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.

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.

Functional characterization of compound heterozygosity Hb S/Hb Deer Lodge in Brazil.

INTRODUCTION: The Hb Deer Lodge (ß2 His>Arg; HBB:c.8A>G) is a structural hemoglobin variant described in some populations around the world, characterized by increased oxygen affinity, but does not confer clinical symptoms to its carriers. The coinheritance of the Hb Deer Lodge with the most common hemoglobin variant, Hb S, has been reported only once; however, functional data were not described. Here we show a case of the Hb S and Hb Deer Lodge carrier in heterozygosity. METHODS: The Hb S and Hb Deer Lodge association was identified by High-Performance Liquid Chromatography (HPLC), reverse phase HPLC and the ß globin gene sequencing. The functional characterization of this interaction was obtained using the O2 dissociation curve, determination of the cooperativity between the globin chains and the Bohr effect in the presence and absence of organic phosphates. RESULTS: When the Hb S and Hb Deer Lodge were associated, there was a decrease in cooperativity, no significant changes in oxygen affinity and no significant Bohr effect changes. CONCLUSION: Despite these genetic variations, the carrier showed no hematological alterations and no clinical symptoms, possibly due to the high oxygen affinity of the Hb Deer Lodge, which interferes with the Hb S polymerization.

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.

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.

Evaluation of oxidative stress-related genetic variants for predicting stroke in patients with sickle cell anemia.

Overt stroke in adults with sickle cell anemia (SCA) continues to be a major cause of morbidity and mortality, while no evidence-based strategy for prevention has been reached so far. Although transcranial Doppler ultrasonography represents the most important tool for identifying young patients with SCA at risk of primary stroke, strategies for stroke prediction in adulthood remain challenging. Emerging data suggest that oxidative stress may exert a pivotal role in the pathogenesis of ischemic brain injury. Combining these pieces of evidences with the well-known genetic contribution to the development of stroke in SCA, we hypothesized that genetic variants related to the biology of oxidative stress could be used to identify adult patients at higher risk of stroke. Overall, 499 unrelated patients with SCA aged >18 years were genotyped for SOD2 Val16Ala (rs4880), GPX3 T-568C (rs8177404), GPX3 T-518C (rs8177406), GPX3 T-65C (rs8177412), and CAT01 C-262 T (rs1001179) polymorphisms, along with α-thalassemia status and ß-globin gene haplotypes. Of these, only the SOD2 Val16Ala polymorphism was associated with stroke. SOD2 Val16Ala polymorphism was independently associated with risk of stroke (odds ratio: 1.98; 95% confidence interval [CI]: 1.18-3.32; P = .009) and with the long-term cumulative incidence of stroke (hazard ratio: 2.24, 95% CI: 1.3-3.9; P = .004). In summary, we provide evidence that oxidative stress-related genetic variants, in particular, the SOD2 Val16Ala polymorphism, may represent a simple and inexpensive alternative for identifying patients at risk of stroke.

Influence of the ßs haplotype and α-thalassemia on stroke development in a Brazilian population with sickle cell anaemia.

Stroke is a catastrophic complication of sickle cell anaemia (SCA) and is one of the leading causes of death in both adults and children with SCA. Evidence suggests that some genetic polymorphisms could be related to stroke development, but their association remains controversial. Here, we performed genotyping of five published single nucleotide polymorphisms, the α-thalassemia genotype, the G6PD A (-) variant deficiency, and the ß(S) haplotype in a large series of SCA patients with well-defined stroke phenotypes. Of 261 unrelated SCA patients included in the study, 67 (9.5 %) presented a documented, primary stroke event. Markers of haemolysis (red blood cell (RBC) counts, p = 0.023; reticulocyte counts, p = 0.003; haemoglobin (Hb) levels, p < 0.001; indirect bilirubin levels, p = 0.006; lactate dehydrogenase (LDH) levels, p = 0.001) were associated with stroke susceptibility. Genetically, only the ß(S) haplotype (odds ratio (OR) 2.9, 95 % confidence interval (CI) 1.56 to 4.31; p = 0.003) and the α(3.7kb)-thalassemia genotype (OR 0.31, 95 % CI 0.11 to 0. 83; p = 0.02) were associated with increased and decreased stroke risk, respectively. In multivariate analysis, the ß(S) haplotype was independently associated with stroke development (OR 2.26, 95 % CI 1.16 to 4.4; p = 0.016). Our findings suggest that only the ß(S) haplotypes and the α(3.7kb)-thalassemia genotype modulate the prevalence of stroke in our SCA population. Genetic heterogeneity among different populations may account for the irreproducibility amongst different studies.

Polymorphism in the HMOX1 gene is associated with high levels of fetal hemoglobin in Brazilian patients with sickle cell anemia.

The aim of this study was to investigate the association between three polymorphisms involved in the oxidative stress pathway and fetal hemoglobin (Hb F) levels in patients with sickle cell anemia in a Brazilian population. One hundred and seven patients with sickle cell anemia were recruited for genomic DNA extraction. The levels of Hb F, sex and age were evaluated. Three polymorphisms, rs4673:T>C and rs9932581:G>A in the CYBA gene and rs2071746:A>T in the HMOX1 gene, were identified through direct sequencing. Hb F levels were not associated with sex, age, or the polymorphisms rs4673:T>C and rs9932581:G>A. However, the TT genotype of the rs2071746:A>T polymorphism was associated with increased levels of Hb F (p value = 0.0131). We observed an association between the TT genotype of the rs2071746:A>T polymorphism, present in the HMOX1 gene, and increased levels of Hb F, indicating the presence of a new marker related to Hb F levels in sickle cell anemia patients.

MTHFR polymorphic variant C677T is associated to vascular complications in sickle-cell disease.

Vaso-occlusion is a determinant for most signs and symptoms of sickle-cell anemia (SCA). The mechanisms involved in the pathogenesis of vascular complications in SCA remain unclear. It is known that genetic polymorphisms associated with thrombophilia may be potential modifiers of clinical features of SCA. The genetic polymorphisms C677T and A1298C relating to the enzyme methylenetetrahydrofolate reductase (MTHFR), a clotting Factor V Leiden mutation (1691G→A substitution of Factor V Leiden), and the mutant prothrombin 20210A allele were analyzed in this study. The aim was to find possible correlations with vascular complications and thrombophilia markers in a group of SCA patients in Pernambuco, Brazil. The study included 277 SCA patients, divided into two groups: one consisting of 177 nonconsanguineous SCA patients who presented vascular manifestations of stroke, avascular necrosis, leg ulcers, priapism, and acute chest syndrome (group 1); and the other consisting of 100 SCA patients without any reported vascular complication (group 2). Molecular tests were done using either polymerase chain reaction (PCR) restriction fragment length polymorphism or allele-specific PCR techniques. Comparisons between the groups were made using the χ(2) test. The 677 CT and TT genotypes showed a significant risk of vascular complications (p=0.015). No significant associations between the groups were found when samples were analyzed for the MTHFR A1298C allele (p=0.913), Factor V G1691 (p=0.555), or prothrombin G20210A mutation (p=1.000). The polymorphism MTHFR C677T seemed to be possibly predictive for the development of some vascular complications in SCA patients among this population.