Β-thalassemia Disease Research Articles

Frequency of human hemochromatosis (HFE) gene mutations in Egyptians with β-thalassemia

Background Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Two point mutations have been described and are referred to as H63D and C282Y. On the other hand, iron overload is a well-documented complication in thalassemia syndromes. Interactions between thalassemia and hemochromatosis may further increase iron overload. This work aimed at studying the frequency of the H63D and C282Y mutations of the HFE gene in an Egyptian population with β-thalassemia (thalassemia major, intermedia, and minor) by comparing it with normal individuals without hemoglobinopathies. Participants and methods This study included 86 patients with β-thalassemia; 40 of these patients had β-thalassemia major and intermedia and the other 46 patients had β-thalassemia minor (carriers). In addition, 70 individuals were included in the study and served as controls. All the populations studied were screened for H63D and C282Y mutations of the HFE gene using the PCR-restriction fragment length polymorphism (PCR-RFLP) method. Results The allelic frequencies found for H63D and C282Y mutations in this study were 18.6 and 0%, respectively, among the total alleles of individuals with β-thalassemia and 12.8 and 1.4% among controls without hemoglobinopathies. On comparing thalassemia cases, thalassemia carriers, and the control group, the allele frequency of the H63D mutation was significantly higher among β-thalassemia carriers (24%) compared with patients with β-thalassemia (12.5%) and controls (12.8%) (P=0.049). Our study also found an almost significantly higher frequency of the heterozygote genotype for the H63D mutation in patients with β-thalassemia (30.2%) than the controls (17.1%) (Fisher’s test, P=0.06). Compound heterozygosis for H63D and C282Y was found in one of the individuals in our control group. Conclusion The frequent occurrence of β-thalassemia disease with the H63D gene mutation raises the possibility of genetic interactions and emphasizes the value of screening for HFE mutations in thalassemias to detect early cases of iron overload and to modify its treatment modalities.

A Single Nucleotide Polymorphism in the HBBP1 Gene in the Human β-Globin Locus is Associated with a Mild β-Thalassemia Disease Phenotype

The rs2071348 (g.5264146A>C) polymorphism on the HBB pseudogene, namely HBBP1, previously emerged as a variant significantly associated with a milder disease phenotype in Asian β0-thalassemia/hemoglobin (Hb) E (β0-thal/Hb E [β26(B8)Glu→Lys, GAG>AAG]) patients. In this study, we aimed to explore the possible association of rs2071348 with β-thalassemia (β-thal) disease severity in a group of β-thal major (β-TM) patients (severe phenotype) and β-thal intermedia (β-TI) patients (mild phenotype) of Hellenic origin and compare the results with normal (non thalassemic) individuals of the same origin. In addition, we explored whether this single nucleotide polymorphism (SNP) can be exploited as a pharmacogenomic marker to predict the outcome of Hb F-augmenting therapy in β-thal patients receiving hydroxyurea (HU). Our data suggest that the rs2071348 polymorphism is associated with higher Hb F levels and a milder β-thal disease phenotype. However, the rs2071348 polymorphism in the HBBP1 gene does not correlate with response to HU treatment.

Phenotype and Genotype Frequency of β-Thalassemia and Sickle Cell Disease Carriers in Halkidiki, Northern Greece

A decade of screening (years 2000 to 2010) for hemoglobinopathies in 3,931 patients was performed at the General Hospital of Poligiros, Halkidiki, Northern Greece. Among the patients examined, 10.8% heterozygotes for β-thalassemia (β-thal) were found, as well as 4.1% with sickle cell disease and 1.2% with double β-thal/Hb S [β6(A3)Glu→Val] heterozygosity. Iron deficiency was observed in 23.4%.The geographical distribution in the region revealed a substantial incidence of hemoglobinopathies even in mountainous areas. This pattern did not follow the typical distribution according to the malaria hypothesis, as incidence did not dovetail with swamp locations recorded in the past.The HBB gene mutations for 85 patients were also analyzed. Most prevalent in Halkidiki, Northern Greece, was the codon 39 (C>T) mutation (27.1%) followed by the IVS-I-110 (G>A) mutation (22.4%); this was in direct contrast to the current distribution of the same mutations seen in the rest of Greece (Greek National Genetic Database, GNGD). This frequency inversion was statistically significant, with the difference from the GNGD being 20.6% for the IVS-I-110 mutation (p <0.0005) and 7.6% for the codon 39 mutation (p = 0.0238). The history of Halkidiki, denoting a clear example of geographical isolation from the rest of the country, may possibly account for a potentially diverse genetical identity of the disease in this region.

Normalized coagulation markers and anticoagulation proteins in children with severe β-thalassemia disease after stem cell transplantation

The hypercoagulable state is well recognized in patients with severe β-thalassemia disease. One of the mechanisms of chronic hypercoagulable state is the abnormal expression of phosphatidylserine on red blood cells (RBC). This study aimed to determine the coagulable state in patients with severe β-thalassemia disease following successful stem cell transplantation (SCT). Subjects were classified into three groups: normal controls (NC), β-thalassemia disease receiving regular transfusion (Thal-RT) and β-thalassemia disease post SCT (Thal-SCT). Sixty eight subjects, aged 3–17years, consisting of 21 NC, 28 Thal-RT and 19 Thal-SCT were enrolled. After SCT, the annexin V level in Thal-SCT was normalized. At the median follow-up time of 70.3 (50.9-84.2) months after SCT, the levels of coagulation markers (thrombin antithrombin complex, prothrombin fragment and D-dimer) and anticoagulation proteins (protein C, S and antithrombin activities) returned to the levels similar to controls.

Lack of association of G779A ZHX-2 gene variant with HbF levels in β-thalassemia major

The inherited disorders of hemoglobin synthesis are the most common monogenic disorders worldwide. They include thalassemias, hemoglobin variants, and hereditary persistence of fetal hemoglobin. β-thalassemia is the most common monogenic disorder in India. Clinical manifestations of β-thalassemia are extremely variable in severity. The reasons for this heterogeneity are not very well understood. Previous studies have shown that the genetic variants that modulate HbF levels have a very strong impact on ameliorating the clinical phenotype. In the present study, 5570 blood samples from suspected cases were analyzed using HPLC, amplification refractory mutation system-PCR and reverse dot blot techniques. Of 5570 individuals, we found 676 cases of β-thalassemia disease. Molecular analysis revealed the presence of different β-thalassemia mutations in the population under study. Patients with β-thalassemia were classified into mild, moderate, and severe according to severity score based on Hb level, age of onset, age at which patients received their first blood transfusion, degree of growth retardation and splenectomy. Patients with β-thalassemia were analyzed for zinc finger and homeoboxes 2 (ZHX2) G779A polymorphism, and the association between ZHX2 gene polymorphism and severity of β-thalassemia was evaluated. We did not find a significant difference in genotypic and allelic frequency of ZHX2 gene between mild and moderate, mild and severe, and moderate and severe cases. There was no significant difference in high and low percentage of HbF in GG, GA, and AA bearing individuals showing that ZHX2 gene variant has no role in ameliorating the severity of β-thalassemia major in the South Indian population from Andhra Pradesh.

Spectrum of β-Thalassemia Mutations in the Eastern Province of Saudi Arabia

β-Thalassemias comprise a group of heterogeneous hemoglobin (Hb) disorders characterized by the absence or reduced synthesis of the β-globin chain with a variable clinical presentation. The Al-Qatif and Al-Ahsa oases in the Eastern Province of Saudi Arabia are regions known for the high prevalence of these disorders. This study was conducted to provide a more precise picture of the β-thalassemia (β-thal) mutations prevalent in these regions and to estimate their frequencies. One hundred and 96 subjects with transfusion-dependent β-thalassemia (β-thal) disease were included in this study. A total of 14 β-thal mutations were identified with five mutations accounting for more than 80% of the total β-thal mutations identified. Of the 196 patients, 164 were homozygous for a β-thal mutation, while 32 were compound heterozygotes. We report here the novel identification of two mutations, namely, the Tunisian splice site IVS-I-130 (G→C) and the Mediterranean cryptic splice site IVS-I-110 (G→A), which have not been previously reported in the population of the Eastern Province. However, 15 patients (46.9%) with compound heterozygosities carried one of the β-thal mutations and the sickle cell mutation [Hb S or β6(A3)Glu→Val]. These patients were less frequently transfused than the patients who were homozygous for the β-thal mutations and presented with fewer complications. A more comprehensive overview of the genetic heterogeneity of the β-thal mutations in the Eastern Province of Saudi Arabia is presented in this article. This study will contribute to the establishment of an effective prevention program, including premarital screening.

Prevention of severe thalassemia in northeast Thailand: 16 years of experience at a single university center

To demonstrate the performance of thalassemia prevention in northeast Thailand during 1993-2008. Retrospective data from 1422 at-risk couples who attended from January 1993 to December 2008 were studied. All couples were suspected at-risk couples based on initial screening using standard protocols. Three thalassemia carrier types including alpha(0)-thalassemia, beta-thalassemia and hemoglobin E were identified using standard methods. Data on prenatal diagnosis were collected. Of the 1422 positive-screened couples, 1254 (88.2%) were diagnosed as true-positive couples. After DNA analysis, 968 of 1254 (77.2%) resulted at risk for three types of severe conditions being hemoglobin E-beta-thalassemia disease (640/968, 66.1%), homozygous alpha(0)-thalassemia (304/968, 31.4%) and homozygous beta-thalassemia (11/968, 1.1%). The remaining 1.3% of the couples were at risk for more than one disease. After genetic counseling, prenatal diagnosis was performed on 756 couples (78.1%). The proportions of affected fetuses, thalassemia carriers and unaffected fetuses were 26.9, 50.0 and 23.0%, respectively. Implementation of a prevention and control program accompanying with a referral system for prenatal diagnosis is technically feasible in northeast Thailand and a large number of severe thalassemia diseases have been prevented during the past 16 years of operation.

Optimal response to thalidomide in a patient with thalassaemia major resistant to conventional therapy.

Beta-thalassaemia major is a hereditary anaemia resulting from defects in β-globin production. The coexistence of hereditary persistence of foetal haemoglobin (HbF) during adult life in patients with β-thalassaemia reduces the severity of the disease; these patients have a mild disorder, sometimes not even requiring chronic transfusions. The clinical benefit of increased HbF, first inferred in 19761, is due to a decrease in the imbalance between β and non-β-chains and the consequent reduction of haemolysis. Many drugs have been studied as inducers of HbF for patients with β-thalassaemia and sickle cell disease. Hydroxyurea is currently used in moderate to severe forms of sickle cell disease2,3 and in some cases of thalassaemia intermedia4,5. Other inducers of HbF synthesis, such as butyrate6, 5-azacytidine7 and, more recently, decitabine, have also been shown to induce HbF in patients with sickle cell disease8. However, these HbF inducers have shown only a modest effect in the majority of β-thalassaemia patients as well as some degree of toxicity. As a result, they have not been used routinely in clinical practice. Thalidomide, a drug known for its immunomodulating and anti-angiogenic properties, has recently been demonstrated to induce γ-globin gene expression and to increase the proliferation of erythroid cells9,10. Only one patient affected by β-thalassaemia major treated successfully with thalidomide has been described so far11. We report here the case of a young girl with β-thalassaemia (β+/β°) in a very severe clinical condition who could not be given any further transfusions because of the occurrence of severe post-transfusion reactions and who showed an outstanding response to thalidomide.

Splenectomy Exacerbates the Hypercoagulable State and Causes Significant Organ Dysfunction in Patients with Hemoglobin E/β-Thalassemia.

Abstract 2014Poster Board I-1036 Introduction:Clinical manifestations of blood hypercoagulability, such as pulmonary arterial hypertension (PAH) and ischemic stroke, are well known in patients with thalassemia. We herein present evidence that the hypercoagulable state in β-Thalassemia disease patients is to a significant extent the result of prior splenectomy. Patients and Methods:One hundred and ten clinically stable hemoglobin E/β-Thalassemia outpatients, on no medication aside from folic acid and who received no blood transfusion in the preceding 4 weeks were studied. All gave written informed consent, and study protocol was approved by the institution ethics committee on studies in humans (#0774/2548). Echocardiogram was used to estimate systolic PA pressure (SPAP). Clinical features and laboratory data were stratified according to the presence or absence of the spleen, and statistical analysis was done by STATA version 10 (Stata Corp, Texas), considering a P value <0.05 as statistically significant. Results:Of the 110 patients, 61 were asplenic and females comprised 51% in both groups. Selected statistically significant results are shown in the table.ResultsAsplenic (61 Patients)Non-splenectomized (49 Patients)P valueAge at 1st transfusion (median)4.7 years17 years<0.001Total transfusions (median)35 units PRBC13 units PRBC0.005Systolic pulmonary artery pressure37.1 +/- 14.6 mm Hg28.8 +/- 7.6 mm Hg<0.001Cardiomegaly50.9%27.3%0.013Extramedullary hematopoiesis50.9%27.3%0.013RBC count3.19 +/- 0.57 × 106/μL3.87 +/- 1.09 × 106/μL<0.001Corrected reticulocyte count (median)5.2%1.4%<0.001Nucleated RBC (median)581/100 WBCs5/100 WBCs<0.001Corrected WBC count9.72 +/- 4.2 × 103/μL6.71 +/- 2.3 × 103/μL<0.001Platelet count726 +/- 215 × 103/μL243 +/- 115 × 103/μL<0.001Serum ferritin (median)1,892 ng/mL1,050 ng/mL0.001ALT (median)65.5 u/L44.5 u/L0.004AST (median)64 u/L36 u/L0.001Free hemoglobin (median)4.2 mg/dL2.5 mg/dL0.001C-reactive protein (high sens) (median)2.7 mg/L1.2 mg/L0.012Thrombin-antithrombin complexes3.5 μg/L1.1 μg/L<0.001Soluble P-selectin113.4 +/- 31.2 ng/mL56.4 +/- 32.1 ng/mL<0.001Soluble E-selectin43.3 +/- 21.5 ng/mL29.5 +/- 18.4 ng/mL0.003 Conclusions:In comparison with non-splenectomized hemoglobin E/β-Thalassemia patients, asplenic ones have higher SPAP, more severe hemolysis, more liver impairment and chronic iron overload, significant chronic low grade inflammation, and evidence of coagulation activation. Splenectomy should be more judiciously applied than heretofore, and if performed, measures to blunt or prevent a subsequent hypercoagulable and inflammatory state, which could lead to PAH should be initiated. Disclosures:No relevant conflicts of interest to declare.

Efficacy and Safety of Deferasirox

Deferasirox represents a new class of once-daily iron chelators. It was first licensed in 2005 for the treatment of adult and pediatric patients with chronic iron overload due to blood transfusions. These approvals were based on data from the core program of one-year clinical trials, which involved more than 1,000 patients in what is the largest and most rigorous prospective clinical evaluation of any iron-chelating agent to date. Deferasirox has been studied in adults and children with a wide range of conditions, including β-thalassemia, myelodysplastic syndromes, and sickle cell disease. Recent data have highlighted the importance of timely deferasirox dose adjustments and described the efficacy and safety of deferasirox at doses &gt;30mg/kg/day. The pharmacokinetic profile of deferasirox across all age groups shows it to be well absorbed, with a mean elimination half-life of eight to 16 hours that supports once-daily dosing. In clinical trials, deferasirox has demonstrated consistent dose-dependent efficacy, producing sustained reductions in serum ferritin, labile plasma iron, and cardiac iron load. The safety profile of deferasirox presents mostly as mild adverse events (primarily gastrointestinal symptoms, skin rash, and increases in serum creatinine) that resolve rapidly with no reports of progressive renal, hepatic, or bone marrow effects. This adverse event profile is seen to be manageable with appropriate clinical monitoring. Data from the extension phases of these studies are beginning to accumulate, with experience of up to seven years of deferasirox therapy now available. Deferasirox represents a significant development in the treatment of iron overload and has further potential applications.

Stem Cell Transplantation in Hemoglobinopathies

β-Thalassemia (thal) and sickle cell disease are the most common genetic diseases worldwide. Although supportive therapies such as regular transfusion and chelation for β-thal and hydroxyurea (HU) for sickle cell disease have significantly improved clinical manifestations and the quality of life, they cannot eliminate disease and therapy-related complications. Today, hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with hemoglobinopathies. The first successful HSCT was reported in 1981. At that time, more than 1,000 patients underwent HSCT in Pesaro, Italy. Sixty β-thal patients underwent HSCT at the Department of Pediatric Hematology-Oncology, Akdeniz University School of Medicine, Antalya, Turkey between 1998 and 2006. We found stable mixed chimerism in 14 patients out of 45 (31%) in our transplanted thalassemia patients. The thalassemia free survival and overall survival rates were found to be 84.0 and 91.0%, respectively. The literature and our results indicate that HSCT can offer a cure for hemoglobinopathy patients.

Transfusion of multiple units of Js(b+) red blood cells in the presence of anti-Jsb in a patient with sickle β-thalassemia disease and a review of the literature

Abstract Jsb is a high-frequency antigen. Anti-Jsb is a rare alloantibody, and its clinical significance is poorly documented. We report a case in which a 12-year-old boy of Nigerian descent with sickle β-thalassemia presented with multiple alloantibodies, including a panagglutinin and acute chest syndrome, necessitating the emergent transfusion of five units of phenotype-similar, crossmatch-incompatible RBCs, four of which were given during an exchange transfusion. The patient was later found to have anti-Jsb. In addition to routine serologic methods to study the patient’s RBCs and plasma, a monocyte monolayer assay (MMA) was performed on the patient’s samples obtained 2 and 9 days after transfusion of the Js(b+) RBCs to determine the potential clinical significance of the anti-Jsb. Various laboratory parameters including quantitative hemoglobin fraction analyses were used to monitor for increased RBC destruction. The MMA reactivity of the patient’s anti-Jsb increased from 2.3 percent on day 2 after transfusion to strongly positive at 88 percent and 66.5 percent (with and without the addition of fresh serum) 1 week later. MMA reactivity of greater than 5 percent is associated with increased RBC destruction. There was no clinical or laboratory evidence of increased hemolysis above baseline. However, decreased RBC survival was suggested by the relatively brisk decrease of the HbA1 fraction after the transfusions. The current case and others reported in the literature suggest that anti-Jsb may have limited potential for causing overt hemolysis. However, in patients with underlying hematologic disease, even mildly increased RBC destruction may pose problems clinically, and thus transfusion of Js(b+) RBCs should be avoided. In emergent situations, the potential of adverse effects of transfusing incompatible units should be balanced against the risk of withholding transfusion. Family members, especially siblings, should be considered as potential designated donors for patients with antibodies directed against high-frequency antigens. Available reports on anti-Jsb in the literature are also reviewed. Immunohematology 2007;23:75–80.

Stem Cell Transplantation from HLA-Identical Siblings Versus Alternative Donors in β-Thalassemia Diseases.

Background: Allogeneic hematopoietic stem cell transplantation (SCT) has been established as a curative therapy for β-thalassemia major which is prevalent among Thais. Donors are usually patients' siblings but the chance to find an optimal HLA-identical is just about 30%. Thus clinical trial using alternative donors such as partially-mismatched related donors or matched unrelated donors could be a good choice.Methods: To study the efficacy and results, we retrospectively review our experience with β-thalassemia pediatric patients undergoing transplantation from July 1999 to July 2006.Results: There were 43 patients categorized into HLA-identical siblings group (n=26, 13 male and 13 female) and alternative donors group (n=17, 14 male and 3 female). Median age and weight were 7.2 years (1.2–14.8 years), 19.8 kg (9.8–34 kg) and 7.7 years (1.4–14.8 years), 21.5 kg (11–33 kg), respectively. Amongst 26 matched siblings there were 17 bone marrow (BM), 4 peripheral blood stem cell (PBSC), and 5 cord blood (CB) donors. Of the alternative group stem cells derived from 12 matched unrelated, 2 two-antigen mismatched sibling CB, 2 one-antigen mismatched unrelated CB, and 1 one-allele mismatched unrelated BM donors. Myeloablative conditioning regimen consisted of busulfan (oral or intravenous) and cyclophosphamide for matched siblings SCT. Anti-thymocyte globulin was added for alternative donors SCT. Fludarabine was also used in addition for unrelated donors SCT. Graft-vs-host disease (GvHD) prophylaxis consisted of cyclosporine plus methotrexate except for unrelated SCT that tacrolimus plus methotrexate were used instead. Median numbers of infused CD34+ cells of the matched siblings group were 11.1x106/kg (3.7–35.4) in BMT (n=17), 9.3x106/kg (7.1–12.8) in PBSCT (n=4), and 0.94x105/kg (0.2–5.3) in CBT (n=5), compared to 5.5x106/kg (1.5–18.1) in BMT (n=10), 11.1x106/kg (11–17.2) in PBSCT (n=3), and 2.1x105/kg (1.5–3) in CBT (n=4) of the alternative group. Median times to neutrophil and platelet engraftments in each groups were 15 and 33 days, compared to 17 and 41 days, respectively. 39 successful donor engraftments were achieved from 38 HLA-matched (25 related and 13 unrelated) donors and from 1 mismatched unrelated CB unit. The remaining 1 of the matched related group and 3 of the alternative group did not engrafted, all but one case subsequently resumed autologous recovery. Acute GvHD occurred in 4 matched-sibling and 5 unrelated SCT recipients. After immunosuppressive therapy the lesions were completely resolved in all but two patients, one who suffered from severe grade IV intestinal GvHD and another who later developed extensive chronic GvHD which required treatment longer than 4 years. There were 4 mortalities only in the alternative group: 3 patients died post-engraftment due to severe fatal GvHD (n=1), cerebral aspergillosis (n=1), severe veno-occlusive disease with liver failure (n=1; Pesaro class 3); and one died from neutropenic sepsis 3-week after 4/6 matched related CBT. Median follow-up time for surviving patients was 53.5 months (2–84 months). Overall and disease-free survival in HLA-matched SCT (n=38) recipients were 94.7% (n=36) and 92.1% (n=35), compared to in HLA-mismatched (n=5) 60% (n=3) and 20% (n=1), respectively.Conclusions: SCT for β-thalassemia major using HLA-matched donors, either related or unrelated, had superior results than HLA-mismatched. In situation of unavailable matched sibling donors, the search for an appropriate complete HLA-matched unrelated volunteer donor using high resolution DNA typing is essential for favorable outcomes.

Sickle cell disease and pregnancy in Bahrain

Objective: To determine the morbidity and outcome of pregnancies complicated by homozygous sickle cell disease (SCD) in Bahrain. Methods: A controlled study was conducted of all the pregnancies of women with SCD who were delivered at Salmaniya Medical Complex and affiliate hospitals in Bahrain between January 1, 1998, and December 31, 2002. Results: There were 351 pregnancies with SCD, giving an incidence of 0.67%. The mean ± S.D. age of women with SCD was 28.8 ± 5.7 years. These women, who were mostly from Shia villages, had more spontaneous abortions than the 351 controls (32% and 12%, respectively). Compared with the control group, the mean ± S.D. number of pregnancies among women with SCD was 5.0 ± 1.9 vs. 5.7 ± 3.1; birth weight, 2965 ± 540 g vs. 3457 ± 497 g; rate of preterm deliveries, 25.9% vs. 12%; incidence of intrauterine growth restriction, 10% to 13% vs. 4% to 7%; Apgar scores at 1 and 10 min, 8.1 ± 0.8 and 8.8 ± 0.8; and cesarean section rate, 19.0% vs. 12.7%. There were no differences in the rates of pre-eclampsia, antepartum and postpartum hemorrhage, or stillbirth. There were 4 maternal deaths directly related to SCD complications. Hemoglobin analysis was performed in 86% of the women with SCD. Of these women, 60.6% had homozygous SCD with raised levels of fetal hemoglobin (more than 5% HbF); 9.6% had sickle cell hemoglobin with β-thalassemia disease; and 1.4% had sickle cell hemoglobin only. Vaso-occlusive crises were the most common cause of hospital admissions during pregnancy (42.2%), while hemolytic and sequestration crises accounted for 28.0% and 0.6% of admissions. Conclusion: Mortality, morbidity, and perinatal loss are still considerable among women with SCD in Bahrain. To reduce mortality and morbidity, there is a need for a multidisciplinary team able to deal with pregnancy complications due to SCD.

The evaluation of arginase activity during acute vasoocclusive sickle cell crisis

Study objectives: Nitric oxide is an endothelium-derived vasodilator that plays a significant role in the pathophysiology of sickle cell acute vaso-occlusive crisis (VOC) and may play an important role in new therapeutic strategies. Nitric oxide is formed from the conversion of the substrate l-arginine to citrulline by the enzyme endothelial nitric oxide synthase (eNOS). Our previous studies have demonstrated low nitric oxide and, more recently, low l-arginine levels during adult VOC. Although it seems intuitive that low substrate levels would explain low product, the low l-arginine levels in our study were still 10-fold higher than the k_m (3 μM) of eNOS and were more than sufficient to saturate eNOS, yet nitric oxide production was diminished, thereby suggesting a derangement in the normal l-arg-nitric oxide pathway. L-arginine supplementation, in other conditions, even in the face of this low but enzyme-saturating concentration, has been shown to enhance nitric oxide production. This phenomenon has been called the "arginine paradox." One explanation for this paradox involves the enzyme arginase. Arginase, a ubiquitous intra- and extracellular enzyme, catalyzes l-arginine to ornithine. Arginase activity has been found to be enhanced in certain pathologic states and may thus compete for available l-arginine, lessening l-arginine available to NOS. A pediatric study previously demonstrated enhanced arginase activity, as demonstrated by increased ornithine levels during VOC, which has not been examined in adult VOC. We hypothesize that arginase activity is enhanced during adult VOC and may thus play an important role in the l-arginine-nitric oxide pathway during adult VOC. The specific aim of this study is to measure arginase activity by l-arginine and ornithine measurement in VOC. Methods: Included were patients older than 18 years and with a chief complaint of typical VOC pain (defined as patient-described pain consistent in duration, severity, quality, and distribution with previous episodes of VOC and no evidence of coexistent illness), sickle cell patients not in VOC, and healthy controls. Subjects with sickle cell SS, SC, and β-thalassemia disease as documented by previous hemoglobin electrophoresis were eligible. Five milliliters of blood was obtained by standard venipuncture for l-arginine and ornithine analysis before standard analgesic therapy. Blood was immediately centrifuged at 10,000 RPM at 4°C for 10 minutes. The serum was separated and stored at –20°C. L-arginine and ornithine levels were measured by Molecular Structure Facility (University of California, Davis, CA) for an α equal to .05 and power of 0.9, and based on an l-arginine level expected SD of 3.0 μM/L. Results: The study included 42 sickle cell patients in typical VOC, 14 sickle cell controls not in crisis, and 8 healthy controls. As in our previous study, l-arginine was significantly low in VOC (median 23.160 μM/L, <i>P</i><.05) versus healthy sickle cell controls (median 38.560 μM/L) and healthy controls (median 73.010 μM/L). There was no significant difference between citrulline measurements in any of the groups. When ornithine was measured, a statistically significant difference (<i>P</i><.5) was found between sickle cell patients in VOC (median 22.610) and sickle cell healthy controls (36.780). No statistical difference was found between either sickle cell group and healthy controls (median 26.910). Conclusion: We found low ornithine levels in healthy sickle cell controls and VOC subjects. This finding does not support increased arginase activity in sickle cell disease. Interestingly, ornithine levels are significantly lower in VOC than in the healthy sickle cell controls, which may simply be explained by the low l-arginine levels already found in VOC. The significance of this, however, is unknown.

Prenatal Diagnosis in Beta-Thalassemia: An Indian Experience

Objectives: Thalassemia is the most common single gene disorder and is widely distributed in Asian Indians with an average prevalence rate of 4%, with a high prevalence among Sindhis, Punjabis, Gujratis and Bengalis. Prevention and control of β-thalassemia disease require the accurate diagnosis of carriers and proper genetic counseling. Method: Prenatal diagnosis can be performed in the first or second trimester of pregnancy by DNA analysis using polymerase chain reaction. Since there are 17 mutations as well as rare ones causing β-thalassemia in Asian Indians, the point mutation detection by reverse dot blot (RDB) allele-specific oligonucleotide hybridization for common mutations along with the amplification refractory mutation system (ARMS) technique was developed for prenatal diagnosis. Maternal contamination of fetal DNA was ruled out by the variable number of tandem repeat analysis using apolipoprotein B site. Results: Using both techniques (RDB and ARMS) we were able to offer complete diagnosis in 53 pregnancies. On molecular analysis 23% were found to be normal, 48.0% were carriers, and 29.0% were affected with β-thalassemia. Parents were counseled to continue the pregnancy when the fetuses were either normal or had traits whereas in the case of an affected fetus, the parents opted for termination of the pregnancy. Conclusion: Prenatal diagnosis of β-thalassemia by the RDB or ARMS technique can prevent the birth of an affected child in developing countries in which β-thalassemia is quite prevalent.

Bone histomorphometry in children and adolescents with beta-thalassemia disease: iron-associated focal osteomalacia.

Thalassemia/hemoglobinopathy is a hereditary disease that causes chronic anemia and increased erythropoiesis. Consequently, an expansion of bone marrow spaces may contribute to osteopenia/osteoporosis. However, the pathogenesis of bone changes is not yet known. We, therefore, carried out the study on bone histomorphometry and biochemical and hormonal profiles in children and adolescents with suboptimally treated beta-thalassemia disease with the hope of gaining some new insight into the cellular and structural alterations of thalassemic bone. Seventeen patients underwent iliac crest bone biopsy for histomorphometric analyses. Bone mineral density (BMD) measurements were performed by dual energy x-ray absorptiometry. Most patients had growth retardation and delayed bone age. BMD was low especially at the lumbar spine. Serum IGF-I levels were almost always low. Bone histomorphometry revealed increased osteoid thickness, osteoid maturation time, and mineralization lag time, which indicate impaired bone matrix maturation and defective mineralization. In addition, iron deposits appeared along mineralization fronts and osteoid surfaces. Moreover, focal thickened osteoid seams were found together with focal iron deposits. Dynamic bone formation study revealed reduced bone formation rate. These findings indicate that delayed bone maturation and focal osteomalacia are the pathogenesis of bone disease in suboptimally blood-transfused thalassemics with iron overload. Iron deposits in bone and low circulating IGF-I levels may partly contribute to the above findings.

Introductory Speech for Charles Scriver

Introductory Speech for Charles Scriver

Molecular Bases of β‐Thalassemia in the Eastern Province of Saudi Arabia

β-thalassemia is a group of heterogeneous recessive disorders common in many parts of the world. Al-Qatif and Al-Hassa oases in the Eastern Province of Saudi Arabia are regions known for high frequency of these disorders. Using two molecular methods, based on multiplexing-amplification refractory system and reverse hybridization principles, the spectrum of β-thalassemia in the region was studied. Sixty-nine subjects with known β-thalassemia disease and volunteers with high hemoglobin A2(HbA2) and low mean corpuscular volume (MCV) were included in this study. Ten mutations were detected in 91% of the subjects under study. Six of these mutations had previously been observed while the other four mutations are reported here for the first time. In addition, four of the mutations accounted for 76.8% of the subjects studied. IVSII-1 (G > A), IVSI-5 (G > A), and codon 39 (C > T) mutations were found to be the most frequent. However, the frequencies of different mutations reported here are slightly different from those reported earlier. A number of these mutations were also found in the neighboring countries, which can be explained in terms of gene flow.

Prenatal diagnosis of βthalassaemia by reverse dot‐blot hybridization

Thalassaemia is the most common genetic disease and is a public health problem of Thailand. Prevention and control of β-thalassaemia diseases need accurate diagnosis of carriers and proper genetic counselling. Prenatal diagnosis is needed to prevent birth of the thalassaemic offspring in the couple at risk. This can be performed in the first trimester of pregnancy by DNA analysis using the polymerase chain reaction (PCR). Since there are more than 20 mutations causing β-thalassaemia in Thailand, the point mutation detection by reverse dot-blot allele-specific oligonucleotide (ASO) hybridization was developed using two sets of ASO probes. The first battery of ASO probes has been designed to detect 10 common β-globin gene mutations including codon 26, G→A (Hb E); codons 41/42, -TCTT; codon 17, A→T; IVS 2 nt 654, C→T; IVS 1 nt 1, G→T; IVS 1 nt 5, G→C; codon 19, A→G (Hb Malay); codon 35, C→A; codons 71/72, +A and -28 ATA, A→G. The second set of ASO probes detect 14 uncommon β-thalassaemia mutations. We applied this reverse dot-blot hybridization technique to perform prenatal diagnosis in 105 pregnancies at risk of having severe β-thalassaemia diseases. 36 fetuses (34 per cent) were found to be affected with homozygous β-thalassaemia or β-thalassaemia/Hb E disease in which one was twin pregnancy. The others included 31 fetuses with heterozygous β-thalassaemia, 22 heterozygous Hb E, 1 homozygous Hb E and 16 normal fetuses. The common set of ASO probes detected about 95 per cent of cases which suggests that prenatal diagnosis for β-thalassaemia disease can be easily carried out by this approach. Copyright © 1999 John Wiley & Sons, Ltd.