Prevalence of α-Thalassemia in the Egyptian Population

Abstract

Hemoglobinopathies are the most common monogenic diseases in the world, causing many health problems worldwide. In Egypt, thalassemia is the most common cause of chronic hemolytic anemia and correlated with significant morbidity and mortality. One thousand Egyptian newborns were screened to detect α-thalassemia (α-thal) deletions using polymerase chain reaction (PCR)-based DNA analysis of cord blood samples. Ninety-one cases (9.1%) of the studied samples were proved to have at least one of the α genes deleted and 851 cases (85.1%) were normal by PCR analysis, while 58 samples (5.8%) failed to be amplified so further DNA analysis could not be done. In the studied group with α gene deletions, we found different types including silent carriers with only one α-globin gene deleted (3.1%), α-thal trait with two α-globin genes deleted (4.2%), Hb H disease with three α-globin genes deleted (1.8%) and no cases carrying Hb Bart’s disease with loss of four α-globin genes. We determined the deletional spectrum of α-thal, which might be used in the future for molecular investigations of the disease in susceptible patients in our population.     

Association between the α-adducin gene and hypertension in the HyperGEN study

This report from the HyperGEN Study, one of four networks participating in the NHLBI-sponsored Family Blood Pressure Program, presents the results of an association study based on 822 white and 572 black subjects (cases and controls) participating in the HyperGEN Network from five geographically diverse field centers. All cases met the Joint National Committee on Detection and Treatment of High Blood Pressure (JNC VI) criteria for hypertension (Stage I or higher). Each subject was clinically examined for risk factors for hypertension as well as genotyped for the point mutation Gly460Trp at the α-adducin locus on chromosome 4p. In the white group, the prevalence of genotypes with one or more Trp alleles was 26% in normotensives, versus 33% in hypertensives randomly selected from the population, and 39% among the multiply affected hypertensive sibships. Overall, in whites, the Trp allele significantly increased the odds of hypertension (P = .0056), with an odds ratio (OR) of 1.73 (95% confidence interval [CI] = 1.17, 2.54). The α-adducin gene remained a significant independent predictor of hypertension in a multivariate logistic model even after correcting for other risk factors for hypertension, including gender, age, body mass index (BMI), smoking, LDL cholesterol, triglycerides, urine sodium (Na), and urine potassium (K), (OR = 1.55, 95% CI = 1.03, 2.34). Through the use of regression trees, several gene-by-environment interactions were implicated, suggesting that α-adducin appears to be a particularly important risk factor (OR = 4.2) for older (age > 60.5 years), less lean (BMI < 25.8 kg/m²) subjects with moderately high triglycerides (between 145.5 and 218.5 mg/dL). In the black group, the relationship was less clear. Overall, it was protective against hypertension. The prevalence of genotypes with one or more Trp alleles was 24% among normotensive versus 11% in hypertensive black subjects randomly selected from the population, and 13% among multiply affected hypertensive sibships, resulting in an OR of 0.48 (P = .0231; 95% CI = 0.25, 0.90). However, the Trp genotype was no longer a significant independent predictor of hypertension risk in the multivariate logistic model (OR = 0.79; 95% CI = 0.37, 1.67), suggesting that it may be operating through one or more of these other factors. Thus, we conclude that the α-adducin gene is a significant, independent risk factor for hypertension in whites, but not in blacks, and may play a particularly important role for subjects with certain constellations of other risk factors.     

Two novel mutations in the gene for human α-mannosidase that cause α-mannosidosis

Mutation analysis performed on two Italian patients with alpha-mannosidosis allowed the identification of two new mutations, IVS20-2A>G and 322-323insA. The patients were both homozygous for these mutations. The first mutation causes skipping of exon 21, whereas the second causes a frameshift introducing a stop codon at position 160 of the amino acid sequence.     

Growth factor signalling in the regulation of α-cell fate

Glucagon plays critical roles in regulating glucose homeostasis, mainly by counteracting the effects of insulin. Consequently, the dysregulated glucagon secretion that is evident in type 2 diabetes has significant implications in the pathophysiology of the disease. Glucagon secretion from pancreatic α-cells has been suggested to be modulated by blood glucose, signals from the nervous system and endocrine components. In addition to these regulators, intraislet factors acting in a paracrine manner from neighbouring β-cells are emerging as central modulator(s) of α-cell biology. One of the most important of these paracrine factors, insulin, modulates glucagon secretion. Indeed, the α-cell-specific insulin receptor knockout (αIRKO) mouse manifests hypersecretion of glucagon in the postprandial stage and exhibits defective secretion in fasting-induced hypoglycaemia, together mimicking the α-cell defects observed in type 2 diabetes. Interestingly, αIRKO mice display a progressive increase in β-cell mass and a concomitant decrease in α-cells. Lineage trace analyses reveal that the new β-cells originate, in part, from the insulin receptor-deficient α-cells indicating a critical role for α-cell insulin signalling in determining β-cell origin. Our studies also reveal that glucagon-like peptide-1 (GLP-1) treatment of αIRKO mice suppresses glucagon secretion despite absence of functional insulin receptors precluding a role for insulin in GLP-1 action on α-cells in this model. These findings highlight the significance of insulin signalling in the regulation of α-cell biology.     

Low-dose α/β blockade in the treatment of essential hypertension

Despite the recent emphasis on combination drug therapy for hypertension, little attention has been given to α/β blockade using agents other than labetalol. The purpose of this study was to 1) compare the efficacy of low-dose α/β blockade using doxazosin + betaxolol, versus monotherapy with an angiotensin converting enzyme inhibitor (quinapril) and a diuretic (hydrochlorothiazide [HCTZ]), and 2) assess the efficacy of low-dose doxazosin. In a crossover study, 21 hypertensive subjects were treated for 3 weeks each with HCTZ, 12.5 to 25 mg/day, quinapril, 10 to 40 mg/day, and a combination of doxazosin, 1 to 4 mg + betaxolol, 5 to 10 mg daily. Doses were titrated to achieve a systolic pressure <130 mm Hg, as assessed by self-recorded home measurements. Home blood pressure decreased 11.5/7.5 mm Hg after HCTZ, 12.9/8.8 mm Hg after quinapril, and 21.2/16.5 mm Hg after doxazosin + betaxolol (P < .001/< .001 v HCTZ and P < .002/< .001 v quinapril). The target systolic pressure was achieved by 33%, 43%, and 71% of subjects, respectively (P = .04 v HCTZ, and .03 v quinapril). Among the 8 subjects in whom doxazosin dosage was increased to the maximum of 4 mg, the mean blood pressure achieved at 4 mg did not differ from that achieved at 2 mg (136/87 v 136/88 mm Hg). We conclude that oral α/β blockade is superior to monotherapy with an angiotensin converting enzyme inhibitor or a diuretic and that maximal or near maximal efficacy can be achieved at a 2-mg dose of doxazosin. Low-dose oral α/β blockade merits greater consideration in the drug therapy of essential hypertension.     

Molecular Characterization of α-Thalassemia in the Dohuk Region of Iraq

The molecular basis of α-thalassemia (α-thal) has been addressed by several studies from the eastern Mediterranean region, but not from Iraq. To address this issue, we studied 51 individuals with unexplained hypochromia and/or microcytosis, as well as nine patients with documented Hb H disease from the Dohuk region in northern Iraq. We used multiplex gap-polymerase chain reaction (gap-PCR), reverse hybridization, and sequencing for this purpose. It was found that the most common genotypes in those with unexplained hypochromia and/or microcytosis were ?α^3.7/αα, followed by ? ?^MED-I/αα, then ?α^3.7/?α ^3.7, respectively, detected in 84.3% of the above individuals. Other genotypes identified sporadically were ?α^4.2/αα, α^{poly A1}α/αα (AATAAA>AATAAG), α^Adanaα/αα [Hb Adana, codon 59 (Gly→Asp) or HBA1:c.179G>A], and α^Evanstonα/αα [Hb Evanston, codon 14 (Trp→Arg) or HBA1:c.43 T>C]. Three cases (5.88%) remained uncharacterized even after sequencing. All nine Hb H cases carried the ?α^3.7/? ?^MED-I genotype. Such findings are rather different from those in other eastern Mediterranean populations, particularly with relevance to an Hb H molecular basis.     

Diethylnitrosamine- and partial hepatectomy-induced decrease in α2u-globulin mRNA level in the rat liver

Changes in gene expression in the rat liver were investigated by analyzing cDNA libraries for liver mRNAs from adult male rats injected with a chemical carcinogen diethylnitrosamine (DEN). Differential screening using normal and DEN-treated liver cDNAs as probes demonstrated that some of the mRNA species had noticeably lower abundance in the DEN-treated liver than in the untreated liver. Surprisingly, most of those clones were found to code for _2u-globulin (A2uG), an abundant protein in the male rat liver. Further analysis by in situ hybridization revealed that the decrease in the A2uG mRNA level occurred in the area where liver cells were proliferating due to DEN treatment and/or partial hepatectomy (PH). The findings indicate coincidence of cell proliferation with a decrease in the A2uG gene expression in the adult male rat liver, implying that the A2uG-related change favors chemical carcinogen-induced cell growth.Abbreviations A2uG _2u-globulin - DEN diethylnitrosamine - PH partial hepatectomy     

Mutations in the paralogous human α-globin genes yielding identical hemoglobin variants

The human α-globin genes are paralogues, sharing a high degree of DNA sequence similarity and producing an identical α-globin chain. Over half of the α-globin structural variants reported to date are only characterized at the amino acid level. It is likely that a fraction of these variants, with phenotypes differing from one observation to another, may be due to the same mutation but on a different α-globin gene. There have been very few previous examples of hemoglobin variants that can be found at both HBA1 and HBA2 genes. Here, we report the results of a systematic multicenter study in a large multiethnic population to identify such variants and to analyze their differences from a functional and evolutionary perspective. We identified 14 different Hb variants resulting from identical mutations on either one of the two human α-globin paralogue genes. We also showed that the average percentage of hemoglobin variants due to a HBA2 gene mutation (α2) is higher than the percentage of hemoglobin variants due to the same HBA1 gene mutation (α1) and that the α2/α1 ratio varied between variants. These α-globin chain variants have most likely occurred via recurrent mutations, gene conversion events, or both. Based on these data, we propose a nomenclature for hemoglobin variants that fall into this category. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Assessing the severity of the small inframe deletion mutation in the α-subunit of β-hexosaminidase A found in the Turkish population by reproducing it in the more stable β-subunit

GM(2) gangliosidoses are a group of panethnic lysosomal storage diseases in which GM(2) ganglioside accumulates in the lysosome due to a defect in one of three genes, two of which encode the alpha- or beta-subunits of beta- N -acetylhexosaminidase (Hex) A. A small inframe deletion mutation in the catalytic domain of the alpha-subunit of Hex has been found in five Turkish patients with infantile Tay-Sachs disease. To date it has not been detected in other populations and is the only mutation to be found in exon 10. It results in detectable levels of inactive alpha-protein in its precursor form. Because the alpha- and beta-subunits share 60% sequence identity, the Hex A and Hex B genes are believed to have arisen from a common ancestral gene. Thus the subunits must share very similar three-dimensional structures with conserved functional domains. Hex B, the beta-subunit homodimer is more stable than the heterodimeric Hex A, and much more stable than Hex S, the alpha homodimer. Thus, mutations that completely destabilize the alpha-subunit can often be partially rescued if expressed in the aligned positions in the beta-subunit. To better understand the severity of the Turkish HEXA mutation, we reproduced the 12 bp deletion mutation (1267-1278) in the beta-subunit cDNA. Western blot analysis of permanently transfected CHO cells expressing the mutant detected only the pro-form of the beta-subunit coupled with a total lack of detectable Hex B activity. These data indicate that the deletion of the four amino acids severely affects the folding of even the more stable beta-subunit, causing its retention in the endoplasmic reticulum and ultimate degradation.     

The Spectrum of α-Thalassemia Mutations in the Lak Population of Iran

α-Thalassemia (α-thal) is one of the most common genetic disorders worldwide. The aim of this study was to investigate for the first time the α-thal mutation spectrum in the Lak population living in Lorestan Province, Iran. One hundred and seventy-six α-thal carriers participated in the study. Multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR and direct sequencing were used for the detection of different mutations on the α-globin (HBA1 and HBA2) genes. A total of 11 different mutations was identified. The –α^3.7 (rightward; NG_000006.1: g.34164_37967del3804) deletion was observed most frequently (56.35%), followed by α^?5?ntα (HBA2: c.95+2_95+6delTGAGG), α^polyA2α (HBA2: c.*92A>G) and – –^{MED I} (NG_000006.1: g.24664_41064del16401), with frequencies of 15.47, 9.39, and 6.08%, respectively. These four mutations accounted for more than 87.0% of the total mutated alleles. Moreover, 19 different genotypes were identified. The types and distribution pattern of the mutations identified in this study, in comparison with other studies conducted in Iran, was most similar to the Kurdish population of Kermanshah Province, Iran. Due to the lack of information on α-thal in Lorestan Province, it was not possible to compare the mutation spectrum in the Lur and Lak populations. In conclusion, our results may help in setting up a strategy for an α-thal screening program and genetic counseling in the Lak people.     

Mutation I810N in the α3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na⁺,K⁺-ATPase α3 isoform inactive. Total Na⁺,K⁺-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na⁺,K⁺-ATPase α3 by transgenesis, which also rescued Na⁺,K⁺-ATPase activity. Our findings reveal the functional significance of the Na⁺,K⁺-ATPase α3 isoform in the control of epileptiform activity and seizure behavior.     

Identification ofN-acetyl-α-aminoadipic acid in the urine of a patient with α-aminoadipic and α-ketoadipic aciduria

Summary A 3.5-year-old Japanese boy with a mild speech disturbance excreted a large amount of -aminoadipic acid into the urine. The amino acid analysis using an amino acid analyser confirmed the presence of -aminoadipic acid in both urine and plasma.We detected -aminoadipic acid in the hydrolysate of the effluent and washwater fraction through cation exchange resin. This suggested the presence of acetylated derivatives and we identifiedN-acetyl--aminoadipic acid using liquid chromatography-atmospheric pressure ionization mass spectrometry (LC-API-MS). The concentrations of -aminoadipic acid andN-acetyl--aminoadipic acid in the urine of a patient with -aminoadipic aciduria were 376.9 nmol/mg creatinine and 18.1 nmol/mg creatinine, respectively. We also detected -ketoadipic acid in the urine of this patient using LC-API-MS.     

Characterization of α,α-trehalase released in the intestinal lumen by the probiotic Saccharomyces boulardii

Objective. Trehalose intolerance due to α,α-trehalase deficiency has scarcely been studied. The purpose of this study was to measure α,α-trehalase activity in intestinal biopsy samples from 200 consecutive patients over a period of 6 months, and to characterize α,α-trehalase released by the probiotic Saccharomyces boulardii (S. boulardii). Material and methods. Enzyme activities were measured in human and rat intestinal mucosal samples using the micromethod of Messer & Dalqvist. α,α-trehalase from S. boulardii was immunoprecipitated and Western blotted using an IgG purified antibody raised against a 23 amino acid peptide of α,α-trehalase of S. cerevisiae. Results. Among 200 patients, most of whom complained of abdominal symptoms and diarrhoea, 18 (9%) had total α,α-trehalase deficiency (0–12 U/g mucosa) and 39 had partial deficiency (3–12 U/g mucosa). Only 4 patients (2%) presented selective α,α-trehalase deficiency with otherwise normal disaccharidases. Expressed per gram of powder, α,α-trehalase from S. boulardii delivered in vitro an activity 175 times higher than that of human trehalase per gram of intestinal mucosa. V_max (22±0.43 µmol) and K_m (5 mM) were close to that of the human enzyme, whereas Western blot revealed a signal of two subunits of 82 kDa. Finally, treatment of rats with S. boulardii resulted in increases in α,α-trehalase activities of 25 to 45% (p<0.01) in endoluminal fluid and intestinal mucosa compared with in controls. Conclusions. Our data suggest that α,α-trehalase deficiency is more common than is believed and that oral administration of S. boulardii could be beneficial in patients with digestive symptoms caused by trehalose intolerance.     

Spectrum of Common α-Globin Deletion Mutations in the Southern Region of Vietnam

The common deletion mutations of α-globin genes in the Vietnamese population is not well known. Here we report the presence of five deletional mutations of Southeast Asia in the southern region of Vietnam. The – –^SEA (NG_000006.1: g.26264_45564del19301) mutation is the most common type of deletion (87.35%), followed by the –α^3.7 (rightward) (NG_000006.1: g.34164_37967del3804) deletion (9.64%), –α^4.2 (leftward) (AF221717) deletion (2.41%) and – –^THAI (NG_000006.1: g.10664_44164del33501) (0.6%) mutation in this region. The – –^FIL (NG_000006.1: g.11684_43534del31581) mutation was not detected in this study. This result provided a view of the distribution of common α-globin gene mutations in Vietnam and could serve as a baseline for further investigations into these genetic defects.