Molecular and Hematological Analysis of Alpha- and Beta-Thalassemia in a Cohort of Mexican Patients.

Introduction: Alpha- and beta-thalassemia are caused by reduced or absent synthesis of hemoglobin (Hb) subunits α and/or ß. HBA2, HBA1, and HBB mutations are the main cause of thalassemias. The aim of this article is to analyze molecular and hematological features of α- and ß-thal in a cohort of Mexican patients. Methods: One hundred forty-one thalassemia patients were studied. Peripheral blood was collected for blood cell count, electrophoresis, Hb quantification, and molecular testing. Molecular screening was performed by Gap-PCR, ARMS-PCR, Sanger sequencing, and MLPA. Results: Fifty-four patients had α-thal, 75 ß-thal, and 12 patients were complex cases, we observed 13 α- and 18 ß-thal alleles in 43 genotypes, -α3.7/αα and ßCd39C>T/ß were the most frequent. Four α-thal deletions (-Mex4 included HBA2 and HBA1, whereas (αα)Mex5, Mex6 and Mex7 involved MCS-R), a hereditary persistence of fetal hemoglobin-2 like (HPFH-2 like) deletion and six alleles not previously reported in Mexicans (α-59C>Tα, -α4.2, αPlasenciaα, ß-32C>T, ßInitCdA>C and ßFSCd71/72+A) were identified. Conclusion: The observed alleles denote the high heterogeneity and multiple origin admixture of Mexican population. Hematological data are consistent with genotypes, variability in simple carriers, from asymptomatic forms to mild or moderate anemia, was ascertained. We emphasize the importance to consider hematological parameters to establish adequate molecular screening strategies.

Early development of decreased ß-cell insulin secretion in children and adolescents with hemoglobin H disease and its relationship with levels of anemia.

BACKGROUND: Diabetes mellitus (DM) associated with iron overload has been reported among adults with transfusion-dependent thalassemia and those with non-transfusion-dependent thalassemia (NTDT), especially in ß-thalassemia disease. However, little is known about glucose metabolism and how early its dysregulation can develop in α-thalassemia hemoglobin H (Hb H) disease, which is one of the most common types of NTDT worldwide. PROCEDURE: We prospectively calculated glucose metabolism index in 40 patients (aged 10-25 years) with Hb H disease. Glucose metabolism data were compared between patients with deletional versus nondeletional Hb H, and between patients with normal versus abnormal insulin secretion/sensitivity. RESULTS: Despite normal glucose tolerance in all patients, 52.5% had abnormal insulinogenic index indicating decreased ß-cell insulin secretion. Patients with functional hemoglobin < 8 g/dL had significantly higher percentages of abnormal insulinogenic index. There was no significant difference in abnormal insulinogenic index between deletional and nondeletional Hb H. CONCLUSION: Decreased ß-cell insulin secretion is highly prevalent among children and adolescents with Hb H disease, and it is associated with levels of functional anemia at baseline, but not with the type of Hb H disease. This result warrants heightened awareness among hematologists due to potentially increased risk of DM later in life.

CRISPR/Cas9 gene correction of HbH-CS thalassemia-induced pluripotent stem cells.

Haemoglobin (Hb) H-constant spring (CS) alpha thalassaemia (- -/-αCS) is the most common type of nondeletional Hb H disease in southern China. The CRISPR/Cas9-based gene correction of patient-specific induced pluripotent stem cells (iPSCs) and cell transplantation now represent a therapeutic solution for this genetic disease. We designed primers for the target sites using CRISPR/Cas9 to specifically edit the HBA2 gene with an Hb-CS mutation. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm the mutation correction, we verified that the purified clones retained full pluripotency and exhibited a normal karyotype. This strategy may be promising in the future, although it is far from representing a solution for the treatment of HbH-CS thalassemia now.

Iranian patients with hemoglobin H disease: genotype-phenotype correlation.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α3.7/--MED (45%) followed by Homozygote αPoly A2 (17.5%). A total of ten different alpha-globin (α-globin) mutations were observed in patients which --MED, being the most common mutation (26.27%), followed by -α3.7 (24.37%) and αpolyA2(A>G) (18.12%). Hematological parameters such as Hb, MCV, MCH and HbH were assessed and results showed that they varied significantly among genotypes, adjusted to age and gender. This study reveals a highly diverse range of HbH patients different from what was thought in terms of both genotype and phenotype in the Khuzestan region of Iran. These findings could contribute to improve the thalassemia managing policies in this province.

miR-144 regulates oxidative stress tolerance of thalassemic erythroid cell via targeting NRF2.

Thalassemia has a high prevalence in Thailand. Oxidative damage to erythroid cells is known to be one of the major etiologies in thalassemia pathophysiology. Oxidative stress status of thalassemia is potentiated by the heme, nonheme iron, and free iron resulting from imbalanced globin synthesis. In addition, levels of antioxidant proteins are reduced in α-thalassemia and ß-thalassemia erythrocytes. However, the primary molecular mechanism for this phenotype remains unknown. Our study showed a high expression of miR-144 in ß- and α-thalassemia. An increased miR-144 expression leads to decreased expression of nuclear factor erythroid 2-related factor 2 (NRF2) target, especially in α-thalassemia. In α-thalassemia, miR-144 and NRF2 target are associated with glutathione level and anemia severity. To study the effect of miR-144 expression, the gain-loss of miR-144 expression was performed by miR inhibitor and mimic transfection in the erythroblastic cell line. This study reveals that miR-144 expression was upregulated, whereas NRF2 expression and glutathione levels were decreased in comparison with the untreated condition after miR mimic transfection, while the reduction of miR-144 expression contributed to the increased NRF2 expression and glutathione level compared with the untreated condition after miR inhibitor transfection. Moreover, miR-144 overexpression leads to significantly increased sensitivity to oxidative stress at indicated concentrations of hydrogen peroxide (H2O2) and rescued by miR-144 inhibitor. Taken together, our findings suggest that dysregulation of miR-144 may play a role in the reduced ability of erythrocyte to deal with oxidative stress and increased RBC hemolysis susceptibility especially in thalassemia.

Iron Metabolism and Oxidative Status in Patients with Hb H Disease.

To evaluate the iron metabolism and oxidative status in patients with Hb H disease, we investigated 43 patients with Hb H disease, including eight deletional Hb H disease patients and 35 nondeletional Hb H disease patients and 20 healthy controls. The levels of hematological parameters, serum ferritin, hepcidin, superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (TAC), were examined. We found higher serum ferritin levels and lower hepcidin, MDA and TAC levels in Hb H disease patients than in controls. The hepcidin level in Hb H disease patients was positively correlated with MDA and TAC levels but not with serum ferritin and SOD levels. The patients with nondeletional Hb H disease showed higher serum ferritin and Hb H concentrations than those patients with deletional Hb H disease. However, no statistically significant differences in SOD, MDA and TAC levels were found in patients with deletional and nondeletional Hb H disease. Oxidative stress and antioxidant defense were related to hepcidin levels. Our study indicated that hepcidin might be an important parameter for monitoring the iron metabolism and oxidative status of Hb H disease patients.

Potential new approaches to the management of the Hb Bart's hydrops fetalis syndrome: the most severe form of α-thalassemia.

The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart's hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ4) known as Hb Bart's. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζ2γ2), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients.

The super sickling haemoglobin HbS-Oman: a study of red cell sickling, K+ permeability and associations with disease severity in patients heterozygous for HbA and HbS-Oman (HbA/S-Oman genotype).

Studying different sickle cell genotypes may throw light on the pathogenesis of sickle cell disease (SCD). Here, the clinical profile, red cell sickling and K+ permeability in 29 SCD patients (15 patients with severe disease and 14 with a milder form) of HbA/S-Oman genotype were analysed. The super sickling nature of this Hb variant was confirmed. The red cell membrane permeability to K+ was markedly abnormal with elevated activities of Psickle , Gardos channel and KCl cotransporter (KCC). Results were consistent with Ca2+ entry and Mg2+ loss via Psickle stimulating Gardos channel and KCC activities. The abnormal red cell behaviour was similar to that in the commonest genotype of SCD, HbSS, in which the level of mutated Hb is considerably higher. Although activities of all three K+ transporters also correlated with the level of HbS-Oman, there was no association between transport phenotype and disease severity. The super sickling behaviour of HbS-Oman may obviate the need for solute loss and red cell dehydration to encourage Hb polymerisation, required in other SCD genotypes. Disease severity was reduced by concurrent α thalassaemia, as observed in other SCD genotypes, and represents an obvious genetic marker for prognostic tests of severity in young SCD patients of the HbA/S-Oman genotype.

Establishment of MUi009 - A human induced pluripotent stem cells from a 32year old male with homozygous ß°-thalassemia coinherited with heterozygous α-thalassemia 2.

The thalassemias are a group of genetic disorders characterized by a deficiency in the synthesis of globin chains. In this study the MUi009-A human induced pluripotent stem cell line was successfully generated from peripheral blood CD34+ haematopoietic progenitors of a 32year old male who had coinherited a homozygous ß°-thalassemia mutation at codon 41/42 (-TCTT) and a heterozygous α-thalassemia 4.2 deletion. The MUi009-A cell line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into the three germ layers. The cell line may provide a tool for drug testing and gene therapy studies.

Derivation of the human induced pluripotent stem cell line MUi017-A from a patient with homozygous Hemoglobin Constant Spring.

Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

Evaluation of bone mineral density in patients with hemoglobin H disease.

OBJECTIVES: This study was conducted to assess bone mineral density (BMD) and bone mineral content (BMC) of patients with hemoglobin H (HbH) disease. METHODS: BMD and BMC were measured by dual energy X-ray absorptiometry of the lumbar spines and femur neck in 21 patients with Hb H disease over the age of 10 years. An association of BMD with sex, age, hemoglobin, calcium, phosphorus, and serum ferritin level was also evaluated. RESULTS: Prevalence of BMD below the expected range for age in the lumbar spine and femur neck region in patients with HbH disease were 33.3 and 14.3 %, respectively. Lumbar BMD was significantly lower in the patients compared to healthy individuals (median (min-max) 0.725 (0.595-0.924) vs. 1.061 (0.645-1.238), P < 0.001)). There was no significant relationship between BMD in the lumbar and femur neck with any of the evaluated variables (P value >0.05). CONCLUSION: Data regarding bone density in HbH disease is limited; osteoporosis as a common complication of ß-thalassemia intermedia syndrome should be considered even in HbH which shows its prevalence is less than ß-thalassemia intermedia.

Retinal interneuron survival requires non-cell-autonomous Atrx activity.

ATRX is a chromatin remodeling protein that is mutated in several intellectual disability disorders including alpha-thalassemia/mental retardation, X-linked (ATR-X) syndrome. We previously reported the prevalence of ophthalmological defects in ATR-X syndrome patients, and accordingly we find morphological and functional visual abnormalities in a mouse model harboring a mutation occurring in ATR-X patients. The visual system abnormalities observed in these mice parallels the Atrx-null retinal phenotype characterized by interneuron defects and selective loss of amacrine and horizontal cells. The mechanisms that underlie selective neuronal vulnerability and neurodegeneration in the central nervous system upon Atrx mutation or deletion are unknown. To interrogate the cellular specificity of Atrx for its retinal neuroprotective functions, we employed a combination of temporal and lineage-restricted conditional ablation strategies to generate five different conditional knockout mouse models, and subsequently identified a non-cell-autonomous requirement for Atrx in bipolar cells for inhibitory interneuron survival in the retina. Atrx-deficient retinal bipolar cells exhibit functional, structural and molecular alterations consistent with impairments in neuronal activity and connectivity. Gene expression changes in the Atrx-null retina indicate defective synaptic structure and neuronal circuitry, suggest excitotoxic mechanisms of neurodegeneration, and demonstrate that common targets of ATRX in the forebrain and retina may contribute to similar neuropathological processes underlying cognitive impairment and visual dysfunction in ATR-X syndrome.

Incidence of ATRX mutations in myelodysplastic syndromes, the value of microcytosis.

Acquired α-thalassemia myelodysplastic syndrome (MDS) (ATMDS) is an acquired syndrome characterized by a somatic point mutation or splicing defect in the ATRX gene in patients with myeloid disorders, primarily MDS. In a large MDS patient series, the incidence of ATMDS was below 0.5%. But no large series has yet assessed the incidence of ATMDS in microcytic MDS. In this study, we focused on patients with MDS and unexplained microcytosis, which was defined as absence of iron deficiency, inflammatory disease, or history of inherited hemoglobinopathy. Our data confirm the low frequency of ATRX mutations in MDS: 0% in an unselected clinical trial cohort of 80 low risk MDS, 0.2-0.8% in a multicenter registry of 2,980 MDS and 43% of MDS with unexplained microcytosis in this same registry. In addition, we reported four novel mutations of the ATRX gene in ATMDS. This study further determines the frequency of ATRX mutations and highlights the importance of microcytosis to detect ATRX mutations within MDS patients.

ATRX promotes gene expression by facilitating transcriptional elongation through guanine-rich coding regions.

ATRX is a chromatin remodeling protein involved in deposition of the histone variant H3.3 at telomeres and pericentromeric heterochromatin. It also influences the expression level of specific genes; however, deposition of H3.3 at transcribed genes is currently thought to occur independently of ATRX. We focused on a set of genes, including the autism susceptibility gene Neuroligin 4 (Nlgn4), that exhibit decreased expression in ATRX-null cells to investigate the mechanisms used by ATRX to promote gene transcription. Overall TERRA levels, as well as DNA methylation and histone modifications at ATRX target genes are not altered and thus cannot explain transcriptional dysregulation. We found that ATRX does not associate with the promoter of these genes, but rather binds within regions of the gene body corresponding to high H3.3 occupancy. These intragenic regions consist of guanine-rich DNA sequences predicted to form non-B DNA structures called G-quadruplexes during transcriptional elongation. We demonstrate that ATRX deficiency corresponds to reduced H3.3 incorporation and stalling of RNA polymerase II at these G-rich intragenic sites. These findings suggest that ATRX promotes the incorporation of histone H3.3 at particular transcribed genes and facilitates transcriptional elongation through G-rich sequences. The inability to transcribe genes such as Nlgn4 could cause deficits in neuronal connectivity and cognition associated with ATRX mutations in humans.

The roles of cohesins in mitosis, meiosis, and human health and disease.

Mitosis and meiosis are essential processes that occur during development. Throughout these processes, cohesion is required to keep the sister chromatids together until their separation at anaphase. Cohesion is created by multiprotein subunit complexes called cohesins. Although the subunits differ slightly in mitosis and meiosis, the canonical cohesin complex is composed of four subunits that are quite diverse. The cohesin complexes are also important for DNA repair, gene expression, development, and genome integrity. Here we provide an overview of the roles of cohesins during these different events as well as their roles in human health and disease, including the cohesinopathies. Although the exact roles and mechanisms of these proteins are still being elucidated, this review serves as a guide for the current knowledge of cohesins.

[Prevalence and molecular analysis of α-thalassemia in preschool children in Chongqing city].

OBJECTIVE: To investigate the incidence and the gene mutation frequencies and patterns of α-thalassemia in preschool children in Chongqing city. METHODS: Cluster random sampling was used. A total of 1057 preschool children in three areas of Chongqing were screened by using routine blood test and hemoglobin electrophoresis analysis. Molecular analysis carried out for all the samples. RESULTS: Of the 1057 samples, 55 cases were diagnosed as being carriers of α-thalassemia, which included 80 allele genes. Therefore, the frequency of α-thalassemia carriers in Chongqing was 5.20%. Of the 55 α-thalassemia carriers, five different deletions of α-thalassemia were identified, the three most common deletion types and proportions were 54.55% for the -α(3.7) deletion, 18.18% for --(SEA) deletion, and 9.08% for the -α(4.2) deletion, respectively; eight types of nondeletion defects were determined, containing one case of Hb Quong Sze and seven novel mutations of a-globin gene. Furthermore, 24 cases of α-Triplication were detected with the α-Triplication carrier rate of 2.55%. In addition, in this study we also found two cases of abnormal hemoglobin disorders occurred on α-globin gene, Hb J-Wenchang-Wuming and Hb Arya. Hb Arya was characterized in the Chinese population for the first time confirmed by literature retrieval. CONCLUSION: In this study, we have clarified the carrier frequency and molecular spectrum of α-thalassemia in Chongqing, and we first reported the carrier incidence of α-Triplication in Chongqing. The materials obtained from this study would be of valuable reference for genetic counseling and the examination instruction of children in this area.

α-Thalassemia does not seem to influence erythrocyte deformability in sickle cell trait carriers.

Studies dealing with rheological red blood cell (RBC) behavior in sickle cell trait carriers are scarce. Moreover, the association with α-thalassemia (α-thal), which also modifies erythrocyte behavior, has not always been taken into account. We analyzed erythrocyte deformability by means of a shear stress diffractometer, along with hematological and biochemical parameters (glucose and plasma lipids), given their possible influence on erythrocyte deformability, in 14 sickle cell trait carriers and 23 healthy controls. Nine patients were also α-thal carriers and five were not. Among the thalassemia carriers, eight were heterozygous and one was homozygous. When compared with controls, sickle cell trait carriers showed no differences for any of the biochemical parameters analyzed (p > 0.05), but significantly lower hemoglobin (Hb) (p = 0.003), mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) (p < 0.001) levels, although no differences in erythrocyte deformability were observed at any of the shear stresses tested (p > 0.05). When comparing sickle cell trait carriers, with and without α-thal, no differences in erythrocyte deformability were observed (p > 0.05), in spite of the former showing lower MCV and MCH (p < 0.05) levels. Carriers of α-thal had lower Hb S [ß6(A3)Glu → Val; HBB: c.20A > T] levels (p = 0.013) than non carriers. The existence of a compensating mechanism seems reasonable because, despite presenting lower erythrocyte indices, which could worsen erythrocyte deformability, this rheological property improves when the percentage of Hb S is lower.

Metabolic pathways related to oxidative stress in patients with hemoglobin h disease and iron overload.

BACKGROUND: Iron overload is a major complication in patients with hemoglobin H (Hb H) disease and causes damage of tissues. METHODS: We investigated 26 Hb H patients and 75 controls to evaluate their oxidative stress and antioxidant statuses. RESULTS: There were significantly increased levels of superoxide anion in leucocytes, nitrite (NO2-), and malondialdehyde (MDA) in plasma, and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GRx) and oxidized glutathione (GSSG) in erythrocytes, decreased levels of nitrate (NO3-) and vitamin C in plasma, and reduced glutathione (GSH) in erythrocytes, in addition to the abnormal iron status in the patients when compared with those in the controls. Meanwhile, levels of serum ferritin were positively correlated with serum iron, plasma MDA, and erythrocyte SOD in the patients. In addition, the activities of SOD were positively correlated with those of GPx and GRx, and the levels of GSSG and MDA, but negatively correlated with those of GSH. Furthermore, the levels of MDA were negatively correlated those of vitamin C. CONCLUSIONS: These results demonstrate the presence of oxidative stress and decreased levels of antioxidants; moreover, the related metabolic antioxidant pathway is active in Hb H patients with iron overload.

Interaction of hemoglobin Grey Lynn (Vientiane) with a non-deletional α(+)-thalassemia in an adult Thai proband.

Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a substitution of Phe for Leu at position 91 of α1-globin chain, originally described in individual of unknown ethnic background. This article addresses the interaction of Hb Grey Lynn with a non-deletional α(+)-thalassemia found in Thailand, a hitherto un-described condition. The proband was adult Thai woman referred for investigation of mild anemia with Hb 90 g/L. Hb analyses using low pressure liquid chromatography raised a suspicion of abnormal Hb presence, which was failed to demonstrate by cellulose acetate electrophoresis and capillary electrophoresis. DNA sequencing identified a CTT (Leu) to TTT (Phe) mutation at codon 91 corresponding to the Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1) on α1-globin gene and a C deletion between codons 36 and 37 on α2-globin gene causing α(+)-thalassemia. As compared to those observed in a compound heterozygote for Hb Grey Lynn / α(0)-thalassemia reported previously, higher MCV (81.7 fL) and MCH (26.3 pg) values with a lower level of Hb Grey Lynn (19.7%) were observed in the proband. The normochromic normocytic anemia observed could be due to the interaction of Hb Grey Lynn with α(+)-thalassemia. The two mutations could be identified using PCR-RFLP and allele-specific PCR assays developed.