Alpha-globin Gene Research Articles

TNFSF/TNFRSF cytokine gene expression in sickle cell anemia: Up-regulated TNF-like cytokine 1A (TL1A) and its decoy receptor (DcR3) in peripheral blood mononuclear cells and plasma

BackgroundSickle cell anemia (SCA), a disorder with an important inflammatory component, where vasoocclusion is major contributor to the disease pathophysiology. Pro-inflammatory cytokines play an important regulatory role in the process of inflammation. We investigated the expression TL1A/DR3/DcR3 cytokine signaling pathway in peripheral blood mononuclear cells (PBMC) and their corresponding plasma levels in SCA subjects who presented with acute painful episodes. Materials and methodsPBMC were isolated from the blood of SCA subjects and normal healthy controls. RNA isolated from PBMC was used for real time gene expression of TL1A/DR3/DcR3. Gene expression was compared in subgroups within SCA subjects with co-inherited fetal hemoglobin (HbF) or alpha-globin gene deletions. Plasma prepared from blood was used for determination of TL1A/DR3/DcR3 proteins by ELISA assays. ResultsIn the PBMC of SCA subjects, expression of TL1A and DcR3 is elevated, while DR3 expression is lowered in comparison to normal control PBMC. In SCA subjects with HbF > 10%, TL1A/DcR3 expression is lower, while HbF < 10% is associated with increased TL1A/DcR3 expression. Moreover, subjects with HbF > 10% appear to have significantly fewer pain episodes in comparison to those with HbF < 10%. Deletion of alpha-globin genes appears to have no significant effect on TL1A/DR3/DcR3 expression. Circulating levels of TL1A, DR3 and DcR3 in plasma were significantly elevated in SCA subjects. ConclusionsElevated TL1A and DcR3 expression in PBMC of SCA subjects during painful vasoocclusive crisis, suggest an altered TL1A expression may contribute to the pathophysiology of vasoocclusive crisis in SCA. HbF > 10% appears to moderate TL1A elevation, while HbF < 10% exacerbates TL1A/DcR3 responses. Furthermore, subjects with HbF > 10% have significantly lower pain episodes reported as compared to subjects with HbF < 10%.

Three Mexican Families with β thalassemia intermedia with different molecular basis.

Beta thalassemia (β-thal) is a frequent monogenic disease, is clinically and molecularly heterogeneous. This study described molecular and laboratory findings for three Mexican patients with β-thal intermedia phenotype and their relatives. Three Mexican families were studied for presenting β-thal intermedia, ARMS-PCR and Gap-PCR were performed to screen for common mutations, Sanger sequencing for rare or new alleles, and MLPA for identifying deletions and or duplications. In all three families we observed, in heterozygote condition, the mutation c.118C > T (p.Gln39*) also known as codon 39(C > T) in the β globin gene (HBB) associated with a novel molecular defect: a new duplication of the alpha globin gene cluster, a new deletion that includes the loss of exon 3 of HBB and finally a novel mutation in the 3’UTR of HBB (HBB: c.*132C > A). We report three Mexican families with beta thalassemia intermedia due to different molecular basis; a new single nucleotide mutation involving the last nucleotide of the β-globin chain transcript; and two possible new DNA rearrangements, an α cluster duplication, and a partial β gene deletion.

Role of Alpha Hemoglobin Stabilizing Protein Expression in Beta Thalassemia Patients

AbstractBackground: Beta (b)-Thalassemia is an autosomal reces-sive inherited disease causing variable degrees of anemia. The molecular defects are owing to point mutations or small deletions leading to diminution or absence of b-globin chain synthesis. The unmatched a-Hb is destructive to itself and other cellular components, resulting in ineffective erythropoi-esis and hemolysis. Alpha-Hemoglobin Stabilizing Protein (AHSP) is a scavenger protein of erythroid that fastly and reversibly attach the a subunit of hemoglobin molecule preventing its precipitation and subsequent hemolysis.Aim of Work: Is to assess AHSP gene expression in thalassemia major and intermedia and its relation to clinical features, laboratory investigations and to determine its role on the severity of thalassemia.Subjects and Methods: This case-control study is consisted of 120 studied subjects categorized into 40 thalassemia major patients, 40 thalassemia intermedia patients and 40 age healthy individuals as a control group. All subjects underwent the following; history, clinical picture, complete blood picture and Hb electrophoresis, serum ferritin and measurement of AHSP, alpha globin and beta globin genes expression by real time PCR.Results: AHSP gene in thal. intermedia group was higher than both thal. major (p<0.001). Beside thal. intermedia group had higher a globin/AHSP ratio versus thal. major group (p=0.011). b globin/AHSP ratio was not significant between both thal. groups. AHSP in thal.major group correlated with a globin and b globin expression and negatively correlated with organomegally, ferritin and no. of blood transfusion. Whereas in thal. intermedia group, was positively correlated with Hb, HCT, MCV, MCH, a globin gene expression, b globin gene expression and b globin/a globin genes ratio.Conclusion: AHSP expression was higher in thalassemia intermedia group than thalassemia major group. Consequently, AHSP can be considered genetic modifier in b thalassemia.

Expression of the Human Alpha-Globin Cluster in the Absence of the Major Regulatory Element Mcs-R2

The alpha-globin genes are located in the subtelomeric region of the short arm of chromosome 16. Expression of each allele is controlled by 4 distal cis-acting enhancers (MCS-R1-4) located 48, 40, 33 and 10kb upstream of the duplicated structural alpha genes . Experimental and clinical studies have shown that R1 and R2 are the most important enhancers of alpha-globin production. Here we present an individual with alpha thalassemia who inherited one copy of chromosome 16 from which both alpha genes have been deleted (–SEA). This individual's other copy of chromosome 16 harbours a small deletion of R2. Thus all alpha-globin expression in this individual originates from one copy of the alpha cluster in which R1 drives expression of the two alpha-globin genes in cis. Remarkably, this patient maintains a relatively normal degree of oxygenation despite a severe degree of alpha thalassemia.This female patient was born in Surinam and her mother and father were of Indian and Indonesian origins respectively. She most recently presented, aged 26 yrs, with tiredness and shortness of breath on exertion. She had no previously diagnosed medical conditions, but had received two blood transfusions as a child, both prompted by concurrent infections. Her growth and development were normal. She had suffered one miscarriage at 13 weeks gestation aged 25 yrs. There was nothing of note in the family history and the patient is currently employed as a clerical worker. Cardiovascular examination was normal with no hepatosplenomegaly. Her CBC was consistent with HbH disease, with a Hb of 73g/L, Hct 25%, MCV 45fl, MCH 13.2pg with a blood film showing marked microcytosis anisopoikilocytosis, target cells and 30.7% HbH inclusions on Brilliant Cressyl Blue staining. HPLC showed evidence of a fast-moving band, HbA 88.4% and HbA2 1.3%, HbE 5.1%. MLPA analysis showed that the father and proband both carried heterozygous deletions of R2, and the mother and proband both carried the –SEA deletion on one allele. Due to the severity of the anaemia and the striking red cell indices it was highly likely that she had intact regulatory elements in cis with the –SEA deletion on one allele, and on the other, a regulatory network missing R2 lying upstream of the two intact alpha-globin genesTo investigate this further, CD34+ cells were extracted from the patient's peripheral blood and differentiated using a liquid culture system (Trakarnsanga et al. Nature Comms. 2017). The alpha/non-alpha-globin synthetic ratio of mRNA as measured by qPCR at peak globin-production was markedly reduced compared to WT cells and those of a patient carrying the –SEA deletion on one allele alone: WT 1.26, SEA 0.65, Patient 0.11.The upstream enhancers are associated with regions of open chromatin that can be detected by chromatin accessibility assays. To see whether the deletion of R2 had caused new regulatory elements to appear or whether accessibility of the existing elements was altered, ATAC-seq was performed on Day 13 of differentiation. This showed a reduction in the peak at R2 in keeping with its deletion on one allele, but showed no change in the peak height of R1 compared to that in the individual with the –SEA deletion alone or WT.To assess chromatin interactions across the domain Capture-C, a sensitive assay to detect interactions between selected regions of chromatin, was performed (Davies et al. Nature Methods 2016). Compared to WT, when capturing from the alpha-globin promoters interactions with R1, R3 and R4 were observed with no interactions with R2 detected, in keeping with the hypothesised distribution of deletions. No interactions in trans between the intact R2 and alpha-globin genes were detected.Previous cases of deletions of the upstream alpha-globin regulatory element have either involved deletions of the regulatory elements on one allele only, or involved both alleles but with a full complement of alpha genes. Our findings show that whilst a severe HbH phenotype was observed, it is possible to maintain transfusion independence with only R1, R3, R4 and two alpha-globin genes. Considering the ATAC-seq and Capture-C findings, as well as comparison with the homologous locus in mouse it is likely that the bulk of regulation in this patient occurs via R1. The observed phenotype is consistent with redundancy within the regulatory elements of the alpha gene locus, which may enable continued expression of key cell-specific genes in the event of adverse genotypic events. Disclosuresvan Beers:RR Mechatronics: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Pfizer: Research Funding.

Reassessing the Need for Pre-Operative Transfusions in Sickle Cell Disease Patients with a High Baseline Hemoglobin

Background: Patients with sickle cell disease (SCD) are more likely to require surgical procedures, and to have post-operative complications compared to the general population. The TAPS trial demonstrated that pre-operative transfusion is associated with a 3.8-fold reduction in peri-operative complications in patients with SCD. Pre-operative exchange transfusion has not been shown to have benefit over simple top-up transfusion.Patients with SCD may have baseline hemoglobin levels higher than the usual 60-80 g/L for a variety of reasons including; non-hemoglobin SS genotype SCD, co-inheritance of deletion(s) in alpha globin genes, hereditary persistence fetal hemoglobin, and hydroxyurea (HU) use. It is less clear whether patients with pre-operative hemoglobin levels > 90 g/L would also benefit from pre-operative transfusions. Previous studies of pre-operative transfusions in SCD have largely not captured these patients, in part due to low HU uptake at the time of the study and exclusion of non-hemoglobin SS SCD.We conducted a retrospective cohort study to assess the role of pre-operative transfusion in patients with SCD and a high baseline hemoglobin.Methods: 1304 patients seen at The Hospital for Sick Children, Toronto between 2007 and 2017 were assessed for eligibility. Patients were included if they: had a baseline hemoglobin ≥ 90 g/L, were 1-18 years of age at the time of surgery, had a diagnosis of hemoglobin SS, SC, Sβ+-thalassemia or Sβ0-thalassemia SCD subtypes, and had a low or medium risk elective surgery under a general anesthetic. Surgeries were classified according to the Co-operative Study of Sickle Cell Disease. Post-operative complications were defined as one or more of the following within 30 days of surgery: fever, vaso-occlusive crisis (VOC), infection, bleeding requiring transfusion, acute chest syndrome (ACS), stroke, intensive care admission (ICU), emergency room visit after discharge, readmission to hospital after discharge, or death. The incidence of postoperative complications for those with a baseline hemoglobin ≥90 g/L was compared between those who received a transfusion and those who did not. To estimate the adjusted effect of pre-operative transfusion on the risk of developing post-operative complications, a multi-variable logistic regression model was fitted using the change-in-estimate procedure, where variables with the strongest influence on the crude (unadjusted) estimate were included as model covariates (i.e. key confounders).Results: 117 patients with a hemoglobin ≥90 g/L underwent a total of 137 procedures. The most frequent procedures included were: tonsillectomies/adenoidectomies (26), cholecystectomies (25), splenectomies (20), and umbilical hernia repairs (11).There were 22 procedures (16%) where a pre-operative transfusion was administered. All patients received simple top-up transfusions. Of these, 11 (50%) encountered at least one post-operative complication. In contrast, 22/115 (19.1%) procedures without a pre-operative transfusion experienced a post-operative complication. There was an increased risk of post-operative complications in the group that was transfused (p=0.003, OR=4.2, 95% CI 1.6-11). Adjusting for two key confounders identified during the modeling process (splenectomy and prior ACS), pre-operative transfusion was again found to be associated with an increased risk of post-operative complications (p=0.017, OR=3.6, 95% CI 1.2-9.2). The characteristics of these patients and the incidence and distribution of post-operative complications are shown in Table 1.Conclusion: Patients with SCD and a baseline hemoglobin ≥90 g/L who receive a pre-operative top-up transfusion have an increased risk of post-operative complications compared to those who are not transfused. In low and medium risk surgeries, a policy of withholding transfusions for such patients may be considered. Prospective studies validating these findings are needed. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

KLF1 Mutations Are Not Common in Israel but Can Explain Occasional Cases of Elevated HbA2 or Very Elevated Fetal Hemoglobin

Introduction: Mutations in KLF1 (Kruppel Like Factor1) have been noted to cause a number of different phenotypes of erythrocyte abnormalities, as KLF1 is known as a master regulator of many genes expressed in red blood cells. One important manifestation caused by KLF1 mutations is the upregulation of γ- or δ-globin genes, with associated microcytosis, thus mimicking beta thalassemia (β-thal) trait. Such findings cause difficulty in counseling couples referred for prenatal diagnosis of β-thal. KLF1 mutations have been reported to be more frequent in geographic regions where β-thal is common. Therefore we undertook to analyze for the presence of KLF1 mutations in Israeli individuals.Materials and Methods: We selected 100 individuals for analysis belonging to one of 4 different groups: 1. Individuals with isolated elevated HbA2 (n=14), or isolated elevation of HbF (n=13) or elevation of both HbA2 and HbF (n=6), who are not carriers of a β-thal mutation by sequence analysis. 2. Individuals with β-thal trait (n=19) who have a higher HbF (and/or HbA2) than is expected for β-thal trait, who do not have β-thal intermedia on clinical criteria (blood count, peripheral smear, spleen size). These individuals carried one of 8 mutations known to cause β-thal in our region 3. Two patients (pts) with a history of transfusions (one with massive splenomegaly and sickle trait, suspected to have coinherited CDA, and one with β-thal intermedia with HbF elevation unexpectedly high for his mutation (TATA box -28 A to C; HBB:c.-78A>C). This patient had HbF levels ranging from 23-33% and HbA2 ranging from 7.8-8.9% over years of followup from age 39-50 years. 4. Anonymous controls (n=46) who are pts with a hematological malignancy not suspected of carrying a hemoglobinopathy. KLF1 (exons 1, 2 and 3) was amplified using PCR (exon 1: 564 base pair product and exons 2 and 3: 1703 base pair product). PCR products were subjected to DNA sequence analysis using an Applied Biosystems ABI apparatus. The sequence obtained was analyzed using BLAST alignment and deviations from the published sequence were analyzed using the Mutation Taster program.Results: Two pts were found to carry substitutions with possible or proven clinical significance. One pt is heterozygous for a substitution at c.972C>A (codon 324 exon 3, E324D, aspartic acid to glutamine) which has not been previously reported. According to Mutation Taster this substitution may have clinical significance. This pt, who has normal hematological parameters, had isolated HbA2 elevation (4.1%) with no β-thal mutation identified. The second pt was found to be heterozygous for a substitution at c.901C>T (codon 301 exon 2, R301C, arginine to cysteine). This substitution has been reported by Gallienne et al, 2012, to be associated with elevated Hb F. This young pt was found to have 28% Hb F with a low HbA2 level of 1.4% and MCV of 63.8. His father also had 28% HbF. No β-thal mutation was identified in the pt or his father but the patient was found to be homozygous for a single alpha globin gene deletion. In addition to these 2 substitutions, many pts and controls carried known polymorphisms in KLF1. These polymorphisms are: c.304T>C, exon 2 residue 102 (S102P) (found in 24 pts and 19 controls); -148(G>A), in the upstream noncoding region (found in 11 pts and 8 controls); c.544T>C codon 182 exon 2 (F182L) (found in 3 pts and 4 controls); and c.115A>C, codon 39 in exon 2, M39L (found in 1 pt and 3 controls). One rare, previously unreported substitution was found in heterozygous form in the thal intermedia pt, located at c.259C>G codon 87 exon 2, P87A (proline to alanine). This was not found in the controls, however according to the location in the gene, and to Mutation Taster, this is not suspected to be a functional variant.Conclusions: Functional variants in KLF1 were rarely found in this group of patients. One out of 14 (7%) of individuals with isolated elevated HbA2 and 1/13 patients with isolated HbF elevation (7.7%) were identified as carrying heterozygous KLF1 functional variants. Two novel substitutions were found: E324D which is suspected to have clinical significance and P87A which is not presently suspected to be a functional variant. We conclude that KLF1 analysis may explain occasional individuals with high HbA2, or marked HbF elevation, in the absence of β-thal trait in our patient population. Larger studies are needed to confirm these findings. DisclosuresNo relevant conflicts of interest to declare.

The phenotypic and molecular diversity of hemoglobinopathies in India: A review of 15 years at a referral center

The hemoglobinopathies pose a significant health burden in India. Apart from the β thalassemias and sickle cell disorders, α thalassemias and structural hemoglobin variants are also common. Here we have reviewed the phenotypic and molecular diversity of hemoglobinopathies encountered at a referral center in western India over a period of 15years. Screening for hemoglobinopathies was done using HPLC and cellulose acetate electrophoresis. Molecular characterization was done using Covalent Reverse Dot Blot Hybridization (CRDB), Amplification Refractory Mutation System (ARMS), GAP PCR and direct DNA sequencing. The study includes 31075 individuals who were referred for diagnosis of hemoglobinopathies and prenatal diagnosis. Of these 14423 individuals showed various hemoglobin abnormalities. Beta genotyping in 5615 individuals showed the presence of 49 β thalassemia mutations. 143 β thalassemia heterozygotes had normal or borderline HbA2 levels. We identified three δ gene mutations (HbA2 Pellendri, HbA2 St.George, HbA2 Saurashtra) in β thalassemia heterozygotes leading to normal HbA2 levels. The commonest defects among the raised Hb F determinants were Gγ(Αγδβ)0 Indian inversion and the HPFH-3 Indian deletion. A total of 312 individuals showed the presence of α thalassemia, of which 12.0% had a single α gene deletion (-α/αα). HbH disease was identified in 29 cases with 10 different genotypes. Alpha globin gene triplication was seen in 2.1% of β thalassemia heterozygotes with a thalassemia intermedia phenotype. Seven unusual α chain variants and eight uncommon β chain variants were identified. The repertoire of molecular defects seen in the different globin genes will be valuable for management and control of these disorders both in India as well as in other countries where there is a huge influx of migrant populations from India.

Phenotypes of Hb Okayama Mutation

Introduction:Since its first detection in the year of 1983 very little has been reported about Hb Okayama mutation. Hb Okayama, reported as a silent mutation (Globin Gene Server Hb Var ID: 220, dbSNP rs713040), happened to be detected in the process of HPLC analysis for the measurement of HbA1c in Japanese diabetic patient [1,2,3]. Till date only few Hb Okayama has been reported from Japanese and Austrian ethnicity [2,3,4]. Its phenotypes and co-inheritance with other beta globin gene mutation is not yet known. In this study we report phenotypes of Hb Okayama in heterozygous as well as compound heterozygous state when it is co-existing with a common beta globin gene mutation. To the best of our knowledge no such study has been reported in world literature.Methodology:Two groups of patients- beta carriers requiring blood transfusion; and patients with low MCV, MCH but normal in HPLC were further investigated by molecular analysis. ARMS PCR analysis for beta globin gene mutation detection, GAP PCR for alpha deletion and triplication analysis and DNA sequencing (ABI 3500 Genetic Analyzer) to identify rare beta mutations were carried out.Result:Among around 300 patients subjected to molecular investigation over last three years there were 118 cases of thalassaemia trait (by HPLC and confirmed to carry a heterozygous common beta mutations by ARMS PCR analysis), but were requiring blood transfusion or behaving like intermedia. They were subjected to alpha globin gene triplication analysis by GAP PCR. There were 84 cases found to carry absence of alpha triplication with heterozygous beta mutation. Beta globin gene sequencing analysis of these 84 patients revealed that there were 4 patients carrying Okayama heterozygous mutation [ Figure 1, Figure 2 ] along with a common heterozygous beta gene mutation ( IVS 1-5 G>C mutation). HPLC report of these patients carrying Hb Okayama along with IVS 1-5 mutations in compound heterozygous state showed increased HbA2 values like that of beta trait [Table 1 ]. Another group of patients with low MCV, MCH but Normal in HPLC were subjected to alpha deletion analysis after ruling out low Ferritin level. There were 72 patients showing absence of alpha deletion by GAP PCR analysis and carried low MCV, MCH value with Normal HPLC parameters were also subjected to beta globin gene sequencing which revealed the presence of Hb Okayama mutation among two patients in heterozygous state [Figure- 1, Figure 2], who had HbA2 values in normal or border line range with low MCV, MCH levels [Table-2].Discussion:HbA2 measurement is used as the marker for screening of beta trait. Silent beta-thalassaemia carriers represent normal HbA2 level which makes their identification difficult. Okayama mutation or Hb Okayama is known to be a silent mutation [1-4] with normal HPLC. Hb Okayama is structural beta variant with a change of amino acid ( His > Gln) at Codon 2 (CD 2). Change in the nucleotide sequence at 70603 position from T to A or C (CAT>CAA or CAG) of beta globin gene (NG_000007.3) causes this mutation. The phenotypic associations of Hb Okayama in thalassaemia have very little been known till date. There has been report of very high expression of HbF(70%) value in HPLC resulting from compound heterozygous mutations one of which being silent (Cap+1) [ 5] ; where as there are also reports where the phenotypes of compound heterozygous including a silent mutation showing normal HPLC parameters [ 6 ]. However, in those studies no information about the clinical history and blood transfusion is described. In this study Hb Okayama heterozygous co-inheriting with IVS1-5(G>C) heterozygous mutation showed beta trait like HPLC parameters though all the patients carrying these compound heterozygous mutations required blood transfusion. The silent feature of Hb Okayama was evident in the case of the patients carrying only Hb Okayama mutation who showed absolutely normal HPLC parameters with low MCV, MCH and none of them requiring blood transfusion.Conclusion:Beta globin gene expression analysis to understand the association of Hb Okayama mutation in heterozygous and compound heterozygous states will enable to explain the mechanism of its phenotypes. How this mutation interferes with the expression of HbF or switching of delta globin gene is also to be understood as the HbF levels in HPLC was found to be like beta traits in this study. DisclosuresNo relevant conflicts of interest to declare.

Development and evaluation of a transfusion medicine genome wide genotyping array.

Many aspects of transfusion medicine are affected by genetics. Current single-nucleotide polymorphism (SNP) arrays are limited in the number of targets that can be interrogated and cannot detect all variation of interest. We designed a transfusion medicine array (TM-Array) for study of both common and rare transfusion-relevant variations in genetically diverse donor and recipient populations. The array was designed by conducting extensive bioinformatics mining and consulting experts to identify genes and genetic variation related to a wide range of transfusion medicine clinical relevant and research-related topics. Copy number polymorphisms were added in the alpha globin, beta globin, and Rh gene clusters. The final array contains approximately 879,000 SNP and copy number polymorphism markers. Over 99% of SNPs were called reliably. Technical replication showed the array to be robust and reproducible, with an error rate less than 0.03%. The array also had a very low Mendelian error rate (average parent-child trio accuracy of 0.9997). Blood group results were in concordance with serology testing results, and the array accurately identifies rare variants (minor allele frequency of 0.5%). The array achieved high genome-wide imputation coverage for African-American (97.5%), Hispanic (96.1%), East Asian (94.6%), and white (96.1%) genomes at a minor allele frequency of 5%. A custom array for transfusion medicine research has been designed and evaluated. It gives wide coverage and accurate identification of rare SNPs in diverse populations. The TM-Array will be useful for future genetic studies in the diverse fields of transfusion medicine research.

Clinical Course of Homozygous Hemoglobin Constant Spring in Pediatric Patients.

Hemoglobin (Hb) Constant Spring is an alpha-globin gene variant due to a mutation of the stop codon resulting in the elongation of the encoded polypeptide from 141 to 172 amino acid residues. Patients with homozygous Hb Constant Spring are usually mildly anemic. We retrospectively describe clinical manifestations, diagnosis, laboratory investigations, treatment, and associated findings in pediatric patients with homozygous Hb Constant Spring followed-up at Srinagarind Hospital. Sixteen pediatric cases (5 males and 11 females) were diagnosed in utero (N=6) or postnatal (n=10). Eleven cases were diagnosed with homozygous Hb Constant Spring, 4 with homozygous Hb Constant Spring with heterozygous Hb E, and 1 with homozygous Hb Constant Spring with homozygous Hb E. Three cases were delivered preterm. Six patients had low birth weights. Clinical manifestations included fetal anemia in 6 cases, hepatomegaly in 1 case, hepatosplenomegaly in 2 cases, splenomegaly in 1 case. Twelve cases exhibited early neonatal jaundice, 9 of which required phototherapy. Six cases received red cell transfusions; 1 (3), >1 (3). After the first few months of life, almost all patients had mild microcytic hypochromic anemia and an increased reticulocyte count with a wide red cell distribution (RDW), but no longer required red cell transfusion. At 1 to 2 years of age, some patients still had mild microcytic hypochromic anemia and some had normocytic hypochromic anemia with Hb around 10 g/dL, increased reticulocyte count and wide RDW. Associated findings included hypothyroidism (2), congenital heart diseases (4), genitourinary abnormalities (3), gastrointestinal abnormalities (2), and developmental delay (1). Pediatric patients with homozygous Hb Constant Spring developed severe anemia in utero and up to the age of 2 to 3 months postnatal, requiring blood transfusions. Subsequently, their anemia was mild with no evidence of hepatosplenomegaly. Their Hb level was above 9 g/dL with hypochromic microcytic blood pictures as well as wide RDW. Blood transfusions have not been necessary since then.

New Challenges in Diagnosis of Haemoglobinopathies: Migration of Populations

The current influx of economic migrants and asylum seekers from countries with a high prevalence of haemoglobinopathies creates new challenges for health care systems and diagnostic laboratories. The migration of carriers introduces new and novel haemoglobinopathy mutations to the diagnostic repertoire of a laboratory, often creating new pressures to improve and update the carrier screening technology and diagnostic scope. For antenatal screening programmes, the marriage of partners from different ethnic groups can lead to the risk of compound heterozygote children being born novel mutation combinations, creating problems in the provision of accurate advice regarding the expected phenotype of the thalassaemia or haemoglobinopathy disorder. In the UK, the impact of immigration required the National Haemoglobinopathy Reference laboratory to change the strategy and techniques used for the molecular diagnosis of thalassaemia and the haemoglobinopathies. In 2005, due to the increasingly large range of β-thalassaemia mutations that needed to be diagnosed, the laboratory switched from a three-step screening procedure using ARMS-PCR to a simpler but more expensive one-step strategy of DNA sequencing of the beta and alpha globin genes for all referrals. After ten years of employing this strategy, a further 57 novel thalassaemia and haemoglobionpopthy alleles were discovered (11 new β-chain variants, 15 α-chain variants, 19 β-thalassaemia mutations and 12 α+-thalassaemia mutations), increasing further the extremely heterogeneous spectrum of globin gene mutations in the UK population.

Which Alpha Globin Gene Is Primarily Expressed in the Vascular Endothelium?

Alpha globin was recently discovered in the endothelium of resistance arteries, where it interacts with endothelial nitric oxide synthase (NOS3) to regulate the diffusion of nitric oxide. People with Sickle Cell Disease (SCD) who co‐inherit one or two deletions of the alpha globin gene (a condition called alpha thalassemia) are protected against cerebrovascular and renal complications of SCD, raising the question of how endothelial alpha globin deletion modulates vascular pathophysiology. Alpha globin is expressed by two distinct loci, HBA1 and HBA2, with HBA2 contributing the majority (~65%) of total alpha globin in human red blood cells. However, the expression profile of each locus in the vasculature is currently unknown. Determining total and relative locus‐specific expression of alpha globin in resistance arteries is critical to understanding the consequences of HBA1 or HBA2 deletion.To measure expression of vascular alpha globin, perfused vessels (conduits‐ carotid arteries, thoracic aorta; resistance vessels‐ middle cerebral arteries, gracilis arterioles, 3rd order mesenteric arteries) and whole blood were collected from C57Bl/6J mice. Total mRNA was isolated from vessels and whole blood (vessels‐ RNeasy Kit, Qiagen; blood‐ RiboPure Blood Kit, Ambion), and converted to cDNA (SuperScript IV VILO, Invitrogen). Absolute expression of Hba‐a1 (mouse homolog of HBA2), Hba‐a2 (mouse homolog of HBA1), Nos3, and Ae1 (erythrocyte anion exchanger) was quantified by digital droplet PCR (BioRad).In C57Bl/6J mice, abundant expression of Hba‐a1 and Hba‐a2 was observed in whole blood, as well as in all vascular tissues. In whole blood, the Hba‐a1/Hba‐a2 ratio was (2.55:1), consistent with human blood. However, in all vascular tissue, the Hba‐a1/Hba‐a2 ratio was inverted (0.60:1). Ae1 was highly expressed in whole blood, but not in vascular tissue; Nos3 was expressed in all vessels, but not in whole blood. These data indicate that expression of alpha globin is differentially regulated in the vasculature compared to whole blood, with Hba‐a2 being predominant within the vasculature.To further interrogate locus‐specific expression of alpha globin, absolute gene expression was quantified in Hba‐a1(−/−) global knockout (KO) and inducible endothelium‐specific Hba‐a1 KO mice. In global Hba‐a1(−/−), there was a significant reduction in the Hba‐a1/Hba‐a2 ratio in whole blood [ratio (0.069:1)] and in absolute Hba‐a1 expression in all vessels [range (80%–99%) decrease], while expression of Hba‐a2 and Nos3 remained consistent with control. In inducible endothelium‐specific Hba‐a1 KO mice, absolute Hba‐a1 expression was significantly decreased in vascular tissue; however, Hba‐a2 and Nos3 expression also decreased significantly, with Hba‐a1/Hba‐a2 ratio remaining equal to control. These findings suggest that expression of both alpha globin loci, as well as Nos3, may be co‐regulated within the vasculature.Overall, we report robust, locus‐specific expression of alpha globin in the vasculature. Further investigation into the physiologic role of endothelial alpha globin may provide insight into how deletion of alpha globin modulates vascular pathologies associated with SCD and other diseases.Support or Funding InformationNHLBI, NIAID Intramural FundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Haemoglobin variants, iron status and anaemia in Sri Lankan adolescents with low red cell indices: A cross sectional survey

Iron deficiency complicates the use of red cell indices to screen for carriers of haemoglobin variants in many populations.In a cross sectional survey of 7526 secondary school students from 25 districts of Sri Lanka, 1963 (26.0%) students had low red cell indices. Iron deficiency, identified by low serum ferritin, was the major identifiable cause occurring in 550/1806 (30.5%) students. Low red cell indices occurred in iron-replete students with alpha-thalassaemia including those with single alpha-globin gene deletions. Anaemia and low red cell indices were also common in beta-thalassaemia trait. An unexpected finding was that low red cell indices occurred in 713 iron-replete students with a normal haemoglobin genotype.It is common practice to prescribe iron supplements to individuals with low red cell indices. Since low red cell indices were a feature of all forms of α thalassaemia and also of iron deficiency, in areas where both conditions are common, such as Sri Lanka, it is imperative to differentiate between the two, to allow targeted administration of iron supplements and avoid the possible deleterious effects of increased iron availability in iron replete individuals with low red cell indices due to other causes such as α thalassaemia.

Case study – Reproductive risk of clinically relevant thalassaemia despite mild microcytosis and normal thalassaemia screen

Alpha thalassaemia is an autosomal recessive disorder, commonly caused by a deletion in the alpha-globin gene cluster encoded by the HBA1 and HBA2 genes on chromosome 16p13.3. Individuals with one or more deleted (or dysfunctional) alpha globin gens exhibit a wide spectrum of haematological and clinical phenotypes. MLPA is a copy number assay that detects common deletions associated with 90% of alpha thalassaemia cases.

Genome Editing Strategies to Treat Beta-Hemoglobinopathies

Genetic forms of anemia caused by HBB gene mutations that impair beta globin production are extremely common worldwide. The resultant disorders, mainly sickle cell disease (SCD) and beta-thalassemia, cause substantial morbidity and early mortality. Treatments for these diseases include medical therapies and bone marrow transplantation (BMT), which can be curative. However, medical therapies are suboptimal and BMT is associated with serious toxicities, particularly because HLA-matched allogeneic sibling donors are not available for most patients. Thus, new therapies are urgently needed for millions of affected individuals. Gene therapy offers great promise to cure SCD and beta thalassemia and emerging genome editing technologies represent a new form of gene therapy. Approaches to cure SCD and beta-thalassemia via genome editing include: 1) Correction of HBB mutations by homology directed repair (HDR); 2) use of non-homologous end joining (NHEJ) to activate gamma globin production and raise fetal hemoglobin (HbF) levels; 3) NHEJ to disrupt alpha-globin genes (HBA1 or HBA2) and thereby alleviate globin chain imbalance in intermediately severe forms of beta thalassemia. Challenges for these approaches include selection of the most effective genome editing tools, optimizing their delivery to hematopoietic stem cells (HSCs), improving specificity and better understanding potential off target effects, particularly those that are biologically relevant. Technologies for genome editing are advancing rapidly and being tested in preclinical models for HBB-mutated disorders. Ultimately, however, the best strategies can only be identified in clinical trials. This will require close collaborations between basic/translational researchers who study genome editing, clinical hematologists and collaboration between experts in academia and the bio-pharmaceutical industry. Disclosures No relevant conflicts of interest to declare.

Prevalences of inherited red blood cell disorders in pregnant women of different ethnicities living along the Thailand-Myanmar border.

Background: Inherited red blood cell disorders are prevalent in populations living in malaria endemic areas; G6PD deficiency is associated with oxidant-induced haemolysis and abnormal haemoglobin variants may cause chronic anaemia. In pregnant women, microcytic anaemia caused by haemoglobinopathies mimics iron deficiency, complicating diagnosis and treatment. Anaemia during pregnancy is associated with morbidity and mortality. The aim of this study was to characterise the prevalence of G6PD deficiency and haemoglobinopathies among the pregnant population living along the Thailand-Myanmar border. Pregnant women attending antenatal clinics in this area belong to several distinct ethnic groups. Methods: Data were available for 13,520 women attending antenatal care between July 2012 and September 2016. Screening for G6PD deficiency was done by fluorescent spot test routinely. G6PD genotyping and quantitative phenotyping by spectrophotometry were analysed in a subsample of women. Haemoglobin variants were diagnosed by HPLC or capillary electrophoresis and molecular methods. The prevalence and distribution of inherited red blood cell disorders was analysed with respect to ethnicity. Results: G6PD deficiency was common, especially in the Sgaw Karen ethnic group, in whom the G6PD Mahidol variant allele frequency was 20.7%. Quantitative G6PD phenotyping showed that 60.5% of heterozygous women had an intermediate enzymatic activity between 30% and 70% of the population median. HbE, beta-thalassaemia trait and Hb Constant Spring were found overall in 15.6% of women. Only 45.2% of women with low percentage of HbA 2 were carriers of mutations on the alpha globin genes. Conclusions: Distribution of G6PD and haemoglobin variants varied among the different ethnic groups, but the prevalence was generally high throughout the cohort. These findings encourage the implementation of an extended program of information and genetic counselling to women of reproductive age and will help inform future studies and current clinical management of anaemia in the pregnant population in this region.

Prevalences of inherited red blood cell disorders in pregnant women of different ethnicities living along the Thailand-Myanmar border

Background: Inherited red blood cell disorders are prevalent in populations living in malaria endemic areas; G6PD deficiency is associated with oxidant-induced haemolysis and abnormal haemoglobin variants may cause chronic anaemia. In pregnant women, microcytic anaemia caused by haemoglobinopathies mimics iron deficiency, complicating diagnosis and treatment. Anaemia during pregnancy is associated with morbidity and mortality. The aim of this study was to characterise the prevalence of G6PD deficiency and haemoglobinopathies among the pregnant population living along the Thailand-Myanmar border. Pregnant women attending antenatal clinics in this area belong to several distinct ethnic groups. Methods: Data were available for 13,520 women attending antenatal care between July 2012 and September 2016. Screening for G6PD deficiency was done by fluorescent spot test routinely. G6PD genotyping and quantitative phenotyping by spectrophotometry were analysed in a subsample of women. Haemoglobin variants were diagnosed by HPLC or capillary electrophoresis and molecular methods. The prevalence and distribution of inherited red blood cell disorders was analysed with respect to ethnicity. Results: G6PD deficiency was common, especially in the Sgaw Karen ethnic group, in whom the G6PD Mahidol variant allele frequency was 20.7%. Quantitative G6PD phenotyping showed that 60.5% of heterozygous women had an intermediate enzymatic activity between 30% and 70% of the population median. HbE, beta-thalassaemia trait and Hb Constant Spring were found overall in 15.6% of women. Only 45.2% of women with low percentage of HbA 2 were carriers of mutations on the alpha globin genes. Conclusions: Distribution of G6PD and haemoglobin variants varied among the different ethnic groups, but the prevalence was generally high throughout the cohort. These findings encourage the implementation of an extended program of information and genetic counselling to women of reproductive age and will help inform future studies and current clinical management of anaemia in the pregnant population in this region.

Impact of annotation error in α-globin genes on molecular diagnosis.

BackgroundRecent studies on the variants in duplicated human alpha globin genes (HBA2 and HBA1) actively target the α-globin gene as molecular modulators for the treatment of β-thalassemia major. Identification of the exact position of variant in HBA1, HBA2 or its patchworks is mandatory to support the therapeutic aims in β-thalassemia major, by identifying specific modulators for the reactivation of fetal hemoglobin production. Hence, accurate identification of the variants in α-globin genes is crucial for the proper diagnosis, treatment and genetic counseling.MethodThe objective was to reveal the annotation errors produced in α-globin gene sequence analysis while using different analytic tools. An HBA2 gene sequence with the HBA2:c.95+2_95+6delTGAGG variant and a recently reported HBA12 gene convert have been taken as examples to prove annotation error in α-globin gene from different analytic tools.Results and discussionAlthough various bioinformatics tools used to predict variants are usually of high reliability, the current study using the an alpha globin 2 sequence with the HBA2:c.95+2_95+6delTGAGG variant and a recently reported HBA12 gene convert, has showcased ambiguous outputs among the three bioinformatics tools used and against the manual analytical method adopted.ConclusionsThis report emphasizes the necessity for caution in the usage of DNA sequence analysis tools during molecular diagnosis and the importance of the selection of more appropriate tools for analysis. Furthermore, ethnic specific sequences should be considered as reference sequence for the analysis to bypass sequence dissimilarities among diverse populations.

Rapid detection of alpha-globin gene αααanti-3.7 triplets with droplet digital PCR

To establish a rapid method for detection of alpha-globin gene αααanti-3.7 based on droplet digital PCR (ddPCR) technique. The differential sequence between the X1 and Y1 box of α1 gene was selected as the amplicon of the target gene with β-actin as the reference gene. The specific primers and TaqMan probes were designed, and then a quantitative method for detecting the copy number was established based on ddPCR technique. The sensitivity and accuracy of the method were evaluated by detecting 28 samples of known genotypes and 60 clinical samples. The ddPCR-based method accurately identified the genotypes of all the 28 samples with known genotypes and detected 5 cases of αα/αααanti-3.7 from the 60 clinical samples, and the results were verified by MLPA. The sensitivity and accuracy of this method were both 100% for detecting alpha-globin gene αααanti-3.7. This ddPCR-based method for detecting αααanti-3.7 triplet can be applied for population screening and in routine clinical molecular diagnosis with simple operation, rapid analysis and accurate results.

Activation of the alpha-globin gene expression correlates with dramatic upregulation of nearby non-globin genes and changes in local and large-scale chromatin spatial structure

BackgroundIn homeotherms, the alpha-globin gene clusters are located within permanently open genome regions enriched in housekeeping genes. Terminal erythroid differentiation results in dramatic upregulation of alpha-globin genes making their expression comparable to the rRNA transcriptional output. Little is known about the influence of the erythroid-specific alpha-globin gene transcription outburst on adjacent, widely expressed genes and large-scale chromatin organization. Here, we have analyzed the total transcription output, the overall chromatin contact profile, and CTCF binding within the 2.7 Mb segment of chicken chromosome 14 harboring the alpha-globin gene cluster in cultured lymphoid cells and cultured erythroid cells before and after induction of terminal erythroid differentiation.ResultsWe found that, similarly to mammalian genome, the chicken genomes is organized in TADs and compartments. Full activation of the alpha-globin gene transcription in differentiated erythroid cells is correlated with upregulation of several adjacent housekeeping genes and the emergence of abundant intergenic transcription. An extended chromosome region encompassing the alpha-globin cluster becomes significantly decompacted in differentiated erythroid cells, and depleted in CTCF binding and CTCF-anchored chromatin loops, while the sub-TAD harboring alpha-globin gene cluster and the upstream major regulatory element (MRE) becomes highly enriched with chromatin interactions as compared to lymphoid and proliferating erythroid cells. The alpha-globin gene domain and the neighboring loci reside within the A-like chromatin compartment in both lymphoid and erythroid cells and become further segregated from the upstream gene desert upon terminal erythroid differentiation.ConclusionsOur findings demonstrate that the effects of tissue-specific transcription activation are not restricted to the host genomic locus but affect the overall chromatin structure and transcriptional output of the encompassing topologically associating domain.