EVALUATION OF STRUCTURAL VARIATION OF 12-LIPOOXYGENASE GENE IN RELATION TO VASO-OCCLUSIVE EVENTS IN SICKLE CELL DISEASE

Abstract

Although the genetic variables that affect the severity of sickle cell disease (SCD) are not
fully understood, it is known that inflammation predisposes to vaso-occlusion - a major cause
of organ damage in SCD. The enzyme 12-lipooxygenase catalyses conversion of arachidonic
(fatty) acid to the pro-inflammatory molecule 12-Hydroxyeicosatetraenoic acid (12-HETE).
The gene for 12-lipooxygenase (ALOX12) is known to have single nucleotide polymorphisms
(SNPs) in its structure. Previous studies show that such variation in the structure of a gene
can affect the function and end biological effects of its protein product. In the light of this,
the aim of this study was to find out if the number of vaso-occlusive events in SCD is
affected by two known SNPs in ALOX12 gene.
There were 80 study participants: 20 HbSS individuals with mild SCD, 30 HbSS persons
who have severe SCD, and 30 HbAS or HbAA siblings of the patients with severe SCD. The
participants included 20 children and 60 adults. The mean age was 23.97 + 9.76 (range 6.0 to
52.0) years for patients with severe SCD, 18.75 +8.62 (range 4.0 to 28) years for those with
mild disease, and 24.47 + 11.04 (range 5.0 to 57.0) years for the sibling controls.
DNA samples from the participants were analyzed to determine the nucleotides present at the
positions of two known SNPs in ALOX12 gene: rs2073438 and Gln261Arg. The nucleotide
polymorphisms involve having Guanosine (G) or Adenosine (A) in rs2073438 position, and
either G or A in Gln261Arg position. Plasma concentrations of 12-HETE, tumour necrosis
factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) were measured by enzyme-linked
immunosorbent assay (ELISA); and full blood count determined. The number of vaso
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occlusive events and socio-demographic characteristics of the SCD patients were obtained
from data in the hospital records.
Results showed that the two SNPs: rs2073438 and Gln261Arg were present in all the
study participants. With respect to SNP rs2073438, the proportions of patients with severe
SCD and mild disease did not differ significantly between the homozygous G/G and
heterozygous G/A persons (χ2 = 0.087, p = 0.768). This suggests that neither mild nor severe
SCD is associated with having the nucleotide G or A at the rs2073438 position. Also, at the
Gln261Arg SNP position, there were no significant differences in the proportions of patients
with mild or severe SCD between homozygous CGG/CGG individuals and heterozygous
CAG/CGG persons (χ2 = 1.333, p = 0.248). Both the homozygous and heterozygous
genotypes of rs2073438 and Gln261Arg SNPs were not associated with reduced or increased
numbers of vaso-occlusive events in SCD patients. Median plasma levels of 12-HETE, IL-1β
and TNF-α in patients with severe SCD were significantly higher than in the sibling controls
and in patients with mild disease (χ2 = 61.633, p < 0.001); (χ2 = 16.100, p < 0.001) and (χ2 =
34.047, p < 0.001) respectively. None of the alternative nucleotides (G or A) in the two SNPs
analyzed was significantly associated with high plasma concentrations of 12-HETE, IL-1β or
TNF-α (U = 13.00, p = 0.862) and (U = 108.50, p = 0.933) respectively. Severe SCD patients
had significantly higher leucocyte and platelet counts than their HbAS/HbAA siblings, and
patients with mild SCD.
In conclusion, data from this study suggest that the number of vaso-occlusive events in SCD
is not affected by the SNPs rs2073438 and Gln261Arg in the gene for 12-lipooxygenase.