Prevalence of diabetes mellitus in individuals heterozygous and homozygous for sickle cell anemia

Colorimetric determinations of glycosylated Hb were carried out in a sample (n = 97) of sickle cell anemia patients, and in an age- and sex-matched group of individuals (n = 45) heterozygous for sickle cell anemia, from the Eastern Province of Saudi Arabia. A statistically significant increase in the value of glycosylated Hb was found in sickle cell trait (HbAS) group, when compared with those of sickle cell anemia (HbSS) and normal (HbAA) groups. Since glycosylated Hb is considered a valid indicator of long-term blood glucose, and assuming normal red blood cell survival in HbAS carriers, the increased value of glycosylated Hb may suggest that there exists a higher incidence of undiagnosed diabetes mellitus in individuals with heterozygous inheritance for sickle cell hemoglobin than homozygous sickle cell patients and normal individuals. The mechanism underlying this observation remains to be defined.     

Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India

To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.     

Aspects of sickle cell gene in Saudi Arabia—interaction with glucose-6-phosphate dehydrogenase deficiency

Summary Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and sickle cell haemoglobin (Hb S) are red cell genetic abnormalities that occur at a high frequency in several areas of the world including several areas of Saudi Arabia. Genetic and clinical interactions between these two disorders are reported to occur in some populations.In the present investigations, samples from affected individuals were studied for the prevalence of G-6-PD deficiency and Hb S genes. The results of haematological parameters and common clinical findings in the Hb S homozygotes with and without G-6-PD deficiency are presented and the possibility that the two conditions interact beneficially is discussed.     

Genetic polymorphism of drepanocytosis

The pathophysiological mechanism of sickle cell anemia has been thoroughly studied and is now well understood, in contrast to the extreme clinical heterogeneity of the disease. A possible genetic explanation for this diversity arose from the discovery of an HpaI restriction polymorphism 3' to the beta globin gene, in linkage disequilibrium with the Hb S mutation. This linkage is unequally distributed among ethnic groups in Africa and predominantly found in Central West Africa. A multipolymorphic analysis spanning 60 Kb of the beta globin gene cluster demonstrated that the sickle mutation arose at least 3 times in 3 different geographical areas (Atlantic West Africa, Central West Africa and Equatorial Central Africa) and expanded by malaria selection. Two genetic factors seem to have epistatic effects which differ when comparing the two first groups. The alpha thalassemia gene (-alpha) is distributed equally among African Black control populations (0.10). The frequency is significantly higher in the SS patients of the Benin area (Central West Africa), whereas it is unmodified in the patients of Senegal (Atlantic West Africa). Alpha thalassemia does not seem therefore to have exercised the same selective effect in this latter group. Secondly, fetal hemoglobin is quantitatively and qualitatively different in both groups. A high G gamma phenotype (greater than 60%) is found in Senegal, whereas a low G gamma phenotype is constant in Benin, without overlap between the two series. The total production of fetal hemoglobin is statistically higher, although only moderately, so in the first group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sickle cell-beta 0-thalassaemia in Saudi Arabia

During an extensive investigation to determine the frequency of sickle cell and thalassaemia genes in the Saudi population, 22 cases with S/beta 0-thalassaemia were identified and the haematological, biochemical and clinical findings were compared with those in patients with sickle cell anaemia. The values of mean cell volume, mean cell haemoglobin and packed cell volume were found to be lower while all other haematological parameters including Hb A2 were higher in the S/beta 0-thalassaemia group. No statistically significant difference in the Hb F level was found between the two groups. Biochemical parameters were grouped according to organ function tests. Only slight differences were seen in the values of some parameters. The clinical data showed that, in general, patients with sickle cell anaemia had a more severe condition than the S/beta 0-thalassaemia.     

Beta-globin gene haplotypes in the Saudi sickle cell anaemia patients

The beta S-globin gene haplotypes were investigated using restriction endonucleases Hinc II and Hind III in 22 sickle cell anaemia patients from the eastern province, 67 sickle cell anaemia patients from the south-western province and 4 sickle cell anaemia patients from north-western province. The beta S was found to be mainly linked to the haplotype + + - + + in the eastern province (50% homozygous and 45.45% heterozygous), and - - - - + haplotypes in the south-western (44.77% homozygous and 43.28% heterozygous) and north-western (100% homozygous) provinces. A comparison of the haematological values and clinical manifestations in patients with the two major haplotypes revealed significant differences, with the disease presenting more severely in the south-western compared to the eastern population. The level of Hb F was not significantly different in the two groups and no association could be demonstrated between the beta-globin gene haplotype and Hb F level. These results have led us to suggest that the haplotype + + - + + is in some way linked to a benign sickle cell anemia, though the exact mechanism leading to a benign disease is not clear.     

Glutathione reductase deficiency in association with sickle cell and thalassaemia genes in Saudi populations

Glutathione reductase (GR) deficiency is reported to occur with a variable frequency in some populations of the world. In this study, the populations of two regions of Saudi Arabia which have a high frequency of sickle cell, thalassaemia and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, were screened for GR deficiency. Studies were also carried out to investigate the frequency of GR deficiency with other genetic blood disorders. The frequencies of complete GR deficiency were 0.0065 and 0.006, while those of partial deficiency were 0.146 and 0.074 in Al-Hafouf and Khaiber, respectively. GR deficiency was encountered in combination with the sickle gene, the G-6-PD deficiency gene and the thalassaemia gene in both regions. Individuals with GR deficiency showed slightly reduced haematological parameters. In thalassaemic/GR-deficient subjects, mean cell volume and mean cell haemoglobin were low, while in sickle cell anaemia patients with GR deficiency the haematological parameters were higher than in sickle cell anaemia patients without GR deficiency.     

Xmn I polymorphism in the gamma-globin gene region among Saudis

The gamma-globin chains of haemoglobin F are encoded by two non-allelic genes which synthesise two different gamma-chains referred to as G gamma and A gamma. Xmn I restricts on both sides of the genes and normally produces an 8.1-kilobase (kb) fragment containing both gamma-genes. A polymorphic site on the 5' end of the G gamma genes has been reported in some populations and it results in the production of a 7.0-kb fragment containing both gamma-genes. We investigated the Xmn I polymorphic site in normal individuals, haemoglobin S heterozygotes and sickle cell disease patients from three different regions of Saudi Arabia. In the eastern province, 10% of gamma-genes in normal individuals were linked to the 7.0-kb fragment while in the sickle cell disease patients and haemoglobin S heterozygotes the frequency of the polymorphic site was 0.932 and 0.625, respectively. In the south-western province, the frequency of the polymorphic site in normal individuals was 0.096 (similar to that in the eastern province) but in the sickle cell disease patients and sickle cell heterozygotes the frequency was 0.033 and 0.095, respectively. Finally, only 6 sickle cell disease patients from the north-western province were investigated and all had the gamma-globin gene linked to the 8.1-kb fragment. The results of this study in different regions of Saudi Arabia are presented and the possible significance of Xmn I polymorphism is suggested.     

Analysis of the recombination zone in hereditary persistence of fetal hemoglobin with fetal gene rearrangement of the G gamma-G gamma-A gamma type

An increased synthesis of fetal hemoglobin in adult life is a common feature of the genetically determined severe disorders like beta thalassemia and sickle cell anemia. A continued synthesis of fetal hemoglobin in adults is also characteristic of clinical or subclinical syndromes like respectively delta beta thalassemia or hereditary persistence of fetal hemoglobin (HPFH). These disorders are highly heterogeneous with respect to their molecular defects as well as to the composition of Hb F. We report here a novel case of hereditary persistence of fetal hemoglobin in heterozygous state discovered by chance, in a young perfectly healthy french man. The gamma chain of his fetal hemoglobin was almost entirely composed of G gamma chains. Molecular analysis of the DNA revealed the existence of triplicated gamma genes on one chromosome with the genotype arrangement of G gamma-G gamma-A gamma. A polymorphic Xmn I restriction site (at position -158 5' to the cap site) was present in 5' of both of these G gamma genes. The presence of this site in front of G gamma gene had previously been shown to be associated both with high G gamma phenotype constitutively and also with high fetal hemoglobin level only in case of anemic stress. In the absence of any anemic stress in this individual, the constitutive increase of both fetal hemoglobin and G gamma chains could be due to the presence of a chromosome with triplicated arrangement of gamma genes. The classical triplication (G gamma-A gamma-G gamma-A gamma) does not result in HPFH phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcriptional activity of paired homeobox Pax6 is enhanced by histone acetyltransferase Tip60 during mouse retina development

The pathophysiology of oxidative hemolytic anemia is closely associated with hemoglobin (Hb) stability; however, the mechanism of how Hb maintains its stability under oxidative stress conditions of red blood cells (RBCs) carrying high levels of oxygen is unknown. Here, we investigated the potential role of peroxiredoxin II (Prx II) in preventing Hb aggregation induced by reactive oxygen species (ROS) using Prx II knockout mice and RBCs of patients with hemolytic anemia. Upon oxidative stress, ROS and Heinz body formation were significantly increased in Prx II knockout RBCs compared to wild-type (WT), which ultimately accelerated the accumulation of hemosiderin and heme-oxygenase 1 in the Prx II knock-out livers. In addition, ROS-dependent Hb aggregation was significantly increased in Prx II knockout RBCs. Interestingly, Prx II interacted with Hb in mouse RBCs, and their interaction, in particular, was severely impaired in RBCs of patients with thalassemia (THAL) and sickle cell anemia (SCA). Hb was bound to the decameric structure of Prx II, by which Hb was protected from oxidative stress. These findings suggest that Prx II plays an important role in preventing hemolytic anemia from oxidative stress by binding to Hb as a decameric structure to stabilize it.     

Analysis of hemoglobin electrophoresis results and physicians investigative practices in Saudi Arabia

BACKGROUND AND OBJECTIVES:

Riyadh and central province falls in a moderate prevalent zone of hemoglobinopathies in Saudi Arabia. However, it has been observed that the physicians working in Saudi Arabia invariably advise all cases of anemia for hemoglobin electrophoresis (HE). The present work was carried out to study the yield of the HE in Riyadh and the investigative practices of the physicians advising HE.

SETTINGS AND DESIGN:

The study was carried out in the hospitals of King Saud University from 2009 to 2011 in order to assess the yield of HE in referred cases of clinical anemia.

MATERIALS AND METHODS:

A total of 1073 cases divided in two groups of males and females had undergone complete blood count and red blood cell morphology. Cellulose acetate HE was performed and all the positive results were reconfirmed on the high performance liquid chromatography (HPLC). The results were analyzed for the type of hemoglobinopathies. For statistical analysis Statistical Package for Social Sciences 15 version (SPSS Inc., Chicago, IL, USA) was used.

RESULTS:

A total of 405 males and 668 females blood samples were included in the present study. 116 (28.5%) males and 167 (25%) females showed an abnormal pattern on HE. The incidence of beta thalassemia trait was higher in females while sickle cell trait was predominantly seen in males. Red cell indices were reduced considerably in thalassemias, but were unaffected in sickle cell disorders, except those which had concurrent alpha trait. The total yield of HE was 26.6% which was much less than expected.

CONCLUSION:

The physicians are advised to rule out iron deficiency and other common causes of anemia before investigating the cases for hemoglobinopathies, which employs time consuming and expensive tests of HE and HPLC.     

G gamma and A gamma globin genes are identical from -471 of the promoter midway through gamma IVSII in a Benin beta s haplotype associated with elevated fetal hemoglobin.

In seven kindreds in which sickle cell (SS) patients had elevated (greater than 12%) fetal hemoglobin (Hb F), Milner and colleagues reported that a determinant for elevated Hb F and elevated F cells was linked to the beta s gene. Independently, the Senegal (SEN) beta s haplotype has been found in association with elevated Hb F in SS and beta-thalassemia patients. We have used the kindreds of Milner and colleagues to characterize further the association of haplotype and gamma gene DNA sequence variation with Hb F expression. For the largest kindred, Wi, all four SS had high (greater than 14%) Hb F and both SEN and Benin (BEN) haplotypes. Two AS cases carrying SEN had low Hb F and low F cells, while three AS and one CS carrying BEN had elevated Hb F and elevated F cells; only one AS carrying BEN had low Hb F and low F cells. In order to look for genetic alterations that could account for the elevated Hb F of kindred Wi, we sequenced both the G gamma and A gamma genes of the Wi BEN haplotype. The data showed largely identical G gamma and A gamma genes which may have been generated by two gene conversions: the A gamma promoter was like that of G gamma 3' to -471, while the G gamma IVSII was like that of A gamma in its 5' half. In addition, three new mutations were found in gamma IVSII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.     

Hemoglobin G San José {\text{[}}\beta _{\text{2}} {\text{(A4)Glu}} \to {\text{Gly}}\alpha _{\text{2}} {\text{],}}β thalassemia,and α thalassemia in a sicilian family

A 3-year-old child of Sicilian origin was found to have a severe form of Cooley's anemia. Investigations were extended to other members of her family. In three, a rare beta-chain structural Hb variant, Hb G San José [beta 7 (A4) Glu to Gly], was observed: in the father of the porposita heterozygosity for the abnormal Hb was found to be coexistent with beta o thalassemia; two sisters had lowered MCV and MCH values and levels of the abnormal Hb significantly lower than in other heterozygotes for Hb G San José. The alpha-chain/total beta-chain synthesis ratios suggest an alpha-thalassemic-like effect. Their mother had lowered MCV and MCH values, an Hb A2 level in the upper limit of the normal range, and a balanced alpha-chain/beta-chain synthesis ratio. Therefore, the possibility of coexistence of an alpha thalassemia trait with a beta thalassemia trait in the mother of the proposita and with Hb G San José heterozygosity in the two sisters who had lowered levels of abnormal Hb is discussed.     

Does Gγ/Aγ ratio and Hb F level influence the severity of sickle cell anaemia

Sickle cell anaemia (SCA) exhibits significant variations in clinical presentation in different populations for which several genetic factors including SCA-associated -and -thalassaemias, G-6-PD deficiency and elevated Hb F level have been implicated as possible ameliorating factors. Saudi Arabia is unique in that mild and severe forms of the disease occur at a high frequency. We investigated the G/A ratio and Hb F level and correlated these values with the severity of SCA. The results showed that Hb F level varies significantly in both groups of patients with no evident correlation with the mild clinical manifestations. However, G/A ratio correlated significantly with the disease severity where a high ratio was observed in patients with the mild and a low ratio in patients with the severe disease. The results are evaluated and discussed in the light of correlation studies and regression analysis.     

Genetic modifiers in hemoglobinopathies

Hereditary anemias show considerable variation in their clinical presentation. In some cases, the causes of these variations are easily apparent. In thalassemia (or in HbE/thalassemia), genetic variation is primarily caused by the severity of the thalassemia mutation. However, not uncommonly, there is variation unexplained by the globin gene mutations themselves, which may be caused by genetic modifiers. In sickle cell disease, the primary mutation is the same in all patients. Therefore, variations in disease severity generally are due to genetic modifiers. In most genetic diseases involving beta globin, the most clearcut influence on phenotype results from elevated fetal hemoglobin levels. In addition, alpha globin gene number can influence disease phenotype. In thalassemia major or intermedia, reduction in the number of alpha globin genes can ameliorate the disease phenotype; conversely, excess alpha globin genes can convert beta thalassemia trait to a clinical picture of thalassemia intermedia. In sickle cell disease, the number of alpha globin genes has both ameliorating and exacerbating effects, depending on which disease manifestation is being examined. Unlinked genetic factors have substantial effects on the phenotype of hereditary anemias, both on the anemia and other disease manifestations. Recently, studies using genome-wide techniques, particularly studying QTLs causing elevated HbF, or affecting HbE/thalassemia, have revealed other genetic elements whose mechanisms are under study. The elucidation of genetic modifiers will hopefully lead to more rational and effective management of these diseases.     

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.     

Assessment of erythrocyte deformability with the laser-assisted optical rotational cell analyzer (LORCA)

The erythrocyte deformability of 28 patients with anemia was evaluated with the laser-assisted optical rotational cell analyzer (LORCA), an image analyzer that converts into numerical form the degree of refraction of a laser beam induced by red cells subjected to a range of torsional stresses. The patients were 10 thalassemics, including three with intermediate forms (1 HbC/beta degree, 1 homozygote beta for Orkin's haplotype VI, 1 beta degree/beta delta Sicilian type) and seven heteroygotes for beta Th; six with hereditary spherocytosis (including 2 with structural alteration of the spectrin beta chain); three with type II congenital dyserythropoietic anemia (HEMPAS), two hemizygotes and one heterozygote for G-6PD deficiency, and six with severe hypochromic hyposideremic anemia. Red cell deformability was reduced in intermediate thalassemia, hereditary spherocytosis and HEMPAS, normal in heterozygous beta thalassemia and G-6PD deficiency, and increased in hypochromic hyposideremic anemia. These results show that erythrocyte deformability can be impaired by an Hb chain imbalance, membrane and cyto skeleton structure anomalies and changes in the red cell area/volume ratio.     

Patterns of sickle cell, thalassaemia and glucose-6-phosphate dehydrogenase deficiency genes in north-western Saudi Arabia

This study was conducted on 429 blood samples collected from Saudi males and females from Al-Ula in the north-western province of Saudi Arabia in order to determine the frequency of the sickle cell gene, glucose-6-phosphate dehydrogenase (G6PD) deficiency gene, and alpha- and beta-thalassaemia genes, and to investigate the pattern of their interactions. The frequency of the sickle cell gene was 0.0785, while that of the beta-thalassaemia gene was 0.1195. Heterozygous alpha-thalassaemia 2 (- alpha/alpha alpha) was encountered at a frequency of 0.121, while homozygous alpha-thalassaemia 2 (- alpha/- alpha) occurred at a frequency of 0.0046. HbH disease and hydrops fetalis were not encountered. One case with triple alpha-gene arrangement, alpha alpha alpha anti-3.7, was identified. The G6PD deficiency gene frequency was 0.08 and 0.032 in males and females, respectively. Several cases with 2 abnormal genes were encountered. The haematological and biochemical data from the patients with sickle cell disease suggest that the disease in this population is more severe in comparison with cases reported from the eastern population.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.