Molecular characterization of G6PD mutations reveals the high frequency of G6PD Aures in the Lao Theung population.

BACKGROUND: The prevalence and genotypes of G6PD deficiency vary worldwide, with higher prevalence in malaria endemic areas. The first-time assessment of G6PD deficiency prevalence and molecular characterization of G6PD mutations in the Lao Theung population were performed in this study. METHODS: A total of 252 unrelated Lao Theung participants residing in the Lao People's Democratic Republic (PDR) were recruited. All participant samples were tested for G6PD enzyme activity and G6PD gene mutations. The amplification refractory mutation system (ARMS)-PCR for detecting G6PD Aures was developed. RESULTS: The G6PD mutations were detected in 11.51% (29/252) of the participants. Eight G6PD mutations were detected. The G6PD Aures was the most common mutation identified in this cohort, which represented 58.62% (17/29) of all mutation. The mutation pattern was homogenous, predominantly involving the G6PD Aures mutation (6.75%), followed by 1.19% G6PD Union and 0.79% each G6PD Jammu, G6PD Mahidol and G6PD Kaiping. One subject (0.4%) each carried G6PD Viangchan and G6PD Canton. Interestingly, one case of coinheritance of G6PD Aures and Quing Yan was detected in this cohort. Based on levels of G6PD enzyme activity, the prevalence of G6PD deficiency in the Lao Theung population was 9.13% (23/252). The prevalence of G6PD deficient males and females (activity < 30%) in the Lao Theung population was 6.41% (5/78) and 1.72% (3/174), respectively, and the prevalence of G6PD intermediate (activity 30-70%) was 5.95% (15/252). CONCLUSIONS: The G6PD Aures mutation is highly prevalent in the Lao Theung ethnic group. The common G6PD variants in continental Southeast Asian populations, G6PD Viangchan, Canton, Kaiping, Union and Mahidol, were not prevalent in this ethnic group. The technical simplicity of the developed ARMS-PCR will facilitate the final diagnosis of the G6PD Aures.

Generation of human induced pluripotent stem cells (CHULAi001-A) from peripheral blood mononuclear cells of a glucose-6-phosphate dehydrogenase (G6PD) deficient subject carrying the Viangchan mutation.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic enzyme-defect disorder. In this study, CHULAi001-A was established from peripheral blood mononuclear cells of a G6PD-deficient subject who carried the Viangchan variant (871 G > A). Episomal plasmids expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA against p53 were introduced into parental cells by electroporation and cultured under feeder-free conditions to reprogram iPSCs. Embryonic pluripotency, in vitro differentiation capacity, and episomal vector integration of the established CHULAi001-A were verified. The CHULAi001-A iPSCs retained a normal karyotype.

Prevalence and molecular characterization of glucose-6-phosphate dehydrogenase deficiency in the Lue ethnic group of northern Thailand.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzyme disorders. Prevalence and variant distribution of G6PD deficiency can vary in different regions and among differing ethnic groups. To reveal the G6PD frequency and molecular characterization among the Lue ethnic group of northern Thailand, blood samples of 296 unrelated individuals collecting from 6 Lue villages were analyzed. The observed G6PD enzyme activity ranged from 0.11 to 20.60 U/g Hb. Overall, 13.51% (40/296) of the individuals were identified as having G6PD deficiency status. The prevalence in males was 14.28% (20/140), while that of females was 12.82% (20/156). The most common G6PD variants in the Lue were the Kaiping 1388G > A (5.40%) and Canton 1376G > T (6.42%) types. Observed prevalence and variant types of the G6PD gene in the Lue population are similar to that of the Tai-Kadai speaking ethnic groups in southern China, which is consistent with their historically close line of ancestry. However, the founder effect that occurred during the Lue's transboundary migration from China to Thailand showed its impact upon different patterns of G6PD distribution among each Lue village.

Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases.

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

Molecular prevalence of thalassemia and hemoglobinopathies among the Lao Loum Group in the Lao People's Democratic Republic.

INTRODUCTION: Thalassemias and hemoglobinopathies are the most prevalent inherited anemias detected in South East Asians. These disorders represent not only a clinical health problem but also a socioeconomic problem for this region. Regarding the prevention and control of thalassemias and hemoglobinopathies in the Lao PDR, screening and diagnostic strategies should be strongly considered. The knowledge about the prevalence and molecular genotyping of thalassemias and hemoglobinopathies among the Lao Loum group, which includes the majority of Lao people, is now limited, making the prevention and control of thalassemias difficult. METHODS: This study aimed to determine the prevalence of thalassemia among Lao Loum subjects of reproductive age. Multiplex gap PCR and direct sequencing were used to investigate the mutations of α-globin and ß-globin genes. RESULTS: Thalassemias and hemoglobinopathies were detected in 154 of 354 (43.50%) patients, and 22 different genotypes were identified in this cohort. Remarkably, high frequencies of hemoglobin E, α0 -thalassemia (--SEA ), and α+ -thalassemia (-α3.7 ) were noted. A variety of hematologic features was observed, including co-inheritance of heterozygous HbE and heterozygous α-thalassemia, which was associated with significantly lower levels of MCV and MCH values than those observed in typical HbE heterozygotes. Female participants who were heterozygous for ß0 or co-inheritance of heterozygous ßE with heterozygous α-thalassemia exhibited mild anemia. CONCLUSION: Our data show that thalassemias and hemoglobinopathies have become health problems imposing a serious burden in the Lao PDR. Prevention programs aimed at decreasing the incidence of severe thalassemia diseases should be designed and initiated.

Platelet proteome reveals specific proteins associated with platelet activation and the hypercoagulable state in ß-thalassmia/HbE patients.

A hypercoagulable state leading to a high risk of a thrombotic event is one of the most common complications observed in ß-thalassemia/HbE disease, particularly in patients who have undergone a splenectomy. However, the hypercoagulable state, as well as the molecular mechanism of this aspect of the pathogenesis of ß-thalassemia/HbE, remains poorly understood. To address this issue, fifteen non-splenectomized ß-thalassemia/HbE patients, 8 splenectomized ß-thalassemia/HbE patients and 20 healthy volunteers were recruited to this study. Platelet activation and hypercoagulable parameters including levels of CD62P and prothrombin fragment 1 + 2 were analyzed by flow cytometry and ELISA, respectively. A proteomic analysis was conducted to compare the platelet proteome between patients and normal subjects, and the results were validated by western blot analysis. The ß-thalassemia/HbE patients showed significantly higher levels of CD62P and prothrombin fragment 1 + 2 than normal subjects. The levels of platelet activation and hypercoagulation found in patients were strongly associated with splenectomy status. The platelet proteome analysis revealed 19 differential spots which were identified to be 19 platelet proteins, which included 10 cytoskeleton proteins, thrombin generation related proteins, and antioxidant enzymes. Our findings highlight markers of coagulation activation and molecular pathways known to be associated with the pathogenesis of platelet activation, the hypercoagulable state, and consequently with the thrombosis observed in ß-thalassemia/HbE patients.

A comprehensive ethnic-based analysis of alpha thalassaemia allelle frequency in northern Thailand.

Alpha (α)-thalassaemia is one of the most prevalent hereditary blood disorders, commonly affecting Southeast Asian people, with the highest incidence (30-40%) being seen in northern Thailand. However, this high incidence was estimated without consideration of the variations between ethnic populations and the geographical location of the populations. To address this issue, a total of 688 samples from 13 different northern Thai ethnic groups (30 villages) categorized into three linguistic groups were genotyped for deletional alpha-thalassaemia (-α3.7, -α4.2, --SEA and --THAI) and/or non-deletional alpha-thalassaemia (αCS and αPS) via multiplex gap-PCR and dot-blot hybridization, respectively. Alpha+(-α3.7, -α4.2, αCS and αPS) and alpha°-thalassaemia (--SEA and --THAI) allele frequencies (with 95% Confidence Interval) were the highest in the Sino-Tibetan group [0.13 (0.08-0.18)] and the Tai-Kadai group [0.03 (0.02-0.05)], respectively. With regards to ethnicity, the varying allele frequency of α+ and α°-thalassaemia amongst a variety of ethnic groups was observed. The highest α+-thalassaemia allele frequency was found in the Paluang [0.21 (0.10-0.37)] while α°-thalassaemia allele frequency was the highest in the Yuan [0.04 (0.01-0.10)]. These detailed results of alpha thalassaemia allele frequency and genetic diversity amongst the northern Thai ethnic groups demonstrate the need for ethnicity based thalassaemia prevention programs.

Plasma microRNA-451 as a novel hemolytic marker for ß0-thalassemia/HbE disease.

In Southeast Asia, particularly in Thailand, ß0-thalassemia/hemoglobin E (HbE) disease is a common hereditary hematological disease. It is associated with pathophysiological processes, such as the intramedullary destruction of immature erythroid cells and peripheral hemolysis of mature red blood cells. MicroRNA (miR) sequences, which are short non-coding RNA that regulate gene expression in a suppressive manner, serve a crucial role in human erythropoiesis. In the present study, the plasma levels of the erythroid-expressed miRNAs, miR­451 and miR­155, were analyzed in 23 patients with ß0-thalassemia/HbE and 16 control subjects. Reverse transcription­quantitative polymerase chain reaction analysis revealed significantly higher levels of plasma miR­451 and miR­155 in ß0­thalassemia/HbE patients when compared to the control subjects. Notably, among the ß0­thalassemia/HbE patients, a significant increase in miR­451 levels was detected in severe cases when compared with mild cases. The levels of plasma miR­451 correlated with reticulocyte and platelet counts. The results suggest that increased plasma miR­451 levels may be associated with the degree of hemolysis and accelerated erythropoiesis in ß0­thalassemia/HbE patients. In conclusion, miR­451 may represent a relevant biomarker for pathological erythropoiesis associated with ß0-thalassemia/HbE.

Iron dysregulation in beta-thalassemia.

Iron deficiency anemia and iron overload conditions affect more than one billion people worldwide. Iron homeostasis involves the regulation of cells that export iron into the plasma and cells that utilize or store iron. The cellular iron balance in humans is primarily mediated by the hepcidin-ferroportin axis. Ferroportin is the sole cellular iron export protein, and its expression is regulated transcriptionally, post-transcriptionally and post-translationally. Hepcidin, a hormone produced by liver cells, post-translationally regulates ferroportin expression on iron exporting cells by binding with ferroportin and promoting its internalization by endocytosis and subsequent degradation by lysosomes. Dysregulation of iron homeostasis leading to iron deposition in vital organs is the main cause of death in beta-thalassemia patients. Beta-thalassemia patients show marked hepcidin suppression, ineffective erythropoiesis, anemia and iron overload. Beta-thalassemia is common in the Mediterranean region, Southeast Asia and the Indian subcontinent, and the focus of this review is to provide an update on the factors mediating hepcidin related iron dysregulation in beta-thalassemia disease. Understanding this process may pave the way for new treatments to ameliorate iron overloading and improve the long term prognosis of these patients.

Comparative plasma protein profiling of hemoglobin H disease.

HbH and HbH-constant spring (HbH-CS) are the most common forms of α-thalassemia detected in the Thai population. The accumulation of excess ß globin chains in these diseases results in increased red cell hemolysis, and patients with HbH-CS normally have a more severe clinical presentation than patients with HbH disease. This study aimed to detect alterations in the expression of plasma proteins of HbH and HbH-CS patients as compared to normal plasma. Platelet poor plasma was separated from HbH and HbH-CS and normal subjects and differential plasma proteins were detected using two-dimensional gel electrophoresis and identified using LC/MS/MS. A total of 14 differentially expressed proteins were detected of which 5 proteins were upregulated and 9 were downregulated. Most of the differentially expressed proteins are liver secreted proteins involved in hemolysis, oxidative stress response, and hemoglobin degradation. Seven proteins were found to be differentially expressed between HbH and HbH-CS. Levels of haptoglobin, a hemoglobin scavenging protein, were significantly increased in HbH patients as compared to HbH-CS patients. The identification of differentially expressed proteins may lead to a better understanding of the biological events underlying the clinical presentation of HbH and HbH-CS patients and can have application as hemolytic markers or severity predictors.