Naglaa Fawaz

Varied Prevalence of Factor V G1691A (Leiden) and Prothrombin G20210A Single Nucleotide Polymorphisms Among Arabs

Background: Factor V G1691A (FV-Leiden) and prothrombin (PRT) G20210A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Arabs, we addressed the prevalence of both SNPs in 4 distinct Arab populations (Lebanon, Tunisia, Bahrain, and Saudi Arabia).Methods: Study subjects comprised 698 Lebanese, 313 Tunisian, 194 Bahraini, and 149 Saudi Arabian healthy subjects; genotyping was done by PCR-RFLP using Mnl I and Hind III for FV-Leiden and PRT G20210A, respectively.Results: The prevalence of the mutant A alleles of FV-Leiden and PRT G20210A were significantly higher among Lebanese (0.0788 and 0.0136) and Tunisians (0.0351 and 0.0128), as compared to Bahraini (0.0155 and 0.0052) and Saudi (0.0101 and 0.000) subjects. Higher frequency of the FV-Leiden G/A and A/A genotypes were seen in Lebanon (13.8 and 1.0%), followed by Tunisia (5.8 and 0.6%), Bahrain (3.1 and 0.0%) and Saudi Arabia ((2.0 and 0.0%). All PRT G20210A positive cases were in the heterozygote (G/A) state, and these comprised 3.6% for Lebanon, 2.6% for Tunisia, 1.0% for Bahrain. The carrier rate of FV-Leiden was significantly higher among Lebanese compared to the other populations (p < 0.001), while the difference in the prevalence of FV-Leiden between the other populations was not statistically different. With the exception of Lebanese-Saudi (p = 0.038), the prevalence of PRT G20210A was similar among the study communities. Furthermore, the overall average genetic differentiation between populations (estimated with the FST) was 0.0022 for FV-Leiden and 0.005 for PRT G20210A.Conclusions: These results further confirm the heterogeneity in FV-Leiden and PRT G20210A distribution among Arabs, and recommend potential institution of prophylactic measures for carriers of either or both SNPs.

An Arab selective gradient in the distribution of factor V G1691A (Leiden), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T

Venous thrombosis (VTE) is a multi-factorial disease resulting from the interaction of genetic and environmental risk factors. Among the inherited factors are factor V (FV) G1691A (FV Leiden) [1], prothrombin (PRT) G20210A [2], and methylenetetrahydrofolate reductase (MTHFR) C677T [3] single nucleotide polymorphisms (SNPs). In FV Leiden, an arginine is substituted by glutamine at amino acid residue 506, which renders FVa resistant to degradation by activated protein C [1]. The PRTG20210A SNP, a G to A transition in the 3¢ untranslated region of the PRT gene, is associated with increased PRT levels [2].WhilebothFVLeidenandG20210ASNPsarepresent at varying rates in Caucasians [4], and are virtually absent from Africans and Asians [5], the MTHFR C677T is found in many populations with a marked heterogeneity in its distribution, exemplified by its low incidence in Africa and Indian subcontinent [6], and higher rates in North America, Europe [7], and Japan. Insofar as the incidence of FV Leiden, PRT G20210A, andMTHFRC677T SNPs among Arabs is poorly defined, we assessed the incidence of these three SNPs in four distinct Arab communities: Lebanon, Tunisia, Bahrain, and Saudi Arabia. Study subjects comprised 698 Lebanese (288 males and 410 females), 313 Tunisian (129 males and 184 females), 193 Bahraini (150 males and 43 females), and 149 Saudi (69 males and 80 females) healthy subjects. Genotyping was carried out by PCR-RFLP analysis using MnlI, HindIII, and HinfI digestion for detecting FV Leiden, PRT G20210A and MTHFR C677T, respectively. Allele frequencies were determined using the gene counting method, and Wright’s FST calculations were made using GENALEX software. Additional statistical was performed with SPSS version 12.0.1. The distribution of the three SNPs genotypes was in Hardy– Weinberg equilibrium. The frequencies of FV Leiden (A) and PRT G20210A mutant (A) alleles, together with the G/A and A/A genotypes of FV Leiden, were highest among Lebanese, while Tunisians, Bahraini and Saudi Arabian participants had lower frequencies (Table 1).Higher incidence of FVLeidenwas seen among Lebanese compared with other populations (P < 0.001), while the difference between Tunisians and Bahraini (P 1⁄4 0.157) or Saudi (P 1⁄4 0.073) subjects was not different. All PRT G20210A SNP carriers were in the heterozygous state (G/A); no PRT G20210A SNP carrier was found among Saudi participants (Table 1). With the exception of Lebanese–Saudi (P 1⁄4 0.038), thedistributionofPRTG20210A was similar among study communities. The distribution of the C677T alleles varied; high incidence of the T allele and the T/T genotype was found in Lebanon and Tunisia, and lower rates among Saudi and Bahraini subjects (Table 1). While the incidence of T/T genotype was similar between Lebanese and Tunisian (P 1⁄4 0.542), and Saudi and Bahraini (P 1⁄4 0.470) subjects, higher incidence of T/T genotype was seen among Lebanese vs. Bahraini (P < 0.001) and Saudi (P 1⁄4 0.014), and between Tunisian and Bahraini (P 1⁄4 0.005) subjects.

dRTA and hemolytic anemia: first detailed description of SLC4A1 A858D mutation in homozygous state

Mutations in the anion exchanger 1 (AE1) gene encoding the erythroid and kidney anion (chloride–bicarbonate) exchanger 1 may result in familial distal renal tubular acidosis (dRTA) in association with membrane defect hemolytic anemia. Seven children presenting with hyperchloremic normal anion gap metabolic acidosis, failure to thrive, and compensated hemolytic anemia were studied. Analysis of red cell AE1/Band 3 surface expression by Eosin 5′‐maleimide (E5M) was performed in patients and their family members using flow cytometry. Genetic studies showed that all patients carried a common SLC4A1 mutation, c.2573C>A; p.Ala858Asp in exon 19, found as homozygous (A858D/A858D) mutation in the patients and heterozygous (A858D/N) in the parents. Analysis by flowcytometry revealed a single uniform fluorescence peak, with the mean channel fluorescence (MCF) markedly reduced in cases with homozygous mutation, along with a left shift of fluorescence signal but was only mildly reduced in the heterozygous state. Red cell morphology showed striking acanthocytosis in the homozygous state [patients] and only a mild acanthocytosis in heterozygous state [parents]. In conclusion, this is the first description of a series of homozygous cases with the A858D mutation. The E5M flowcytometry test is specific for reduction in the Band 3 membrane protein and was useful in conjunction with a careful morphological examination of peripheral blood smears in our patient cohort.

Human platelet alloantigens (HPA) 1, HPA2, HPA3, HPA4, and HPA5 polymorphisms in sickle cell anemia patients with vaso-occlusive crisis

Objectives:  Vaso‐occlusive crisis (VOC) is a significant cause of morbidity and mortality in sickle cell anemia (SCA) patients. Insofar as polymorphism in human platelet alloantigen (HPA) exhibit a prothrombotic nature, we hypothesized that specific HPA polymorphic variants are associated with VOC. We investigated the distribution of HPA1, HPA2, HPA3, HPA4, and HPA5 alleles genotypes among VOC and non‐VOC control SCA patients.

Severity ranking of non-deletional alpha thalassemic alleles: insights from an Omani family study

In an Omani family, four different alpha thalassemic alleles, one single‐gene deletional (−α3.7) and three non‐deletional forms (αTSaudi, αΔ5nt, and αΔG), interact in various combinations and result in two distinct hematological phenotypes, with and without HbH inclusions. After excluding the presence of potential genetic modifiers, viz associated β‐thalassemic alleles or functional alpha hemoglobin stabilizing protein (AHSP) polymorphisms, we observed that only the genetic combinations involving αTSaudi mutation are associated with HbH inclusions (a marker of degree of α/β‐chain imbalance) and high reticulocyte count (a marker of ongoing hemolysis). Overall, the αTSaudi mutation is associated with a more severe α‐globin deficiency than the other two (αΔ5nt and αΔG) non‐deletional α0 thalassemic mutations. The likely molecular explanation is that the compensatory increase in the linked α1 globin gene expression is much more compromised in cases with αTSaudi mutation.

Association of the Methylenetetrahydrofolate Reductase A1298C but not the C677T Single Nucleotide Polymorphism with Sickle Cell Disease in Bahrain

The association of methylenetetrahydrofolate reductase (MTHFR) gene mutations, C677T and A1298C, together with changes in homocysteine (Hcy) levels was investigated in 106 sickle cell disease patients and 156 healthy controls from Bahrain. The mutation analysis was done by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). While the frequencies of the mutant alleles C677T and A1298C were comparable between patients and controls, the frequency of the A1298C (C/C) (p = 0.03) but not C677T (T/T) (p = 0.67) genotype, and of the 677T/1298C haplotype were significantly higher in the patients (p = 0.05). Homocysteine levels were normal in all subjects. This suggests that the A1298C, but not C677T, mutation is associated with the genotype of sickle cell disease.

Coexistence of sickle cell disease and severe congenital neutropenia: first impressions can be deceiving

We report an Omani family in whom the propositus had a rare coexistence of sickle cell disease and severe congenital neutropenia associated with a mutation in ELANE. In contrast to his siblings with sickle cell disease, the severity of HbSS‐associated complications such as painful crises and acute chest syndrome was significantly reduced. His course of the disease had markedly worsened after initiating G‐CSF therapy. These clinical observations suggest that neutropenia may ameliorate inflammatory responses and thus display a modulating factor with respect to the clinical course of sickle cell disease.

Idiopathic thrombocytopenic purpura and hypokalaemic dRTA with compensated haemolysis and striking acanthocytosis in a band 3 (SLC4A1/AE1) A858D homozygote

Dear Editor, The pleiotropic effects of mutations of the band 3 (SCL41/ AE1) gene which result in familial distal renal tubular acidosis (dRTA) and red cell abnormalities are now wellestablished [1]. The occurrence of dRTA and haemolytic anaemia in A858D homozygotes was first reported recently in children from India [2] and Oman [3]. This letter describes certain unusual features in an Omani adolescent A858D homozygote. A 17-year-old Omani Arab youth, son of a consanguineous marriage presented with complaints of bleeding gums and easy bruising for a few weeks. Also, he reported experiencing progressively severe muscle weakness which, after several weeks, culminated in his inability to rise out of bed. He was of normal stature and weight with mild scleral icterus, generalised skin purpura, buccal mucosal petechiae and mild splenomegaly; hypotonia, hyporeflexia and quadriparesis were additional findings. Investigations showed a normal haemoglobin (Hb— 131 g/L), haematocrit (0.39) and white blood cell count, but an increased reticulocyte count (373.5×10/L) and a very low platelet count (6.0×10/L). Freshly drawn blood films revealed polychromasia, a striking predominance of acanthocytes (70–85 %), a moderate number of ovalocytes and a few other poikilocytes (Fig. 1). Ahaptoglobinaemia, unconjugated hyperbilirubinaemia, normal serum immunoglobulins and a negative antiglobulin test provided supportive evidence for a non-immune-compensated haemolysis. The bleeding problem was diagnosed as autoimmune idiopathic thrombocytopenic purpura (ITP) despite negative results of platelet and other relevant autoantibody tests [4]. Associated autoimmune diseases such as SLE and Sjogrens syndrome [5] were ruled out. Quadriparesis was due to hypokalaemia (serum K, 2.5 mmol/L) secondary to complete dRTA characterised essentially by hyperchloraemic acidosis (Cl, 117 mmol/ L), reduced serum HCO3 − (18 mmol/L) concentration and normal values of serum anion gap, blood urea, serum creatinine, arterial blood pH and blood gases, coupled with a positive urine anion gap and a persistent urine pH >6.0; the patient refused a urine acidification test. DNA analysis revealed a homozygous A858D (alanine → aspartic acid) mutation of the SLC41/AE1 gene. Following a 3-week course of oral prednisolone, bleeding symptoms ceased, platelet count was normalised (317×10/ L) and muscle weakness improved considerably on oral potassium chloride, sodium bicarbonate and spironolactone. During the ensuing years, because of poor drug compliance, he was hospitalised several times with hypokalaemic limb pareses (serum K levels varying between 2.0 and 3.1 mmol/L), but his blood counts have remained normal throughout. Ultrasonography later revealed bilateral J. U. Rehman : S. Al Zadjali :N. A. Fawaz : S. Al Kindi Department of Haematology, College of Medicine & Health Sciences, Sultan Qaboos University, PO Box 35, Muscat 123, Sultanate of Oman

Immunophenotypic characteristics of T-acute lymphoblastic leukemia in Omani patients: A correlation with demographic factors

Objectives To study and classify the immunophenotypic characteristics of Omani patients diagnosed with T-cell acute lymphoblastic leukemia (T-ALL) and to correlate the results with age and gender as well as biological factors (peripheral and bone marrow blast cells percentage). Methods Fifty cases from both genders and of all ages who fulfilled the inclusion criteria with a diagnosis of T-ALL were included in the study. Correlation of T-ALL subtypes with age, gender, and initial bone marrow and peripheral blood blast cells percentage was assessed using ANOVA. Results Among the 50 T-ALL patients analyzed, 44 were male and six were female giving a male-to-female ratio of 7:1 (p = 0.007). The average age of patients was 19.2 years with no significant differences in the three disease subtypes. No significant association was seen between the peripheral or bone marrow blast cell percentage and the differentiation stages of the neoplastic clone of T-ALL. All female patients were found to express an immature T-ALL phenotype. Conclusions This study reports the subtypes of T-ALL in Oman for the first time. It is hoped that this will lead to a better understanding of the disease outcomes.

Anti-D-induced severe hemolytic disease of the newborn in an Omani newborn born a rhesus-positive mother

Abstract Hemolytic disease of the fetus and newborn (HDFN) due to anti-D antibodies is a well-known complication of rhesus (Rh) incompatibility, encountered in D-positive babies born to alloimmunized D-negative mothers who have been sensitized during previous labor or abortion. Here, we report a case of significant hemolytic disease of the newborn due to the presence of anti-D antibodies in an Rh-positive baby born to an Rh-positive mother. The boy presented at day 1 of life with neonatal jaundice and required intensive phototherapy. His hemoglobin (Hb) concentration gradually dropped from 17 g/dL to 6.6 g/dL. The blood bank workup revealed O Rh-positive blood group, with a positive direct antiglobulin test (DAT) and confirmed the presence of anti-D antibodies. His mother was typed as O Rh positive, with a negative DAT and positive anti-D. He required two blood transfusions, and his Hb stabilized at the age of 7 weeks. Anti-D HDFN is a rare complication of Rh-positive and Rh-weak positive pregnancies. The lack of awareness of this phenomenon is often a source of confusion for clinicians. A literature review of similar cases and possible explanations are discussed.