Haemolysis in sickle cell anaemia: a genotype/phenotype association study

Introduction: Sickle cell anaemia (SCA) is a clinically heterogeneous autosomal recessive monogenic anaemia characterised by chronic haemolysis and recurrent episodes of severe vaso-occlusion and infection. Several environmental and genetic determinants have been suggested to modulate the onset, course and outcome of SCA. The level of chronic haemolysis has been considered a critical measure of SCA severity and a possible proximate cause of some disease complications such as stroke, pulmonary hypertension, priapism, leg ulceration and cholelithiasis. Thus, we proposed to search for genetic modifiers of this sub-phenotype and gain insights into the underlying mechanisms. Patients and Methods: We studied the association between commonly measured haemolysis biomarkers (LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants (34 SNP, 6 indel, 1 STR) of 10 candidate genes in a longitudinally observed series of 99 paediatric homozygous SCA patients (median current age of 9.9 yr) followed up in two general hospitals in Greater Lisboa area (median follow-up per patient of 5.0 yr). Candidate gene genotyping was performed by PCR-RFLP, Sanger sequencing, Gene Scan or Gap-PCR. All genotype distributions were tested for adherence to the Hardy-Weinberg equilibrium. When appropriate, haplotypes were inferred by software PHASE, version 2.1.1 Results: Although in a large number of tests seemingly significant association was observed only the following ones were confirmed upon correction for multiple comparisons: i) an increased serum LDH level was associated with haplotype 7 within VCAM1 gene; ii) a lower total bilirubin was associated with the 3.7-kb deletion at HBA gene, rs2070744_T allele at NOS3 gene, and haplotype 9 within VCAM1 promoter; and iii) a diminished reticulocyte count was associated with the 3.7-kb deletion at HBA, whereas an increased count was associated with rs1984112_G allele at CD36 gene. Conclusion: On the whole, our findings suggest a complex genetic architecture for the SCA haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.Partially funded by FCT: PIC/IC/83084/2007 and PEst-OE/SAU/UI0009/201