Hb H disease associated with compound heterozygosity for --SEA deletion and a novel alpha globin chain variant (HBA2:c.175C>A) on the distal histidine in a Chinese family.

OBJECTIVES: In clinical practice, the majority of α-thalassaemia cases arise from deletions of the α-globin genes. However, a subset of cases is attributed to rare haemoglobin variants, which can manifest with borderline or normal screening results, potentially leading to missed diagnoses in clinical practice. METHODS: Blood samples were collected from family members and underwent haematological, DNA and RNA analysis. RESULTS: The five-month-old proband presented a haematological phenotype consistent with Hb H disease. The mother's haematology profile was consistent with an α-thalassaemia carrier, while the father exhibited a borderline reduction in MCV and MCH. MALDI-TOF identified an abnormal α-chain in the proband. DNA analysis revealed a novel α-globin variant (HBA2:c.175C>A, α58His>Asn, Hb DG-Nancheng) affecting the distal histidine in the family. The father and the mother had α-genotype of --SEA/αα and αDG-Nanchengα/αα, respectively; while the proband inherited both mutant alleles (--SEA/αDG-Nanchengα). Sequencing of cDNA from HBA2 gene identified an equal ratio of normal and mutant alleles. CONCLUSION: This rare case highlighted the importance of identifying rare haemoglobin variant during prenatal screening. The clinical and genetic data provides useful information on the pathogenicity of this variant and further insight into the role of distal histidine residue of α-globin.

[Study of the types of mutations of Thalassemia in Shanghai area].

OBJECTIVE: To analyze the mutations of globin genes among patients suspected for thalassemia from the Shanghai area. METHODS: A total of 4 644 patients diagnosed at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between June 2016 and December 2019 were selected as the study subjects. The patients were tested for common mutations associated with thalassemia gene by Gap-PCR and reverse dot blotting (RDB). Patients were suspected to harbor rare mutations based on the inconsistency between hematological phenotypes and results of common mutation detection, and were further analyzed by Gap-PCR and Sanger sequencing. RESULTS: Among the 4 644 patients, 2 194 (47.24%) were found to carry common thalassemia mutations, among which 701 (15.09%) were α-thalassemia, 1 448 (31.18%) were ß-thalassemia, and 45 (0.97%) were both α- and ß-thalassemia. Forty six samples were found to harbor rare mutations, which included 17 α-globin gene and 29 ß-globin gene mutations. CD77(CCC>ACC) (HBA2: c.232C>A) of the α-globin gene, NG_000007.3: g.70567_71015del449, codon 102(-A) (HBB: c.308_308delA) and IVS-Ⅱ-636 (A>G) (HBB: c.316-215A>G) of the ß-globin gene were previously unreported new types of globin gene mutations. CONCLUSION: Among the 4 644 patients, the detection rate for common thalassemia mutations was 47.24%, whilst 46 samples were detected with rare gene mutations. The type of gene mutation types were diverse in the Shanghai area. The study has provided more accurate results for genetic diagnosis and counseling.

Impact of α-Globin Gene Expression and α-Globin Modifiers on the Phenotype of ß-Thalassemia and Other Hemoglobinopathies: Implications for Patient Management.

In this short review, we presented and discussed studies on the expression of globin genes in ß-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of ß-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of ß-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by ß-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with ß-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

A 6-Year Follow-up of a Chinese Child with Homozygous ß0-Thalaasemia and a Heterozygous KLF1 Mutation.

Patients with the genotype of ß0/ß0 for ß-thalassemia (ß-thal) usually behave as ß-thal major (ß-TM) phenotype which is transfusion-dependent. The pathophysiology of ß-thal is the imbalance between α/ß-globin chains. The degree of α/ß-globin imbalance can be reduced by the more effective synthesis of γ-globin chains, and increased Hb F levels, modifying clinical severity of ß-TM. We report a Chinese child who had homozygous ß0-thal and a heterozygous KLF1 mutation. The patient had a moderate anemia since 6 months old, keeping a baseline Hb value of 8.0-9.0 g/dL. She had normal development except for a short stature (3rd percentile) until 6 years old, when splenomegaly and facial bone deformities occurred. Although genetic alteration of KLF1 expression in ß0/ß0 patients can result in some degree of disease alleviation, our case shows that it is insufficient to ameliorate satisfactorily the presentation. This point should be borne in mind for physicians who provide the genetic counseling and prenatal diagnosis to at-risk families.

Hb Hebei [α20 (B1) His→Leu; HBA2:C.62A > T]: A Novel Hemoglobin Variant Found during Measurement of Glycated Hemoglobin.

We report a novel hemoglobin (Hb) variant found in a 34-year-old Chinese male during a routine measurement of glycated hemoglobin. The variant resulted in a P3 peak of 27.5% of the total Hb on high performance liquid chromatography (HPLC) with a glycated hemoglobin mode. However, no abnormal Hb peaks were observed in capillary electrophoresis (CE) with 3.1% Hb A2 and 96.9% Hb A. The amino acid substitution was determined by Sanger sequencing as α20 (B1) His→Leu; the corresponding DNA mutation was identified as CAC > CTC at the first position of codon 20 of the α-chain. This is the first description of the mutation, and we have named it Hb Hebei for the region of origin of the proband.

Reliability of hemoglobin A2 value as measured by the Premier Resolution system for screening of ß-thalassemia carriers.

OBJECTIVES: Accurate quantification of hemoglobin (Hb) A2 is vital for diagnosing ß-thalassemia carriers. This study aimed to assess the precision and diagnostic utility of HbA2 measurements using the new high-performance liquid chromatography (HPLC) method, Premier Resolution, in comparison to capillary electrophoresis (CE). METHODS: We analyzed 418 samples, previously identified as A2A by CE, using Premier Resolution-HPLC. We compared the results, established correlations, and determined an optimal HbA2 cutoff value for ß-thalassemia screening. Additionally, we prospectively evaluated the chosen cutoff value in 632 samples. Mutations in the ß- and α-globin genes were identified using polymerase chain reaction (PCR) techniques and DNA sequencing. RESULTS: HbA2 levels were consistently higher with Premier Resolution, yet there was a significant correlation with CE in all samples (bias, -0.33; r, 0.991), ß-thalassemia (bias, -0.27; r, 0.927), and non-ß-thalassemia carriers (bias, -0.36; r, 0.928). An HbA2 cutoff value of ≥4.0 % for ß-thalassemia screening achieved 100 % sensitivity and 99.6 % specificity. Further validation yielded sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 97.3 , 99.8, 97.3, 99.8, and 99.7 %, respectively. We also identified a rare ß-Hb variant, Hb La Desirade [HBB:c.389C>T], associated with ß-thalassemia and co-inherited with a single α-globin gene. CONCLUSIONS: The Premier Resolution HPLC is a reliable and accurate method for routine ß-thalassemia carrier screening, aligning with existing CE methods.

Case report: Identification of a novel triplication of alpha-globin gene by the third-generation sequencing: pedigree analysis and genetic diagnosis.

BACKGROUND: Thalassemia, a common autosomal hereditary blood disorder worldwide, mainly contains α- and ß-thalassemia. The α-globin gene triplicates allele is harmless for carriers, but aggravates the phenotype of ß-thalassemia. Therefore, it is particularly crucial to accurately detect the structural variants of α-globin gene clusters. CASE REPORT: We reported a 28-year-old man, the proband, with microcytic hypochromic anemia. From pedigree analysis, his mother and sister had hypochromic microcytosis, and his father was normal. Genetic testing of thalassemia identified a novel α-globin gene triplicate named αααanti4.2del726bp (NC_000016.10:g.170769_174300dupinsAAAAAA) by third-generation sequencing (TGS) in the proband and his father, which was further validated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. The genotypes of the proband's mother and sister were both -α3.7/αα compounded with heterozygous HBB:c.126_129delCTTT. They were categorized as silent α-thalassemia with co-inheritance of ß-thalassemia trait. The proband's genotype additionally had the α-globin gene triplicates compared with his mother and sister, which increased the imbalance between α/ß-globin, so the proband had more severe hematological parameters. The proband's wife was diagnosed as HBA2:c.427T > C heterozygosis, and his daughter had the novel α-globin gene triplicates compounded with HBA2:c.427T > C, therefore the girl might be asymptomatic. CONCLUSION: The identification of the novel α-globin gene triplicates provides more insight for the research of thalassemia variants and indicates that TGS has significant advantages on genetic testing of thalassemia for the reliability, accuracy and comprehensiveness.

The Spectrum of α-Thalassemia Mutations in Syrian Patients.

α-Thalassemia (α-thal) is a globally prevalent genetic disorder of hemoglobin (Hb) structure where the rate of α-globin chain synthesis is reduced or absent due to the presence of α-globin mutation(s). The aim of this study is to define the spectrum of α-globin gene mutations and evaluate their allele frequency in a group of α-thal carriers. A total of 55 individuals with possible α-thal patients were referred from the thalassemia centers in Syria. They have unexplained hypochromia and microcytosis. All patients were genetically tested for 21 common α-globin gene mutations using reverse hybridization kit. Seven different α-globin gene mutations and 13 different genotypes were detected in 55 patients. The two most frequently encountered mutations were -α3.7 deletion (47.1%) and --MED mutation (21.4%). The most commonly observed genotype was -α3.7/αα (40%), followed by --MED/αα genotype (21.8%). We determined the most common α thalassemia mutations in the Syrian patients. α-Thalassemia mutations with deletions were mostly observed in our study.

Super-enhancers include classical enhancers and facilitators to fully activate gene expression.

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from ß-Thalassemia Patients Treated with Sirolimus.

The ß-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the ß-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including ß-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from ß-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for ß-thalassemias.

Hemoglobin profile and molecular characteristics of the complex interaction of hemoglobin Doi-Saket [α9(A7) asn > lys, HBA2:c.30C > a], a novel α2α1 hybrid globin variant, with hemoglobin E [ß26(B8) Glu > lys, HBB:c.79G > A] and deletional α+-thalassemia in a Thai family.

BACKGROUND: An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important. OBJECTIVE: This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α+-thalassemia phenotype in carriers with and without other hemoglobinopathies. MATERIALS AND METHODS: Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism were developed. RESULTS: Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of αDoi-Saket with ßE globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis. CONCLUSIONS: Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management.

The Hb Doi-Saket is a novel α-globin variant mutation occurring in the α2-globin gene in cis to the -α^3.7 kb chromosome.The carrier of Hb Doi-Saket may present slight microcytosis and have severe clinical entities when it interacts with deletions in α-globin genes.Hb analysis with the HPLC system could completely separate Hb Doi-Saket and its derivative from other Hbs.

Molecular understanding of unusual HbE-ß+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels.

BACKGROUND: Low HbF expression in HbE-ß+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the ß- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-ß+-thalassemia from HbE heterozygotes have been investigated. PARTICIPANTS AND METHODS: A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize ß- and α-globin genes. DNA polymorphism at position -158 nt 5' to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-ß+-thalassemia from HbE heterozygotes were determined. RESULTS: Five ß+-thalassemia mutations trans to ßE-gene (ß-87(C>A), ß-31(A>G), ß-28(A>G), ß19(A>G), and ß126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000-1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden's index for differentiating HbE-ß+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-ß+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at -158 nt 5' to Gγ-globin was found to be negatively associated with HbF expression. CONCLUSIONS: HbE-ß+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-ß+-thalassemia diagnosis.

HbE-ß⁺-thalassemia displays a wide range of HbF expression, which may lead to the misdiagnosis of HbE heterozygosity in patients whose Hb analysis shows HbE and HbA. α-Thalassemia may be a major factor associated with decreased secondary activation of HbF expression in the disease.HbE may be a potential indicator for effectively differentiating HbE-ß⁺-thalassemia from HbE heterozygotes.The high proportion and heterogeneity of α-thalassemia mutations found in patients with HbE-ß⁺-thalassemia evoke a complex thalassemia syndrome, requiring complete DNA analysis.

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions.

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify -α3.7, -α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Transfusion requirements and complication rate in ß-thalassemia intermedia due to heterozygous ß-globin gene mutation and triplicated α-globin genes.

INTRODUCTION: The heterozygous condition for ß-thalassemia mutation associated with an extra functional α-globin gene can produce a Thalassemia Intermedia (TI) phenotype. This genotype is the second in frequency in the French Thalassemia Registry NaThalY that prospectively collects laboratory and clinical data. MATERIALS AND METHODS: The present report analyses transfusion needs, iron overload (ferritin, hepatic and cardiac iron concentrations), and complication rates in 45 patients included in NaThalY and presenting a heterozygous ß0 or ß+ -thalassemia mutation associated with a triplication at HBA locus. This cohort was compared to a cohort of patients with TI due to mutations in the beta-globin gene only and included in the French registry. RESULTS: Patients with an extra functional α-globin gene showed a less severe anemia, lower transfusion needs and lower complication rates than those with TI related to the ß-globin gene only. Nevertheless, some of them displayed complications such as cholelithiasis or extramedullary hematopoiesis. In addition, one third of the cohort needed transfusions and another third was under iron chelation. CONCLUSION: The genotype associating a heterozygous ß0 or ß+ -thalassemia mutation with a triplication at HBA locus should be accurately diagnosed as it could lead to symptomatic anemia and to potential iron overload and iron-related complications even in patients with no transfusion need.

Understanding fundamental principles of enhancer biology at a model locus: Analysing the structure and function of an enhancer cluster at the α-globin locus.

Despite ever-increasing accumulation of genomic data, the fundamental question of how individual genes are switched on during development, lineage-specification and differentiation is not fully answered. It is widely accepted that this involves the interaction between at least three fundamental regulatory elements: enhancers, promoters and insulators. Enhancers contain transcription factor binding sites which are bound by transcription factors (TFs) and co-factors expressed during cell fate decisions and maintain imposed patterns of activation, at least in part, via their epigenetic modification. This information is transferred from enhancers to their cognate promoters often by coming into close physical proximity to form a 'transcriptional hub' containing a high concentration of TFs and co-factors. The mechanisms underlying these stages of transcriptional activation are not fully explained. This review focuses on how enhancers and promoters are activated during differentiation and how multiple enhancers work together to regulate gene expression. We illustrate the currently understood principles of how mammalian enhancers work and how they may be perturbed in enhanceropathies using expression of the α-globin gene cluster during erythropoiesis, as a model.

Clinical significance of mutational variants in beta and alpha genes in patients with hemoglobinopathies from two large Greek centers: a complex interplay between genotype and phenotype.

Hemoglobinopathies affect patients in the wider Mediterranean area consisting of 4 distinct subgroups: beta thalassemia major (TM), beta thalassemia intermedia (TI), sickle cell disease (SCD) and hemoglobin H disease (alpha thalassemia). The clinical spectrum varies from mild to severe. Complex interactions between genes and environmental factors form the clinical manifestations. There is an unmet need to clarify these multifactorial mechanisms. This is the first Greek study describing mutational alleles (HBB and HBA1/HBA2 gene variants) in 217 patients with hemoglobinopathies of two large centers in Greece (Larissa and Athens) and associating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). Thus, the complex interplay between corresponding genotypes and phenotypes was investigated. Our results are in accordance with previous national studies with limited variations, due to regional prevalence of specific gene variants, as expected. It is also a description of the prevalence of hemoglobinopathies in the Greek population. The type and prevalence of beta and alpha globin gene variants differ significantly among countries. We also confirm the well-known observation of many studies that in our beta thalassemic or SCD patients, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course, whereas the inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype. In cases in whom the genotype and phenotype did not correlate, factors like the function or modification of possible regulatory genes or additional nutritional-environmental effects should be investigated. KEY MESSAGES: • This is the first Greek study, fully molecularly defining the beta and alpha mutational alleles in 217 patients with hemoglobinopathies of two large centers in Greece and correlating particular genotypes or gene variants with clinical manifestations (transfusion frequency, complications). • In the beta thalassemic or SCD patients of our cohort, co-inheritance of variants in the alpha globin genes, leading to absence or reduction of alpha globin synthesis were associated with milder clinical course (confirmation of a well-known previous observation). • The inheritance of additional alpha genes (triplication) led to a more severe clinical phenotype (confirmation of a well known previous observation). • The function or modification of possible regulatory genes should be investigated in cases in whom the genotype and phenotype did not correlate.

Molecular characterization of a novel 83.9-kb deletion of the α-globin upstream regulatory elements by long-read sequencing.

Inherited deletions of upstream regulatory elements of α-globin genes give rise to α-thalassemia, which is an autosomal recessive monogenic disease. However, conventional thalassemia target diagnosis often fails to identify these rare deletions. Here we reported a family with two previous pregnancies of Hb Bart's hydrops fetalis and was seeking for prenatal diagnosis during the third pregnancy. Both parents had low level of Hemoglobin A2 indicating α-thalassemia. Conventional Gap-PCR and PCR-reverse dot blot showed the father carried -SEA deletion but did not identify any variants in the mother. Multiplex ligation-dependent probe amplification identified a deletion containing two HS-40 probes but could not determine the exact region. Finally, a long-read sequencing (LRS)-based approach directly identified that the exact deletion region was chr16: 48,642-132,584, which was located in the α-globin upstream regulatory elements and named (αα)JM after the Jiangmen city. Gap-PCR and Sanger sequencing confirmed the breakpoint. Both the mother and fetus from the third pregnancy carried heterozygous (αα)JM, and the fetus was normally delivered at gestational age of 39 weeks. This study demonstrated that LRS technology had great advantages over conventional target diagnosis methods for identifying rare thalassemia variants and assisted better carrier screening and prenatal diagnosis of thalassemia.

Loss of miR-144/451 alleviates ß-thalassemia by stimulating ULK1-mediated autophagy of free α-globin.

Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder ß-thalassemia, mutations in the ß-globin gene (HBB) lead to a reduction in the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by Unc-51-like autophagy activating kinase 1 (ULK1)-dependent autophagy and that stimulating this pathway by systemic mammalian target of rapamycin complex 1 (mTORC1) inhibition alleviates ß-thalassemia pathologies. We show here that disrupting the bicistronic microRNA gene miR-144/451 alleviates ß-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through 2 mechanisms. Loss of miR-451 upregulated its target messenger RNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor adenosine monophosphate-activated protein kinase (AMPK). The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. In addition, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1, causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates, and improve hematological indices in ß-thalassemia. The beneficial effects of miR-144/451 loss in ß-thalassemia were inhibited by the disruption of Cab39 or Ulk1 genes. Together, our findings link the severity of ß-thalassemia to a highly expressed erythroid microRNA locus and a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.

Hb Qinzhou [α1 78 (EF7) Asn→Lys (AAC>AAA); HBA1:c.237C>A]: a Novel α-Globin Variant in a Chinese Family.

BACKGROUND: Many new variants are constantly detected by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Here, we described a novel α-globin gene mutation. METHODS: The proband was a 46-year-old male who came to the hospital with his wife for pre-conception thalassemia screening. Hematological parameters were obtained from a complete blood count. Hb analysis was performed by CE and HPLC. Routine genetic analysis was carried out by Gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot-blot (PCR-RDB). Sanger sequencing was used to identify the hemoglobin variant. RESULTS: An abnormal Hb variant was observed at electrophoretic zone 5 and zone 1 on the CE program. HPLC showed a peak of abnormal Hb in the S window. No mutations were detected by Gap-PCR and PCR-RDB. Sanger sequencing revealed an AAC>AAA mutation at codon 78 of the α-globin gene [α1 78 (EF7) Asn→Lys (AAC> AAA); HBA1:c.237C>A]. The pedigree study demonstrated that the Hb variant was inherited from his mother. CONCLUSIONS: It is the first report about the variant, so we named it Hb Qinzhou for the place of origin of the proband. Hb Qinzhou presents a normal hematological phenotype.

Hematological and molecular characteristics of a novel α-globin variant Hb Liangqing (HBA2:c.224A>G).

OBJECTIVES: To report the hematological and molecular characteristics of a novel α-globin variant found among Chinese families. METHODS: This study was done on two unrelated families (F1 and F2). Hematological results were obtained by an automated blood cell analyzer. Hemoglobin (Hb) fraction analysis was carried out using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Gap-PCR and reverse dot blot (RDB) methods were used to detect the common α-thalassemia mutations in the Chinese population. The Hb variants were defined by Sanger sequencing. RESULTS: Hb fraction analysis of F2 cord blood using HPLC showed an abnormal peak (3.5%) of the S-window, but CE presented a 12.2% abnormal peak at zone 5(S). Similar results of CE were observed from the F1 twin's cord blood. Compared with newborns, Hb analysis of F2 father using HPLC demonstrated an abnormal peak (16.9%) of S-window and an unknown peak (0.5%) at a retention time of 4.60 min, respectively. In contrast, CE revealed a high Hb F peak at zone 7 and an unknown peak at zone 1. There was no abnormality detected with Gap-PCR and RDB in these patients. However, Sanger sequencing confirmed the presence of a new heterozygous mutation (GAC>GGC) at codon 74 of the HBA2 gene (HBA2:c.224A>G), resulting in a novel Hb variant. We named it Hb Liangqing for the birthplace of the proband. CONCLUSION: This is the first report of Hb Liangqing detected by HPLC and CE. The normal hematological phenotype suggests that it may be a benign Hb variant.