in the developed world, little is known about how they impact SCA among Nigerians.

We hypothesized that these factors could modulate SCA in the Nigerian population. Hence, we set out to determine whether βs-haplotype, -α3.7deletion, G6PD, and UGT1A1 polymorphism influence the laboratory markers and clinical events in young Nigerians with SCA.

The study was conducted among 101 hydroxyurea-naïve SCA patients aged 2-21years, (median 9 years) in steady state, on regular follow up at the pediatric hematology unit of the Ekiti State University Teaching Hospital (EKSUTH), Ado Ekiti, Southwest Nigeria.

Molecular analysis were conducted to confirm the diagnosis of SCA by polymerase chain reaction (PCR) and direct sequencing; alpha thalassemia by GAP-PCR; to determine UGT1A1 genotypes by Fragment Analysis; and BS haplotypes and G6PD genotypes by TaqMan SNP genotyping assay at the Hemocentro - University of Campinas, Brazil. Automated analysers were used to carry out hematologic and biochemical analyses. Records of the patients were examined for clinical events and steady state laboratory parameters. Serial abdominal ultrasounds were done as clinically indicated to diagnose gallstones. Institutional review board approval was obtained and the study protocols followed the Declaration of Helsinki as revised in 2000.

Four (TA)n alleles (5, 6, 7, and 8) were found with gene frequencies of 0.11, 0.44, 0.40, and 0.05, respectively, grouped into 11 genotypes: TA5/5, 5/6, 5/7, 5/8, 6/5, 6/6, 6/7, 6/8, 7/7, 7/8, 8/8. Low Activity UGT1A1 genotypes (TA7/7, 7/8, 8/8) were found in 25 (24.7%). The Benin/Benin βs-haplotype was found in 94 (93%), Benin/Cameroun 4 (4%), Benin/Bantu 2 (2%), and Benin/Arab 1(1%). Mutation for the African variant of G6PD deficiency (202G>A) was found in 21(20.8%). None had the Mediterranean (563C>T) mutation. The frequency of 3.7 κb α-globin gene deletion was 34 (33.6%) αα /-α3.7 as heterozygous, 7 (7%) -α3.7/-α3.7 homozygous. Additionally, we found one (1%) α-globin triplication (αααanti3.7/αα).

There was significant difference in bilirubin levels of the patients when differentiated by the UGT1A1 genotypes according to expected enzyme activity (low > intermediate > high activity with median unconjugated bilirubin levels of 1.8, 0.8, and 0.6mg/dL, respectively, P <0.0001). The presence of either alpha thalassemia or G6PD deficiency or both did not alter this trimodal pattern of bilirubin levels. Asymptomatic gallstone was found in 6 (5.9%) patients aged 10 to 16 years. Five of them had the low UGT1A1 activity genotypes 4 (TA7/7) & 1(TA7/8). The low genotype was significantly associated with gallstone when compared to other genotype groups 5/25 vs 1/76, P=0.0033. Bilirubin levels of patients with gallstone was significantly higher than age & sex matched subjects without gallstone (4.7 vs 1.2mg/dl, p=0.002) suggesting that the pathway to developing hyperbilirubinemia and gallstone is primarily driven by the influence of UGT1A1 genotype of our study cohorts.

When we evaluated the influence of -α3.7 deletion, we found that participants with alpha thalassemia had higher frequencies of pain crisis in the preceding one year of the study with median episodes of 3 (range 0-6) vs 0 (range 0-6), P <0.0001. The presence of alpha thalassemia was significantly associated with decreased mean corpuscular volume, mean corpuscular hemoglobin, reticulocyte count and with a higher hemoglobin concentration (P <0.05) However, none of the 6 participants with leg ulcers had alpha thalassemia 0/ 42 vs 6/59 ( P=0.039). There were no significant associations of βs-haplotypes and G6PD deficiency with both laboratory parameters and clinical events.

In conclusion, our findings confirmed that both alpha thalassemia and UGT1A1 polymorphism influence the laboratory markers and clinical events of young Nigerians with SCA. It appears the influence of UGT1A1 polymorphism outweighs the modest ameliorating effect of alpha thalassemia on hyperbilirubinemia and cholelithogenesis among the patients.

Disclosures

Fertrin: Alexion Pharmaceuticals: Consultancy.

Author notes

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Asterisk with author names denotes non-ASH members.