BMP6 and VDR gene polymorphisms are associated with osteonecrosis in a sickle cell anaemia cohort.

The occurrence and severity of osteonecrosis in sickle cell anaemia (SCA) vary due to risk factors, including genetic modifiers. Bone morphogenetic proteins (BMPs), particularly BMP6, and the vitamin D receptor (VDR) play key roles in cartilage and bone metabolism, making them potential contributors to orthopaedic outcomes in SCA. Here, we evaluated the association of polymorphisms in BMP6 (rs3812163, rs270393 and rs449853) and VDR (FokI rs2228570 and Cdx2 rs11568820) genes with osteonecrosis risk in a Brazilian SCA cohort. A total of 177 unrelated SCA patients were selected. The AA genotype of BMP6 rs3812163 was independently associated with a lower osteonecrosis risk (p = 0.015; odds ratio (OR): 0.38; 95% confidence interval (CI): 0.18-0.83) and with the long-term cumulative incidence of osteonecrosis (p = 0.029; hazard ratio: 0.56, 95% CI: 0.34-0.94). The VDR rs2228570 TT genotype was independently associated with a lower osteonecrosis risk (p = 0.039; OR: 0.14; 95% CI: 0.02-0.90). In summary, our results provide evidence that BMP6 rs3812163 and the VDR rs2228570 might be implicated in osteonecrosis pathophysiology in SCA and might help identify individuals at high risk.

Sickle Cell Disease in Brazil: Current Management.

Sickle cell disease (SCD) comprises inherited red blood cell disorders due to a mutation in the ß-globin gene (c20A > T, pGlu6Val) and is characterized by the presence of abnormal hemoglobin, hemoglobin S, hemolysis, and vaso-occlusion. This mutation, either in a homozygous configuration or in compound states with other ß-globin mutations, leads to polymerization of hemoglobin S in deoxygenated conditions, causing modifications in red blood cell shape, particularly sickling. Vaso-occlusive crisis (VOC) is the hallmark of the disease, but other severe complications may arise from repeated bouts of VOCs. SCD is considered a global health problem, and its incidence has increased in some areas of the world, particularly the Americas and Africa. Management of the disease varies according to the region of the world, mainly due to local resources and socioeconomic status. This review aimed to describe more recent data on SCD regarding available treatment options, especially in Brazil. New treatment options are expected to be available to all patients, particularly crizanlizumab, which is already approved in the country.

A-296G variant of THBS1 gene (rs1478605) is associated with a lower frequency of stroke in a Brazilian population with sickle cell anemia.

OBJECTIVES: Stroke is a devastating clinical outcome that significantly contributes to the morbidity and mortality of sickle cell anemia (SCA) patients. Despite its advantages in predicting stroke risk, transcranial Doppler screening has limitations that restrict its applicability, highlighting the need for emerging prognostic tools. Thrombospondin-1 plays a crucial role in endothelial injury, platelet adhesion, and nitric oxide metabolism and may be implicated in stroke pathophysiology. Here, we aimed to evaluate the association of THBS1 genetic variations with the occurrence of stroke in SCA patients MATERIALS AND METHODS: By real-time PCR, 512 SCA patients were fully genotyped for THBS1 A-296G (rs1478605) polymorphism RESULTS: THBS1 GG genotype was associated with a lower risk for stroke occurrence [odds ratio (OR): 0.30; 95% confidence interval (CI): 0.11-0.78; P = 0.011], although these findings were not consistent with multivariate logistic regression analysis (OR: 0.73, 95% CI: 0.12 - 4.37; P = 0.736). In agreement, the cumulative incidence of stroke for patients with AG/AA genotypes was higher when compared to the GG genotype (P = 0.018). However, the association was not maintained in the multivariate proportional hazards model (hazard ratio: 0.67, 95% CI: 0.12-3.61; P = 0.643) CONCLUSIONS: In summary, the present study shows that the THBS1 A-296G (rs1478605) polymorphism may be a potential modifier for stroke in SCA.

Polymorphisms and gene expression of metalloproteinases and their inhibitors associated with cerebral ischemic stroke in young patients with sickle cell anemia.

BACKGROUND: Sickle cell anemia (SCA) is a genetic disease with great clinical heterogeneity and few viable strategies for treatment; hydroxyurea (HU) is the only widely used drug. Thus, the study of single nucleotide polymorphisms (SNPs) and the gene expression of MMPs 1, 2, 9, 7 and TIMPs 1 and 2, which are involved in the regulation of extracellular matrix, inflammation, and neuropathies, may provide further insights into the pathophysiology of the disease and elucidate biomarkers and molecules as potential therapeutic targets for patients with SCA. METHODS AND RESULTS: We evaluated 251 young individuals with SCA from northeastern Brazil. The groups were divided according to vaso-occlusive crisis (VOC) and cerebrovascular disease (CVD), compared to control individuals. SNP detection and gene expression assays were performed by real-time PCR, TaqMan system®. Both the expression levels of MMP1 gene, and the SNP MMP1-1607 1G/2G were associated with the risk of cerebral ischemic stroke (IS), and the expression of MMP1 was also associated with a higher frequency of VOC/year. Expression levels of MMP7, TIMP1, and TIMP2 were increased in patients conditioned to IS. The SNP 372T>C (rs4898) TIMP1 T alleles were more frequent in patients with > 5 VOC events/year. The SNP rs17576 of MMP9 showed differences in gene expression levels; it was increased in the genotypes AG, and AG+GG. CONCLUSION: The findings of this study, the SNPs, and expression provide initial support for understanding the role of MMPs-TIMPs in the pathophysiology of SCA in young patients.

Novel Insights into the Pathophysiology and Treatment of Sickle Cell Disease.

The polymerization of hemoglobin under deoxygenation is the main pathophysiological event in sickle cell diseases, described more than 70 years ago. The last two decades have seen a major increase in knowledge about the cascade of events that follow the polymerization of hemoglobin and the ensuing sickling of red blood cells. Several distinctive therapeutic targets have been discovered as a result, and a few drugs with innovative mechanisms of action are already on the market, while several others are the focus of ongoing trials. The aim of this narrative review is to describe some of the more recent data in the SCD literature regarding pathophysiology and novel treatments.

The ratio of ATP11C/PLSCR1 mRNA transcripts has clinical significance in sickle cell anemia.

One of the physiologic mechanisms responsible to maintain asymmetric phospholipid distribution (in particular phosphatidylserine, PS) in human erythrocyte membranes is orchestrated by the balance between enzymes responsible for active transport of PS from the outer to the inner leaflet (ATP11C) and those whose counteracts these activities (PLSCR1). Using quantitative real-time polymerase chain reaction and standard flow cytometry procedures, we hypothesized that the aberrant expression of either or both ATP11C and PLSCR1 transcripts may disrupt the PS internalization/externalization process and become clinically relevant for patients with sickle cell anemia (SCA). Overall, neither ATP11C/PLSCR1 ratio or ATP11C and PLSCR1 (if analyzed separately) had impact on risk to present acute or chronic organ damage in 178 patients with SCA. By collecting a new set of samples from SCA patients during a vaso-occlusive crisis (VOC, crisis state, 13 patients) and comparing with new samples of patients in steady state (15 patients), we noticed that patients in steady state exhibited mean values of ATP11C/PLSCR1 ratio significantly higher (mean value: 18.2, range, 0.3-53) than those who were in crisis (mean value: 3.7, range, 0.5-9) (P = 0.013). Most importantly, there was a strong inverse correlation between PS exposure and ATP11C/PLSCR1 ratio in sickle erythrocytes (Pearson correlation coefficient, r: - 0.78). Based on these findings, it is conceivable that the ATP11C/PLSCR1 ratio may switch from high to low during a VOC, although the underlying reasons require further investigations.

Association of KLOTHO polymorphisms with clinical complications of sickle cell anemia.

The clinical and phenotypic heterogeneity of patients with sickle cell anemia (SCA) is influenced by environmental and genetic factors. Several genetic modifiers, such as the KLOTHO (KL) gene, have been associated with SCA clinical outcomes. The KL gene and its encoded proteins are implicated in important biological pathways, which affect the disease's pathophysiology, such as expression of adhesion molecules VCAM-1 and ICAM-1, oxidative stress, and nitric oxide biology. Here, we evaluated the clinical relevance of two polymorphisms found on the KL gene (rs685417 and rs211239) in 588 unrelated patients with SCA. Genotyping analyses were performed using the TaqMan system. The KL rs211239 was associated with increased number of vaso-occlusive crisis (VOCs) per year (P = 0.001), while KL rs685417 was associated with increased frequency of stroke (P = 0.034), priapism (P = 0.011), number of complications (P = 0.019), and with a lower incidence of priapism (P = 0.036). Additionally, the associations with VOCs, stroke, and priapism remained consistent in multivariate analyses (P < 0.05). Our data highlight the clinical importance of KL in SCA.

Effect of hydroxyurea therapy on intravascular hemolysis and endothelial dysfunction markers in sickle cell anemia patients.

Intravascular hemolysis (IH) contributes to the development of endothelial dysfunction (ED) in sickle cell anemia (SCA), and the effects of hydroxyurea (HU, the only approved drug that decreases the frequency and severity of vaso-oclussive crises) on IH and ED in SCA remain unclear. We evaluated and compared the markers of IH among steady-state adult Brazilians with SCA and HbAA individuals. Overall, this cross-sectional study enrolled 30 SCA patients not receiving HU therapy (HbSS), 25 SCA patients receiving HU therapy (HbSS_HU), and 32 HbAA volunteers (HbAA). The IH markers evaluated were serum Lactate Dehydrogenase (LDH), total heme, plasma hemoglobin (pHb), and soluble CD163 (sCD163). The ED markers analyzed were plasma von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo) levels, antigen of VWF-cleaving protease (ADAMTS13:Ag), thrombospondin-1, endothelin-1 levels, and ADAMTS13 Activity (ADAMTS13:Act). The levels of VWF:Ag, VWF:RCo, total heme, thrombospondin-1, and endothelin-1 were significantly higher in SCA patients (HbSS and HbSS_HU) compared to HbAA individuals. Also, pHb, LDH, and thrombospondin-1 levels were significantly higher in the HbSS group than in the HbSS_HU group. Contrarily, the levels of sCD163, ADAMTS13:Ag, and ADAMTS13:Act were significantly lower in both groups of SCA patients than HbAA controls, and ADAMTS13:Act levels were significantly lower in HbSS compared to HbSS_HU patients. The higher ADAMTS13 activity levels in those on HU therapy may be attributed to lower pHb and thrombospondin-1 levels as previously shown by in vitro studies that thrombospondin-1 and pHb are bound to VWF. Thus, VWF is restrained from ADAMTS13 activity and cleavage.

Influence of UGT1A1 promoter polymorphism, α-thalassemia and ßs haplotype in bilirubin levels and cholelithiasis in a large sickle cell anemia cohort.

Hyperbilirubinemia in patients with sickle cell anemia (SCA) as a result of enhanced erythrocyte destruction, lead to cholelithiasis development in a subset of patients. Evidence suggests that hyperbilirubinemia may be related to genetic variations, such as the UGT1A1 gene promoter polymorphism, which causes Gilbert syndrome (GS). Here, we aimed to determine the frequencies of UGT1A1 promoter alleles, alpha thalassemia, and ßS haplotypes and analyze their association with cholelithiasis and bilirubin levels. The UGT1A1 alleles, -3.7 kb alpha thalassemia deletion and ßS haplotypes were determined using DNA sequencing and PCR-based assays in 913 patients with SCA. The mean of total and unconjugated bilirubin and the frequency of cholelithiasis in GS patients were higher when compared to those without this condition, regardless of age (P < 0.05). Cumulative analysis demonstrated an early age-at-onset for cholelithiasis in GS genotypes (P < 0.05). Low fetal hemoglobin (HbF) levels and normal alpha thalassemia genotype were related to cholelithiasis development (P > 0.05). However, not cholelithiasis but total and unconjugated bilirubin levels were associated with ßS haplotype. These findings confirm in a large cohort that the UGT1A1 polymorphism influences cholelithiasis and hyperbilirubinemia in SCA. HbF and alpha thalassemia also appear as modulators for cholelithiasis risk.

Alpha thalassemia, but not ßS-globin haplotypes, influence sickle cell anemia clinical outcome in a large, single-center Brazilian cohort.

Alpha thalassemia and beta-globin haplotype are considered classical genetic disease modifiers in sickle cell anemia (SCA) causing clinical heterogeneity. Nevertheless, their functional impact on SCA disease emergence and progression remains elusive. To better understand the role of alpha thalassemia and beta-globin haplotype in SCA, we performed a retrospective study evaluating the clinical manifestations of 614 patients. The univariate analysis showed that the presence of alpha-thalassemia -3.7-kb mutation (αα/-α and -α/-α) decreased the risk of stroke development (p = 0.046), priapism (p = 0.033), and cholelithiasis (p = 0.021). Furthermore, the cumulative incidence of stroke (p = 0.023) and cholelithiasis (p = 0.006) was also significantly lower for patients carrying the alpha thalassemia -3.7-kb mutation. No clinical effects were associated with the beta-globin haplotype analysis, which could be explained by the relatively homogeneous haplotype composition in our cohort. Our results reinforce that alpha thalassemia can provide protective functions against hemolysis-related symptoms in SCA. Although, several genetic modifiers can impact the inflammatory state of SCA patients, the alpha thalassemia mutation remains one of the most recurrent genetic aberration and should therefore always be considered first.