Estimated glomerular filtration rate in sickle cell anemia is associated with polymorphisms of bone morphogenetic protein receptor 1B

Renal disease is common in sickle cell anemia. In this exploratory work, we used data from a longitudinal study of the natural history of sickle cell disease to examine the hypothesis that polymorphisms (SNPs) in selected candidate genes are associated with glomerular filtration rate (GFR). DNA samples and clinical and laboratory data were available for 1,140 patients with sickle cell anemia. GFR was estimated using the Cockcroft-Gault and Schwartz formulas for adults and children, respectively. We examined approximately 175 haplotype tagging (ht) SNPs in about 70 genes of the TGFbeta/BMP pathway for their association with GFR using linear regression. Four SNPs in BMPR1B, a bone morphogenetic protein (BMP) receptor gene, yielded statistically significant associations (P values ranging from 0.015 to 0.046). Three haplotypes in this gene were also associated with GFR. The TGF-beta/BMP pathway has been associated with the development of diabetic nephropathy, which has some features in common with sickle cell nephropathy. Our results suggest that, as with other subphenotypes of sickle cell disease, renal function may be genetically modulated.     

Iron metabolism, sickle cell disease, and response to cyanate.

In an attempt to understand the variability of the hematologic response to oral sodium cyanate, iron metabolism was studied in a group of 39 patients with sickel cell disease. Eleven of the 39 patients were found to have no stainable iron in the marrow despite the fact that patients with sickle cell disease are generally considered to have hemosiderosis. The mean per cent saturation and total iron-binding capacity were in the low-normal range in sickle cell patients whether or not stainable iron was present in the bone marrow aspirate. Serum ferritin concentrations, on the other hand, were found to be high in both groups (greater than 500 mu g/liter) when compared to controls (60 mu g/liter). The high serum ferritin levels denoted significant total-body iron deposition which may be unavailable for normal metabolic processes. One patient with no stainable iron in the bone marrow aspirate did respond to iron therapy alone with an increase in hemoglobin concentration. Serum ceruloplasmin levels were also found to be high in sickle cell disease patients. The ability to respond to oral cyanate therapy was correlated with the amount of stainable iron in the bone marrow aspirate. These studies emphasize the necessity of a reevaluation of iron metabolism in the pathophysiology and treatment of sickle cell disease.     

Apoptosis of osteocytes in glucocorticoid-induced osteonecrosis of the hip

An increase in osteoblast and osteocyte apoptosis has been demonstrated in mice and humans receiving glucocorticoids and may be involved in the pathogenesis of the associated osteonecrosis. To examine the spatial relationship between osteocyte apoptosis and glucocorticoid-induced osteonecrosis, we determined the prevalence of osteocyte apoptosis in whole femoral heads obtained from patients who underwent prosthetic hip replacement because of osteonecrosis due to chronic glucocorticoid treatment (n = 5), alcoholism (n = 3), and trauma (n = 1) as well as in femoral neck cores from patients with sickle cell disease (n = 5). Abundant apoptotic osteocytes and cells lining cancellous bone were found juxtaposed to the subchondral fracture crescent in femurs from the patients with glucocorticoid excess. In contrast, apoptotic bone cells were absent from the specimens taken from patients with trauma or sickle cell disease and were rare with alcohol abuse. These results indicate that glucocorticoid-induced osteonecrosis is a misnomer. The bone is not necrotic; instead, it shows prominent apoptosis of cancellous lining cells and osteocytes. Glucocorticoid-induced osteocyte apoptosis, a cumulative and irreparable defect, could uniquely disrupt the mechanosensory function of the osteocyte network and thus start the inexorable sequence of events leading to collapse of the femoral head.     

Sickle cell leg ulcers: associations with haemolysis and SNPs in Klotho, TEK and genes of the TGF-beta/BMP pathway

Cutaneous leg ulcers are common in sickle cell anaemia and their risk might be genetically determined. Sickle cell anaemia patients were studied to examine the relationship of leg ulcers with haemolysis and with single nucleotide polymorphisms (SNPs) in candidate genes that could affect sickle vasoocclusion. Leg ulcer patients had lower haemoglobin levels and higher levels of lactate dehydrogenase, bilirubin, aspartate transaminase and reticulocytes than did control patients with sickle cell anaemia but without leg ulcers. Age-adjusted comparisons showed that sickle cell anaemia-alpha thalassaemia was more frequent among controls than cases. These results strongly suggested that the likelihood of having leg ulcers was related to the intensity of haemolysis. 215 SNPs in more than 100 candidate genes were studied. Associations were found with SNPs in Klotho, TEK and several genes in the TGF-beta/BMP signalling pathway by genotypic association analyses. KL directly or indirectly promotes endothelial nitric oxide (NO) production and the TEK receptor tyrosine kinase is involved in angiogenesis. The TGF-beta/BMP signalling pathway modulates wound healing and angiogenesis, among its other functions. Haemolysis-driven phenotypes, such as leg ulcers, could be improved by agents that reduce sickle erythrocyte density or increase NO bioavailability.     

Osteonecrosis of the Femoral Head: A Proposed New Treatment in Homozygous Sickle Cell Disease

Possibilities for bone reconstruction in osteonecrosis of the femoral head in sickle cell hemoglobinopathies before the end of growth have not been assessed. The aim of our study was to evaluate the morphological and functional results in 11 osteonecrosis of the femoral head because of homozygous sickle cell disease. Surgical treatment consisted of a triple acetabular osteotomy in seven cases, femoral varus osteotomy in two cases and a combination of both in two cases. The severity of the osteonecrosis was evaluated on radiographs and magnetic resonance imaging (MRI), pre- and postoperative, and function of the hip joint was assessed by Harris and Postel Merle d’Aubigné scores. The anesthetic part (pre- and postoperative) and eventual complications were collected and analyzed. The maximum follow-up was between 1 to 9.6 years. All patients were considered to be satisfactory at maximum follow-up. The severity of the initial damage was not associated with any morphological or functional outcome at maximum follow-up. All patients had an objective functional gain. We found no general complications. We proposed a decision tree for the patient’s management of avascular necrosis of the femoral head in sickle cell hemoglobinopathies before bone maturity, and with an Arlet and Ficat’s stage of 3 or 4. The results of this study confirmed the interest of conservative surgical treatment in children with homozygous sickle cell anemia in case of osteonecrosis of the femoral head. Level of evidence: IV (cases series of our department).     

Genetic modifiers of the severity of sickle cell anemia identified through a genome‐wide association study

We conducted a genome‐wide association study (GWAS) to discover single nucleotide polymorphisms (SNPs) associated with the severity of sickle cell anemia in 1,265 patients with either “severe” or “mild” disease based on a network model of disease severity. We analyzed data using single SNP analysis and a novel SNP set enrichment analysis (SSEA) developed to discover clusters of associated SNPs. Single SNP analysis discovered 40 SNPs that were strongly associated with sickle cell severity (odds for association >1,000); of the 32 that we could analyze in an independent set of 163 patients, five replicated, eight showed consistent effects although failed to reach statistical significance, whereas 19 did not show any convincing association. Among the replicated associations are SNPs in KCNK6 a K⁺ channel gene. SSEA identified 27 genes with a strong enrichment of significant SNPs (P < 10^?6); 20 were replicated with varying degrees of confidence. Among the novel findings identified by SSEA is the telomere length regulator gene TNKS. These studies are the first to use GWAS to understand the genetic diversity that accounts the phenotypic heterogeneity sickle cell anemia as estimated by an integrated model of severity. Additional validation, resequencing, and functional studies to understand the biology and reveal mechanisms by which candidate genes might have their effects are the future goals of this work. Am. J. Hematol., 2010. © 2009 Wiley‐Liss, Inc.     

Association of single nucleotide polymorphisms in klotho with priapism in sickle cell anaemia

The complications of sickle cell disease are probably determined by genes whose products modify the pathophysiology initiated by the sickle haemoglobin mutation. Priapism, one vaso-occlusive manifestation of sickle cell disease, affects more than 30% of males with the disease. We examined the possible association of single nucleotide polymorphisms (SNPs) in 44 candidate genes of different functional classes for an association with the occurrence of priapism. One hundred and forty-eight patients with sickle cell anaemia and incident or a confirmed history of priapism were studied, along with 529 controls that had not developed priapism. Polymorphisms in the KLOTHO gene (KL; 13q12) showed an association with priapism by genotypic [reference SNP cluster identifier number (rs)2249358; odds ratio (OR) = 2.6 (1.4-5.5); rs211239; OR = 1.7 (1.2-2.6)] and haplotype analyses [rs211234 and rs211239; OR = 2.3 (1.5-3.4)]. These findings may have broader implications in sickle cell disease, as KL encodes a membrane protein that regulates many vascular functions, including vascular endothelial growth factor expression and endothelial nitric oxide release.     

Identification of genetic polymorphisms associated with risk for pulmonary hypertension in sickle cell disease

Up to 30% of adult patients with sickle cell disease (SCD) will develop pulmonary hypertension (pHTN), a complication associated with significant morbidity and mortality. To identify genetic factors that contribute to risk for pHTN in SCD, we performed association analysis with 297 single nucleotide polymorphisms (SNPs) in 49 candidate genes in patients with sickle cell anemia (Hb SS) who had been screened for pHTN by echocardiography (n = 111). Evidence of association was primarily identified for genes in the TGFβ superfamily, including activin A receptor, type II–like 1 (ACVRL1), bone morphogenetic protein receptor 2 (BMPR2), and bone morphogenetic protein 6 (BMP6). The association of pHTN with ACVRL1 and BMPR2 corroborates the previous association of these genes with primary pHTN. Moreover, genes in the TGFβ pathway have been independently implicated in risk for several sickle cell complications, suggesting that this gene pathway is important in overall sickle cell pathophysiology. Genetic variation in the β-1 adrenergic receptor (ADRB1) was also associated with pHTN in our dataset. A multiple regression model, which included age and baseline hemoglobin as covariates, retained SNPs in ACVRL1, BMP6, and ADRB1 as independently contributing to pHTN risk. These findings may offer new promise for identifying patients at risk for pHTN, developing new therapeutic targets, and reducing the occurrence of this life-threatening SCD complication.     

Fetal hemoglobin in sickle cell anemia: Bayesian modeling of genetic associations

We genotyped single nucleotide polymorphisms (SNPs) in: (1) the beta-globin gene-like cluster, (2) quantitative trait loci (QTL) previously associated with fetal hemoglobin (HbF) concentration on chromosomes 6q, 8q, and Xp, and (3) candidate genes that could effect HbF levels, in sickle cell anemia subjects. HbF concentration was modeled as a continuous variable with values in a finite interval using a novel Bayesian approach. We first tested the associations of SNPs with HbF in a group of 1,518 adults and children (CSSCD study), and validated the results in a second independent group of 211 adults (MSH study). In subjects aged >or=24 years, 5 SNPs in TOX (8q12.1), 2 SNPs in the beta-globin gene-like cluster, 2 SNPs in the Xp QTL, and 1 SNP in chromosome 15q22 were associated with HbF in the CSSCD and also validated in the MSH. Four other SNPs in 15q22 were associated with HbF only in the larger CSSCD data. When patients aged <24 years in the CSSCD were examined, additional genes, including 4 with roles in nitric oxide metabolism, were associated with HbF level. These studies confirm prior analyses using traditional analytical approaches showing associations of SNPs in TOX, GPM6B, and the beta-globin gene-like cluster with HbF levels. We also identified an additional candidate regulatory region in chromosome 15q22 that is associated with HbF level. By stratifying patients by age, our results also suggest that different genes might modulate the rate of decline of HbF and the final level of HbF levels in sickle cell anemia.     

Studies of novel variants associated with Hb F in Sardinians and Tanzanians in sickle cell disease patients from Cameroon

High level of Hb F has been shown to improve survival in sickle cell disease. Among 453 Cameroonians with sickle cell disease, we have investigated 18 selected single-nucleotide polymorphisms (SNPs) in novel and suggestive loci associated with Hb F level identified through a genomewide association study in sickle cell disease patients in Tanzania, and whole-genome sequencing of a population from Sardinia. Seven of 10 variants reported in Sardinians were either monomorphic or very rare in the Cameroonians. No associations were observed with any SNPs and Hb F levels in Cameroonians affected by sickle cell disease. The present study illustrates the complexity of replicating Hb F-promoting variants association results across populations.     

Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes

Hemolysis, long discounted as a critical measure of sickle cell disease severity when compared with sickle vaso-occlusion, may be the proximate cause of some disease complications. New mechanistic information about hemolysis and its effects on nitric oxide (NO) biology and further examination of the subphenotypes of disease requires a reappraisal and deconstruction of the clinical features of sickle cell disease. The biology underlying clinical phenotypes linked to hemolysis may increase our understanding of the pathogenesis of other chronic hemolytic diseases while providing new insights into treating sickle cell disease. The pathophysiological roles of dysregulated NO homeostasis and sickle reticulocyte adherence have linked hemolysis and hemolytic rate to sickle vasculopathy. Nitric oxide binds soluble guanylate cyclase which converts GTP to cGMP, relaxing vascular smooth muscle and causing vasodilatation. When plasma hemoglobin liberated from intravascularly hemolyzed sickle erythrocytes consumes NO, the normal balance of vasoconstriction:vasodilation is skewed toward vasoconstriction. Pulmonary hypertension, priapism, leg ulceration and stroke, all subphenotypes of sickle cell disease, can be linked to the intensity of hemolysis. Hemolysis plays less of a role in the vaso-occlusive-viscosity complications of disease like the acute painful episode, osteonecrosis of bone and the acute chest syndrome. Agents that decrease hemolysis or restore NO bioavailability or responsiveness may have potential to reduce the incidence and severity of the hemolytic subphenotypes of sickle cell disease. Some of these drugs are now being studied in clinical trials.     

Osteonecrosis in sickle cell trait

Osteonecrosis is common in sickle cell anemia but has been rarely reported with sickle cell trait. Two cases of osteonecrosis of the femoral head caused by sickle cell trait are presented. One patient's diagnosis was delayed because the association of osteonecrosis and sickle cell trait was not recognized. The previous reported cases of this association are discussed, emphasizing diagnostic implications for 12 black patients presenting with hip pain.     

Sickle cell disease-related organ damage occurs irrespective of pain rate: implications for clinical practice

In daily clinical practice, the frequency of painful crises (pain rate) is an important parameter of sickle cell disease severity. We assessed the prevalence of sickle cell disease-related organ damage and complications and their relation to pain rate. Organ damage and history of vaso-occlusive complications were obtained via systematic screening of consecutive patients and by chart review. In 104 adult sickle cell patients pain rate was related to a history of acute chest syndromes, avascular osteonecrosis, iron overload, priapism and cholelithiasis. However, major disease-related complications, such as microalbuminuria and pulmonary hypertension, were detected in 23% and 24% respectively of patients without painful crises in the study period underlining the importance of systematic screening for developing organ damage in sickle cell patients irrespective of pain rate.     

Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized,purified hematopoietic stem cells

Although sickle cell anemia was the first hereditary disease to be understood at the molecular level, there is still no adequate long-term treatment. Allogeneic bone marrow transplantation is the only available cure, but this procedure is limited to a minority of patients with an available, histocompatible donor. Autologous transplantation of bone marrow stem cells that are transduced with a stably expressed, antisickling globin gene would benefit a majority of patients with sickle cell disease. Therefore, the development of a gene therapy protocol that corrects the disease in an animal model and is directly translatable to human patients is critical. A method is described in which unmobilized, highly purified bone marrow stem cells are transduced with a minimum amount of self-inactivating (SIN) lentiviral vector containing a potent antisickling beta-globin gene. These cells, which were transduced in the absence of cytokine stimulation, fully reconstitute irradiated recipients and correct the hemolytic anemia and organ pathology that characterize the disease in humans. The mean increase of hemoglobin concentration was 46 g/L (4.6 g/dL) and the average lentiviral copy number was 2.2; therefore, a 21-g/L /vector copy increase (2.1-g/dL) was achieved. This transduction protocol may be directly translatable to patients with sickle cell disease who cannot tolerate current bone marrow mobilization procedures and may not safely be exposed to large viral loads.     

Uncommon case of tibial bone infarction associated with juvenile idiopathic arthritis

Bone infarction refers to aseptic osteonecrosis occurring in the metaphysis and diaphysis of long bones. Hemoglobinopathies, sickle cell disease, and Gaucher’s disease are usually found. Bone infarction is rarely idiopathic. Magnetic resonance imaging has proved to be more specific to identify bone infarction. We report an exceptional case of a unique idiopathic bone infarction of distal tibia, occurring unusually in a patient with juvenile idiopathic arthritis, without major risk factors of osseous infarct, and with atypical clinical and biological abnormalities.     

Elevated hypercoagulability markers in hemoglobin SC disease

Hemoglobin SC disease is a very prevalent hemoglobinopathy; however, very little is known about this condition specifically. There appears to be an increased risk of thromboembolic events in hemoglobin SC disease, but studies evaluating the hemostatic alterations are lacking. We describe the findings of a cross-sectional observational study evaluating coagulation activation markers in adult patients with hemoglobin SC, comparing them with those in sickle cell anemia patients and healthy controls. A total of 56 hemoglobin SC and 39 sickle cell anemia patients were included in the study, all in steady state, and 27 healthy controls. None of the patients was taking hydroxyurea. Hemoglobin SC patients had a significantly up-regulated relative expression of tissue factor, as well as elevations in thrombin-antithrombin complex and D-dimer, in comparison to controls (P<0.01). Hemoglobin SC patients had lower tissue factor expression, and thrombin-antithrombin complex and D-dimer levels when compared to sickle cell anemia patients (P<0.05). Markers of endothelial activation (soluble thrombomodulin and soluble vascular cell adhesion molecule-1) and inflammation (tumor necrosis factor-alpha) were both significantly elevated in hemoglobin SC patients when compared to controls, being as high as the levels seen in patients with sickle cell anemia. Overall, in hemoglobin SC patients, higher hemolytic activity and inflammation were associated with a more intense activation of coagulation, and hemostatic activation was associated with two very prevalent chronic complications seen in hemoglobin SC disease: retinopathy and osteonecrosis. In summary, our results demonstrate that hemoglobin SC patients have a hypercoagulable state, although this manifestation was not as intense as that seen in sickle cell anemia.     

Modifier genes and sickle cell anemia

PURPOSE OF REVIEW: With the completion of the human genome project and HapMap, previously unknown genetic polymorphisms associated with disease have been observed. This review highlights genetic polymorphisms that have provided insight into the pathophysiology underlying the many phenotypes of sickle cell disease. RECENT FINDINGS: The phenotypes of sickle cell disease are likely to be modulated by polymorphisms in genes that are involved in inflammation, cell-cell interaction, and nitric oxide biology. Case-control studies are beginning to define the relationships between single-nucleotide polymorphisms in candidate genes and the many subphenotypes of sickle cell anemia. A common theme emerging from these studies is that single-nucleotide polymorphisms in genes of the transforming growth factor-beta/bone morphogenetic protein and a few other genes such as Klotho are associated with several subphenotypes of sickle cell disease. SUMMARY: Genomic medicine is merging with clinical practice as our understanding of the structure and variability of the human genome increases. Patients with diseases caused by identical mutations in a single gene - sickle cell anemia is a prime example - can have clinical courses very different from one another, and when environmental influences are removed the phenotypic heterogeneity of mendelian single-gene disorders is best explained by single-nucleotide polymorphisms in genes that modulate the disease phenotype. As this field expands, insights will be gained into complex epistatic factors that influence the clinical presentation of sickle cell disease, enabling physicians to better predict and manage the many complications of this disease.     

Leg ulcers in patients with sickle cell disease [see comments]

During the entry examination, leg ulcers were present in 2.5% of 2,075 patients 10 years of age and older with sickle cell disease who entered into the Cooperative Study of Sickle Cell Disease (CSSCD) between 1979 and 1986. Prevalence rates were highest among patients with sickle cell anemia and sickle cell anemia with thalassemia genotypes. Among sickle cell anemia patients free of ulcers at entry, the overall incidence was 5.73 per 100 person years in those having associated alpha-thalassemia and 9.97 for those without. Among sickle cell anemia patients with two alpha genes, the estimated incidence of leg ulcers is 2.38 per 100 person years and 6.12 per 100 person years among sickle cell anemia patients with three alpha genes (P less than .05). In both groups, the incidence was highest among those patients over 20 years of age and considerably higher among males than females (P less than .001). Leg ulcers were nonexistent in patients with sickle beta plus thalassemia and sickle hemoglobin C disease. Low steady-state hemoglobin is associated with a higher incidence of ulcer formation (P less than .0001) in sickle cell anemia patients. The protective effect of hemoglobin F is apparent at all levels of total hemoglobin among sickle cell anemia patients and those with associated alpha-thalassemia.