First report of the spectrum of δ-globin gene mutations in Omani subjects - identification of novel mutations.

INTRODUCTION: Both coinheritance of thalassemic δ-globin mutation and coexistence of iron deficiency anemia (IDA) tend to decrease HbA2 (α2 δ2 ) level and thereby poses a diagnostic conundrum in ß-thalassemia trait. METHODS: We retrospectively studied 78 Omani subjects, presenting with low HbA2 level by high-performance liquid chromatography (HPLC), and their DNA was sequenced for the presence of mutations in the δ-globin gene (HBD). In these subjects, their serum ferritin levels allowed evaluation of the degree of iron deficiency. RESULTS: Overall, six different δ-globin gene mutations were observed in 40 study subjects (51.3%) and IDA in 33 subjects, with the remaining five subjects showing normal HBD sequence and serum ferritin level. Among the subjects with δ-globin gene mutations, seven had an associated IDA confirmed by significantly low serum ferritin levels. Heterozygosity for the delta (+) cd27G-->T mutation (HbA2 -Yialousa; HBD: c.82G>T) was the most common abnormality observed (n = 26, 66.6%) followed by heterozygosity for HBD c.-118C->T (d -68 C->T) (n = 6, 15.4%), for cd16G-->C (n = 4, 10.3%), for cd98G-->A (n = 2, 5.1%), for cd142G-->C (n = 1, 2.6%), and for cd147G-->T (n = 1, 2.6%). CONCLUSIONS: These delta mutations exhibit low HbA2 either due to a shift in the HPLC position or due to their bona fide thalassemic feature. Two mutations, namely cd142 G-->C (GCC to CCC, Ala to Pro) and stop codon cd147 G-->T (stop to Leu with elongation of 15 amino acids), herein first reported are novel. Coexistence of IDA could lead to erroneous diagnostic interpretation unless it is specifically looked for.

Potential pitfalls in the diagnosis of Hb Handsworth in areas with high prevalence of HbS.

Hb Handsworth is a rare α-globin structural variant caused by a missense mutation either on the α2 or α1-globin gene (HBA2 or HBA1: c.55G>C, p.Gly18Arg). This variant might be erroneously diagnosed as HbS unless secondary confirmative tests are carried out. We encountered a child with a prominent peak eluting in the 'S' window on high-performance liquid chromatography (HPLC). Sickle solubility test, gel electrophoresis, and selective direct nucleotide sequencing of α1, α2, and ß globin genes were performed on the patient's sample. In addition, previous HPLC results on a cord blood sample were retrieved. Sickle solubility test was negative. Gel electrophoresis revealed a band migrating at the S region with an extra faint band seen on acid gel electrophoresis. Molecular analysis of α2 globin gene revealed heterozygous state of Hb Handsworth. Hb Handsworth is a rare variant that can mimic HbS on HPLC. Failure to recognize this rare variant in regions where HbS is highly prevalent may result in serious misdiagnosis and subsequent incorrect genetic counseling.

The pneumatic tube system does not affect complete blood count results; a validation study at a tertiary care hospital.

INTRODUCTION: Effect of the pneumatic tube system (PTS) on sample quality is controversial. Herein we aim at evaluating the impact of sample transportation via the PTS on complete blood count (CBC) results. METHODS: Duplicate CBC samples from normal donors and anemic patients were sent in parallel to the laboratory for testing through the PTS and the courier (CO). We used scatter plots, Bland-Altman plots, correlation coefficient (r), and coefficient of determination for the validation. RESULTS: A total of 115 samples (donors: 59, patients: 56) were tested. There was excellent correlation between both methods for red blood cell parameters (r range = 0.9213-0.9958) and platelet count. White blood cell (WBC) count and differential count showed similar results (r range = 0.8605-0.9821) for all, with exception of basophils which showed modest correlation (r = 0.4827 for patients and 0.5758 for normal donors). Most of the differences in measurement of all CBC parameters were within the 95% confidence interval of the mean difference on Bland-Altman plots. CONCLUSION: Modern PTS can be safely used for transporting CBC samples.

Splenectomy and thrombosis: the case of thalassemia intermedia.

BACKGROUND: Hypercoagulability in splenectomized patients with thalassemia intermedia (TI) has been extensively evaluated. However, clinical and laboratory characteristics of patients who eventually develop overt thromboembolic events (TEE) are poorly studied. PATIENTS/METHODS: Three Groups of TI patients (n=73 each) were retrospectively identified from a registry involving six centers across the Middle East and Italy: Group I, all splenectomized patients with a documented TEE; Group II, age- and sex-matched splenectomized patients without TEE; and Group III, age- and sex-matched non-splenectomized patients without TEE. Retrieved data included demographics, laboratory parameters, clinical complications, and received treatments that may influence TEE development, and reflected the period prior to TEE occurrence in Group I. RESULTS: The mean age of Group I patients at development of TEE was 33.1±11.7years, with a male to female ratio of 33:40. TEE were predominantly venous (95%) while four patients (5%) had documented stroke. Among studied parameters, Group I patients were more likely to have a nucleated red blood cell (NRBC) count ≥300×10(6) L(-1) , a platelet count ≥500×10(9) L(-1) and evidence of pulmonary hypertension (PHT), or be transfusion naïve. The median time to thrombosis following splenectomy was 8years. Patients with an NRBC count ≥300×10(6) L(-1) , a platelet count ≥500×10(9) L(-1) , or who were transfusion naive also had a shorter time to thrombosis following splenectomy. CONCLUSION: Splenectomized TI patients who will develop TEE may be identified early on by high NRBC and platelet counts, evidence of PHT, and transfusion naivety.

Hematopoietic stem cell transplantation in Oman.

Hematopoietic SCT (HSCT) is an integral part of the management of patients with hematologic disorders. The Sultanate of Oman, with a population of 2.3 million, has an HSCT program based in the Sultan Qaboos University (SQU) hospital. Initiated in 1995, this two-bed unit continues to be the only program in the country. Between June 1995 and August 2006, a total of 128 patients underwent HSCT in this center, averaging about 10-12 transplants per year. The median age of these patients was 11 years (2 months to 45 years). Hematologic malignancies (49%) and inherited disorders (42%) constituted the major transplant indications, whereas BM failure accounted for the remaining. The majority of transplants carried out so far have been HLA-matched sibling-donor allogeneic HSCTs. Among the inherited disorders, homozygous beta-thalassemia and primary immunodeficiency are important transplant indications in this center. The approximate cost of an uncomplicated transplant in this center is US$50,000. The success of this program has now led to the initiation of a new and larger HSCT complex to provide the opportunity for more patients to benefit from this treatment modality within the country.

Combined therapy with desferrioxamine and deferiprone in beta thalassemia major patients with transfusional iron overload.

Iron overload is the main cause of morbidity and mortality especially from heart failure in patients with beta thalassemia major (TM). Successful iron chelation is therefore essential for the optimal management of TM. Although desferrioxamine (DFX) has been the major iron-chelating treatment of transfusional iron overload, compliance is a major hindrance in achieving optimal therapeutic results. The availability of oral iron chelation with deferiprone (L(1)) since 1987 is useful but showed poor efficacy when used alone as compared to DFX. We therefore decided to compare DFX alone with a prospective combined therapy with DFX and L(1) in beta thalassemia major patients with iron overload. We studied 91 patients with beta thalassemia major (mean age+/-SD, 15.02+/-5.8; range 2-30 years) attending the day care unit for regular transfusional support. They received packed red cells every 3-4 weeks to maintain pretransfusion hemoglobin concentration above 9 g/dl. They had been receiving DFX at a daily dose of 40 mg kg(-1) day(-1) by subcutaneous infusion for 8-10 h on 4-5 nights each week for the past several years. However, due to various reasons, they had developed considerable transfusional iron overload. These patients were allocated to prospectively receive additional therapy with oral iron chelator L(1) at 75 mg kg(-1) day(-1) body weight in three divided doses with food after informed consent and continued to receive treatment with DFX as per the above dosage. Of the 91 patients, six developed severe gastrointestinal (GI) upset, two agranulocytosis, two arthropathy, one persistently raised liver enzymes, two died owing to sepsis, and two received allogeneic bone marrow transplantation. Amongst the remaining 76 patients, 21 were found noncompliant (not taking DFX regularly, but taking L(1) regularly). Thus, in the 55 evaluable patients {6-48 months on combination therapy; mean [(+/-SD)22+/-12 months]}, the mean serum ferritin (+/-SD) fell dramatically from 3,088 (+/-1,299) ng/ml (DFX alone) to 2,051 (+/-935) ng/ml (DFX and L(1); p<0.001). It is interesting to note that there was also a significant improvement in the myocardial function as assessed by the ejection fraction (p<0.004) and fractional shortening (p<0.05) in those patients (n=42) who could be studied after being on combination therapy for a minimum of 1 year. The study emphasizes that beta thalassemia major patients with transfusional iron overload can be successfully treated with a combination of DFX and L(1). Our results also demonstrate a significant statistical improvement after as little as 6 months of combination therapy. Furthermore, these improvements lead to a progressive fall in the mean serum ferritin. Lastly, the study also demonstrates significant improvement in the echocardiographic parameters of myocardial performance in these patients receiving combination therapy.

A community-based study of common hereditary blood disorders in Oman.

We assessed the prevalence of three common hereditary blood disorders (sickle-cell and beta-thalassaemia traits and glucose 6-phosphate dehydrogenase deficiency) among the Omani population. We interviewed a representative sample of 6103 Omani households and blood samples from 6342 children aged 0-5 years were collected. About 27% of Omani males had inherited glucose-6-phosphate dehydrogenase deficiency (compared with 11% of females) while countrywide prevalence rates for the sickle-cell and beta-thalassaemia traits were estimated to be 5.8% and 2.2% respectively and showed no significant gender differences. There was a significant association between all three disorders and region of the country.

A community-based study of common hereditary blood disorders in Oman

We assessed the prevalence of three common hereditary blood disorders [sickle-cell and beta-thalassaemia traits and glucose 6-phosphate dehydrogenase deficiency] among the Omani population. We interviewed a representative sample of 6103 Omani households and blood samples from 6342 children aged 0-5 years were collected. About 27% of Omani males had inherited glucose-6-phosphate dehydrogenase deficiency [compared with 11% of females] while countrywide prevalence rates for the sickle-cell and beta-thalassaemia traits were estimated to be 5.8% and 2.2% respectively and showed no significant gender differences. There was a significant association between all three disorders and region of the country

Primary pseudomonas meningitis in an adult, splenectomized, multitransfused thalassaemia major patient.

A 19-year-old splenectomized, multitransfused female patient with beta-thalassaemia major developed primary meningitis due to P. putida. Her blood cultures were negative. P. putida is an unusual nosocomial organism to cause primary meningitis. Infection due to this organism carries high mortality. However, owing to early diagnosis and energetic treatment this patient survived without any sequelae. A review of serious infections over the last 7 years in patients in our thalassaemia care centre revealed 11 serious infections among our splenectomized patients (n = 46) and none in the non-splenectomized group (n = 106). Surprisingly, all overwhelming infections (23.8% in the splenectomized group) were caused by Gram-negative bacilli like Klebsiella, Pseudomonas, Aeromonas and Campylobacter species. As all our splenectomized patients had prior pneumococcal vaccination and oral penicillin prophylaxis, overwhelming septicaemia due to S. Pneumoniae was successfully prevented, but an increasing incidence of overwhelming sepsis due to Gram-negative bacilli, against which no vaccination or suitable prophylactic antibiotics are available, is now posing a new threat to this vulnerable group of patients.

Genetic epidemiology of HbS in Oman: multicentric origin for the betaS gene.

On the basis of a sample of 117 chromosomes, we have demonstrated the multicentric origin of the sickle mutation in Northern Oman. Three major haplotypes coexist: 52.1% Benin (typical and atypicals), 26.7% Arab-India, and 21.4% Bantu. These haplotypes are not autochthonous to Oman but originated elsewhere and arrived in Oman by gene flow. The distribution of haplotypes is in excellent agreement with the historical record, which establishes clear ancient contacts between Oman and sub-Sahara west Africa and explains the presence of the Benin haplotype; contacts with Iraq, Iran, present-day Pakistan, and India explain the presence of the Arab-India haplotype. More recent contacts with East Africa (Zanzibar/Mombasa) explain the presence of the Bantu haplotype. The pattern of the Arab-India haplotype in the populations of the Arabian peninsula reinforces the hypothesis that this particular mutation originated in the Harappa culture or in a nearby population and in addition reveals that the Sassanian Empire might have been the vehicle by which this Indo-European sickle mutation migrated (gene flow) to the present-day Arabian peninsula, including Oman.

HbS-oman heterozygote: a new dominant sickle syndrome.

Hemoglobin (Hb) S-Oman has two mutations in the beta-chains. In addition to the classic betaS mutation (beta6 Glu --> Val), it contains a second mutation in the same chain (beta121 Glu --> Lys) identical to that of HbOARAB. We have studied a pedigree of heterozygous carriers of HbS-Oman that segregates into two types of patients: those expressing about 20% HbS-Oman and concomitant -/ thalassemia and those with about 14% of HbS-Oman and concomitant -/- thalassemia. The higher expressors of S-Oman have a sickle cell anemia (SS) clinical syndrome of moderate intensity, while the lower expressors have no clinical syndrome, and are comparable to the solitary case first described in Oman. In addition, the higher expressors exhibit a unique form of irreversibly sickled cell reminiscent of a "yarn and knitting needle" shape, in addition to folded and target cells. The CSAT of S-Oman is identical to that of S-Antilles, another supersickling hemoglobin, whose carriers express the abnormal hemoglobin at 40% to 50%, with a very similar clinical picture to HbS-Oman. Because the level of expression is so different and the clinical picture so similar, and based on the hemolysates CSAT's, we conclude that HbS-Oman produces pathology beyond its sickling tendencies. A clue for this additional pathogenesis is found in the fact that homozygous HbOARAB, which has the same second substitution as S-Oman, has a moderately severe hemolytic anemia; when HbOARAB is combined with HbS, it makes the phenotype of this double heterozygote as severe as SS. Properties of HbS-Oman red blood cells (RBCs) include reticulocytes that are much denser than normal (similar to those of SC and CC disease), a decrease in the Km for Ca2+ needed to activate the Gardos' channel (making this transporter more sensitive to Ca2+), increased association of HbS-Oman with the RBC membrane, the presence of dense cells by isopycnic gradient, the presence of folded cells, and abundant nidus of polymerization under the membrane. Other properties include a clear increase in volume and N-ethylmaleimide-stimulated K:Cl cotransport in RBCs expressing more than 20% HbS-Oman. We conclude that the pathology of heterozygous S-Oman is the product of the sickling properties of the beta6 Val mutation which are enhanced by the second mutation at beta121. In addition, the syndrome is further enhanced by a hemolytic anemia induced by the mutation at beta121. We speculate that this pathology results from the abnormal association of the highly positively charged HbS-Oman (3 charges different from normal hemoglobin) with the RBC membrane.

Molecular characterization of G6PD deficiency in Oman.

Screening of unselected university students in the Sultanate of Oman revealed an overall frequency of glucose-6-phosphate dehydrogenase (G6PD) deficiency of 26% in males. Samples from 23 G6PD-deficient individuals (a random sub-sample of the student population), were characterised biochemically and at the molecular level. Of 20 deficient men, 15 had G6PD Mediterranean, 2 had G6PD Chatham, 1 had G6PD A- and in 2 the mutation is not yet known. Of the 3 G6PD-deficient woman, 2 were homozygous for the G6PD Mediterranean mutation and 1 was a genetic compound, G6PD Mediterranean/G6PD A- (the first report of this genotype). Our findings establish that the G6PD Mediterranean mutation accounts for most cases of G6PD deficiency in Oman. The presence of G6PD A- at a polymorphic frequency can be regarded as evidence of significant gene flow from Africa.

Frequency and clinical significance of erythrocyte genetic abnormalities in Omanis.

The frequencies of four malaria associated erythrocyte genetic abnormalities have been established in 1000 Omani subjects. They are: homozygous alpha+ thalassaemia (-alpha/-alpha) 0.45; high Hb A2 beta thalassaemia trait 0.015; sickle trait (Hb A/S) 0.061; and glucose 6 phosphate dehydrogenase deficiency (Gd-): males 0.27, females 0.11. From our data the alpha+ (-alpha/) thal gene (confirmed by Southern blotting) is pandemic in this population. Moreover, in spite of the very high frequency of Gd-, oxidative haemolytic syndromes are very uncommon. Also preliminary data indicate that among the Omani population with sickle cell disease, homozygosity of the alpha+ gene markedly modifies the clinical picture.