Alexandra Dias

Genetic variation in CD36, HBA, NOS3 and VCAM1 is associated with chronic haemolysis level in sickle cell anaemia: a longitudinal study†

Chronic haemolysis stands out as one of the hallmarks of sickle cell anaemia, a clinically heterogeneous autosomal recessive monogenic anaemia. However, the genetic architecture of this sub‐phenotype is still poorly understood. Here, we report the results of an association study between haemolysis biomarkers (serum LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants of ten candidate genes in a series of 99 paediatric SS 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). Although in a large number of tests a seemingly significant (i.e. P < 0.05) association was observed, 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. On the whole, our findings suggest a complex genetic architecture for the sickle cell anaemia haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.

Sickle cell disease severity scoring: a yet unsolved problem

To the editor: Sickle cell disease (SCD) is a monogenic disorder under polygenic and environmental control. This aetiopathogenic architecture leads to marked clinical heterogeneity with the emergence of multiple and diverse subphenotypes, which makes the severity stratification of patients particularly difficult (1, 2). A number of severity scores have been proposed, aiming at the integration of many clinical and laboratory dimensions into a meaningful single synthetic measure of morbidity and/or risk of death within a given period. Following the cross-validation study recently published in this journal (3), we have analysed the correlation between two thoroughly developed scores, namely a disease severity score (DSS) (4) and a paediatric severity score (PSS) (5), in a series of 99 paediatric SS patients followed up in two large general hospitals in Greater Lisbon area, Portugal. The evaluated patients were mostly (97%) of Sub-Saharan African ancestry and presented an M/F ratio of 1.17, a median current age of 9.9 yr and a median follow-up/patient of 5.0 yr. Patients’ severity scores were established by interrogating the clinical database derived from medical records for the specific variables considered in each score. DSS was calculated via the http://www.bu.edu/sicklecell/downloads/Projects website. The same definitions of adverse events were used by all the clinicians who constructed and completed the database. The normality of scores distribution was tested with the Shapiro–Wilk test (a = 0.05), and their statistical relationship was evaluated by Spearman correlation. Interrater agreement regarding the severity groups defined by each score was assessed by weighted kappa measure (j) (6). The statistical analysis was performed with SPSS v20 (IBM Corp. Armonk, NY, USA). This study was approved by INSA’s Ethics Committee as part of a wider research on the development and validation of vasoocclusion early predictors in SCD. Appropriate written informed consent was obtained. Both DSS and PSS displayed a non-normal (P < 0.01) multimodal distribution (see Fig. 1A, B) with cut-off values arbitrarily defined at 0.400 and 40, respectively. The Spear-

Hemorheological alterations in sickle cell anemia and their clinical consequences – The role of genetic modulators

Sickle cell anemia (SCA) is an autosomal recessive disease caused by the HBB:c.20A>T mutation that leads to hemoglobin S synthesis. The disease presents with high clinical heterogeneity characterized by chronic hemolysis, recurrent episodes of vaso-oclusion and infection. This work aimed to characterize by in silico studies some genetic modulators of severe hemolysis and stroke risk in children with SCA, and understand their consequences at the hemorheological level.Association studies were performed between hemolysis biomarkers as well as the degree of cerebral vasculopathy and the inheritance of several polymorphic regions in genes related with vascular cell adhesion and vascular tonus in pediatric SCA patients. In silico tools (e.g. MatInspector) were applied to investigate the main variant consequences.Variants in vascular adhesion molecule-1 (VCAM1) gene promoter and endothelial nitric oxide synthase (NOS3) gene were significantly associated with higher degree of hemolysis and stroke events. They potentially modify transcription factor binding sites (e.g. VCAM1 rs1409419_T allele may lead to an EVI1 gain) or disturb the corresponding protein structure/function. Our findings emphasize the relevance of genetic variation in modulating the disease severity due to their effect on gene expression or modification of protein biological activities related with sickled erythrocyte/endothelial interactions and consequent hemorheological abnormalities.