Various α-Thalassemia Genotype Combinations of the Saudi-Type Polyadenylation Signal Mutation (αT-Saudiα) in the Population of Bahrain: An Update of Genotype-Phenotype Analyses.

The Saudi-type polyadenylation (polyA) signal mutation on the α2-globin gene, HBA2: c.*94A > G; AATAAA>AATAAG; αT-Saudiα, is one of the major α-thalassemia (α-thal) determinants in the population of Bahrain. We determined five different genotype combinations involving this mutation in Bahrain. Here, we report these various genotypes involving the Saudi-type polyA signal mutation and their relevant phenotype presentations. A total of 32 patients were diagnosed with the genotype of αT-Saudiα/αT-Saudiα. These homozygous patients presented with a typical form of Hb H disease and severe anemia with a mean hemoglobin (Hb) level of 8.5 ± 0.7 g/dL. Second, we diagnosed 29 patients carrying the genotype of αT-Saudiα/αHpHα, and they presented with a less severe form of Hb H disease and a mean Hb level of 9.9 ± 0.9 g/dL. Third, we found two genotype combinations of the polyA signal mutation with either the rightward (-α3.7) (n = 46) or leftward (-α4.2) (n = 22) deletions. These patients presented with an α-thal trait phenotype or a mild form of Hb H disease with a mean Hb level of 10.8 ± 1.0 g/dL for the -α3.7/αT-Saudiα, and 10.4 ± 1.4 g/dL for the -α4.2/αT-Saudiα genotypes. We also found 39 patients with the simple heterozygous state of the polyA signal mutation, who presented with a mild form of α-thal trait and a mean Hb level of 12.1 ± 1.7 g/dL. Finally, data from 48 patients carrying the normal α genotype (αα/αα) are presented with a mean Hb level of 12.9 ± 1.7 g/dL. These data could pave the way for accurate genetic counseling and sound clinical management based on precise molecular diagnosis of α-thal.

Silent ß-thalassemia mutations at -101 (C>T) and -71 (C>T) and their coinheritance with the sickle cell mutation in Bahrain.

Silent ß-thalassemia (ß-thal) is a group of mutations affecting the ß-globin gene that cannot be differentiated in heterozygote states from normal conditions by using conventional criteria for the diagnosis of ß-thal trait. Here we report the existence of two silent ß-thal mutations in the population of Bahrain, one at -101 (C>T) and the other at -71 (C>T). We screened 126 healthy individuals with high-normal Hb A2 levels and found a frequency of 23.0% for both of these mutations (8.0% for -71 and 15.0% for -101). Heterozygotes for either of these two mutations have an overlapping level of Hb A2 ranging from 3.1-3.9% (3.7 ± 0.3) with some cases displaying microcytic and hypochromic anemia, mainly attributed to coinherited defective α-thal genes. Interestingly, each of these mutations were discovered in combination with the sickle cell allele displaying distinct high performance liquid chromatography (HPLC) profiles, different from those observed in the typical sickle cell disease or the sickle cell trait conditions. These investigations are invaluable to provide appropriate counseling for partners undergoing premarital counseling and to understand the molecular basis of mild and atypical forms of sickle cell disease.

Loss of the AE3 anion exchanger in a hypertrophic cardiomyopathy model causes rapid decompensation and heart failure.

The AE3 Cl(-)/HCO(3)(-) exchanger is abundantly expressed in the sarcolemma of cardiomyocytes, where it mediates Cl(-)-uptake and HCO(3)(-)-extrusion. Inhibition of AE3-mediated Cl(-)/HCO(3)(-) exchange has been suggested to protect against cardiac hypertrophy; however, other studies indicate that AE3 might be necessary for optimal cardiac function. To test these hypotheses we crossed AE3-null mice, which appear phenotypically normal, with a hypertrophic cardiomyopathy mouse model carrying a Glu180Gly mutation in α-tropomyosin (TM180). Loss of AE3 had no effect on hypertrophy; however, survival of TM180/AE3 double mutants was sharply reduced compared with TM180 single mutants. Analysis of cardiac performance revealed impaired cardiac function in TM180 and TM180/AE3 mutants. TM180/AE3 double mutants were more severely affected and exhibited little response to ß-adrenergic stimulation, a likely consequence of their more rapid progression to heart failure. Increased expression of calmodulin-dependent kinase II and protein phosphatase 1 and differences in methylation and localization of protein phosphatase 2A were observed, but were similar in single and double mutants. Phosphorylation of phospholamban on Ser16 was sharply increased in both single and double mutants relative to wild-type hearts under basal conditions, leading to reduced reserve capacity for ß-adrenergic stimulation of phospholamban phosphorylation. Imaging analysis of isolated myocytes revealed reductions in amplitude and decay of Ca(2+) transients in both mutants, with greater reductions in TM180/AE3 mutants, consistent with the greater severity of their heart failure phenotype. Thus, in the TM180 cardiomyopathy model, loss of AE3 had no apparent anti-hypertrophic effect and led to more rapid decompensation and heart failure.

FIP1L1/PDGFRalpha synergizes with SCF to induce systemic mastocytosis in a murine model of chronic eosinophilic leukemia/hypereosinophilic syndrome.

Expression of the fusion gene FIP1-like 1/platelet-derived growth factor receptor alpha (FIP1L1/PDGFRalpha, F/P) and dysregulated c-kit tyrosine kinase activity are associated with systemic mastocytosis (SM) and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES). We analyzed SM development and pathogenesis in a murine CEL model induced by F/P in hematopoietic stem cells and progenitors (HSCs/Ps) and T-cell overexpression of IL-5 (F/P-positive CEL mice). These mice had more mast cell (MC) infiltration in the bone marrow (BM), spleen, skin, and small intestine than control mice that received a transplant of IL-5 transgenic HSCs/Ps. Moreover, intestinal MC infiltration induced by F/P expression was severely diminished, but not abolished, in mice injected with neutralizing anti-c-kit antibody, suggesting that endogenous stem cell factor (SCF)/c-kit interaction synergizes with F/P expression to induce SM. F/P-expressing BM HSCs/Ps showed proliferation and MC differentiation in vitro in the absence of cytokines. SCF stimulated greater migration of F/P-expressing MCs than mock vector-transduced MCs. F/P-expressing bone marrow-derived mast cells (BMMCs) survived longer than mock vector control BMMCs in cytokine-deprived conditions. The increased proliferation and survival correlated with increased SCF-induced Akt activation. In summary, F/P synergistically promotes MC development, activation, and survival in vivo and in vitro in response to SCF.

Loss of the AE3 Cl(-)/HCO(-) 3 exchanger in mice affects rate-dependent inotropy and stress-related AKT signaling in heart.

Cl(-)/HCO(-) 3 exchangers are expressed abundantly in cardiac muscle, suggesting that HCO(-) 3 extrusion serves an important function in heart. Mice lacking Anion Exchanger Isoform 3 (AE3), a major cardiac Cl(-)/HCO(-) 3 exchanger, appear healthy, but loss of AE3 causes decompensation in a hypertrophic cardiomyopathy (HCM) model. Using intra-ventricular pressure analysis, in vivo pacing, and molecular studies we identified physiological and biochemical changes caused by loss of AE3 that may contribute to decompensation in HCM. AE3-null mice had normal cardiac contractility under basal conditions and after ß-adrenergic stimulation, but pacing of hearts revealed that frequency-dependent inotropy was blunted, suggesting that AE3-mediated HCO(-) 3 extrusion is required for a robust force-frequency response (FFR) during acute biomechanical stress in vivo. Modest changes in expression of proteins that affect Ca(2+)-handling were observed, but Ca(2+)-transient analysis of AE3-null myocytes showed normal twitch-amplitude and Ca(2+)-clearance. Phosphorylation and expression of several proteins implicated in HCM and FFR, including phospholamban (PLN), myosin binding protein C, and troponin I were not altered in hearts of paced AE3-null mice; however, phosphorylation of Akt, which plays a central role in mechanosensory signaling, was significantly higher in paced AE3-null hearts than in wild-type controls and phosphorylation of AMPK, which is affected by Akt and is involved in energy metabolism and some cases of HCM, was reduced. These data show loss of AE3 leads to impaired rate-dependent inotropy, appears to affect mechanical stress-responsive signaling, and reduces activation of AMPK, which may contribute to decompensation in heart failure.