Delta Beta-thalassemia Research Articles

Diagnosing delta-beta thalassaemia in a resource poor setting

An eight-year-old girl presented with mild scleral icterus, pallor and hepatosplenomegaly. Her preliminary investigations revealed low haemo-globin (Hb) (94g/L), high reticulocyte count (3.5%), normal total bilirubin level and normal liver function tests. Her blood picture revealed evidence of chronic haemolysis. High perfor-mance liquid chromatography (HPLC) revealed markedly raised Hb-F (98.3%). Capillary electro-phoresis revealed Hb-F of 99.2%. Acid elution test (Kleihauer test) showed pan-cellular distribution of Hb-F. Her parents were first cousins. Her mother’s profile showed thalassaemic red cell indices with normal Hb-A2 and high Hb-F (13.3%). The father was not available for screening. The paternal grandmother’s HPLC showed normal Hb-A2 and high Hb-F (6.8%). Based on clinical and laboratory findings and family screening results, the child was diagnosed as homozygous delta-beta (αβ)-thalassaemia presenting as thalassaemia intermedia.

Identification of a rare [Gγ(Aγδβ)0] -thalassemia using tandem mass spectrometry.

Thalassemias are a group of inherited monogenic disorders characterized by defects in the synthesis of one or more of the globin chain subunits of the hemoglobin tetramer. Delta-beta (δβ-) thalassemia has large deletions in the β globin gene cluster involving δ- and β-globin genes, leading to absent or reduced synthesis of both δ- and β-globin chains. Here, we used direct globin-chain analysis using tandem mass spectrometry for the diagnosis of δβ-thalassemia. Two cases from unrelated families were recruited for the study based on clinical and hematological evaluation. Peptides obtained after trypsin digestion of proteins extracted from red blood cell pellets from two affected individuals and their parents were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometric analysis revealed a severe reduction in δ, β, and Aγ globin proteins with increased Gγ globin protein in the affected individuals. The diagnosis of Gγ(Aγδβ)0 -thalassemia in the homozygous state in the affected individuals and in the heterozygous state in the parents was made from our results. The diagnosis was confirmed at the genetic level using multiplex ligation-dependent probe amplification (MLPA). Our findings demonstrate the utility of direct globin protein quantitation using LC-MS/MS to quantify individual globin proteins reflecting changes in globin production. This approach can be utilized for accurate and timely diagnosis of hemoglobinopathies, including rare variants, where existing diagnostic methods provide inconclusive results.

Detecting rare thalassemia in children with anemia using third-generation sequencing

ABSTRACTBackgroundIn China, conventional genetic testing methods can only detect common thalassemia variants. Accurate detection of rare thalassemia is crucial for clinical diagnosis, especially for children that need long-term blood transfusion. This study aims to explore the application value of third-generation sequencing (TGS) in the diagnosis of rare thalassemia in children with anemia.MethodsWe enrolled 20 children with anemia, excluding from iron deficiency anemia (IDA). TGS was employed to identify both known and novel thalassemia genotypes, while sanger sequencing was used to confirm the novel mutation detected.ResultsAmong the 20 samples, we identified 5 cases of rare thalassemia. These included β−4.9 (hg38,Chr11:5226187-5231089) at HBB gene, α−91(HBA2:c.*91delT), αCD30(HBA2:c.91-93delGAG), Chinese Gγ+(Aγδβ)0(NG_000007.3: g .48795-127698 del 78904) and delta – 77(T > C)(HBD:c.-127T>C). Notably, the –SEA/α−91α genotype associated with severe non-deletional hemoglobin H disease (HbH disease) has not been previously reported. Patients with genotypes β654/β−4.9 and –SEA/α−91α necessitate long-term blood transfusions, and those with the –SEA/αCD30α, Chinese Gγ+(Aγδβ)0 and delta thalassemia demonstrate mild anemia.ConclusionsTGS demonstrates promising potential as a diagnostic tool for suspected cases of rare thalassemia in children, especially those suspected to have transfusion-dependent thalassemia (TDT).

Homozygous delta beta-thalassemia: A rare case report

Delta Beta (δβ) thalassemia is a rare autosomal recessive hemoglobinopathy. This is caused by either reduced production of δ and β genes (heterozygous state) or complete absence (homozygous state). High-performance liquid chromatography (HPLC) is an important screening and diagnostic tool, to be supported by molecular data and parental studies. A 40-year-old male (propositus), with mild anemia presented in the outpatient department. There were no other definitive causes of anemia with adequate nutritional status and no response after the treatment with nutritional supplements. HPLC was advised to look for any qualitative/quantitative defects. HPLC findings reveal 96% fetal hemoglobin (HbF), a very small quantity of adult hemoglobin (HbA), and an absent HbA2. In view of this data, followed up with parental studies. Both parents had elevated HbF and similar grades of anemia. These supported the presence of a heterozygous state of δβ-thalassemia in parents and homozygous δβ thalassemia in propositus. In δβ-thalassemia, there is a deletion of the δ and β genes on Chromosome 11. Expression of the gamma gene increases to compensate, causing increased production of gamma globulin for HbF (α2 β2). On HPLC or electrophoresis, heterozygosity shows as normal HbA2 and elevated HbF, and as absent HbA2 with almost 100% HbF for homozygous individuals. Screening of hemoglobinopathies by HPLC is that can be further subjected to parental screening for a more definitive diagnosis and narrow down the differentials as compared to molecular testing considering the cost, limited availability, and higher turnaround time.

Delta beta thalassemia, a rare hemoglobin variant

CONTEXT: Fetal hemoglobin (HbF) reduces to <1% few months after birth. However, it can persist beyond infancy into adult life in rare conditions such as delta-beta (δβ) thalassemia and hereditary persistence of HbF. δβ thalassemia is a relatively rare type of thalassemia due to decrease in both beta and delta globin chain production. AIMS: This study aims to assess the hematological and high-performance liquid chromatography (HPLC) findings in δβ thalassemia. SETTINGS AND DESIGN: Cross-sectional observational study. SUBJECTS AND METHODS: The study was conducted in department of Clinical Pathology, PGIMS, Rohtak. On screening of 1197 patients over a period of around 1 year, a total of 13 cases of δβ thalassemia were detected by HPLC. STATISTICAL ANALYSIS USED: Not done. RESULTS: The age of presentation ranged from 1 to 42 years with a mean age of 22.8 years. Eleven cases were diagnosed as heterozygous δβ thalassemia and two as homozygous δβ thalassemia. CONCLUSIONS: δβ thalassemia is an uncommon cause of elevated HbF. Clinical and hematological parameters should be carefully analyzed for accurate diagnosis.

Prevalence of Haemoglobin Variants and Haemoglobinopathies in a Single Paediatric Centre in Southern India: A Retrospective Cross-sectional Study

Introduction: Haemoglobinopathies are qualitative disorders of Haemoglobin (Hb) resulting from structural defects in the amino acid sequence of one of the globin chains, whereas, thalassaemia results from quantitative defects in the synthesis of one or more of the globin chain subunits of the Hb tetramer. Cation Exchange- High Performance Liquid Chromatography (CE-HPLC) is one of the methods for initial screening of Hb variants like HbS, HbD, HbE etc and for quantification of HbF, HbA and HbA2 levels. Aim: To find out the prevalence of haemoglobinopathies in patients of a Government paediatric tertiary care hospital in south India. Materials and Methods: In this retrospective, cross-sectional study, all laboratory requests, in the period from August 2019 to July 2021, for Hb variant analysis by HPLC were collected, irrespective of provisional diagnosis. The Hb variant analysis was carried out by CE-HPLC on the Bio-Rad D-10 analyser. This study was conducted in a Government Paediatric tertiary care hospital in Southern India for patients who had any clinical or familial suspicion of haemoglobinopathies. The Statistical analysis was performed using Microsoft Excel 2010. Results: Total data of 704 laboratory requests for Hb Variant analysis were obtained. Out of 704 laboratory request, 585 were from children younger than 12 years of age and 119 were parental screening. There were 164 abnormal chromatograms. Out of 164 patients, 91 were female patients and 73 were male patients. Out of 164 abnormal chromatograms, 97 (59.15%) were beta- thalassaemia trait, 18 (10.98%) were beta-thalassaemia major, 13 (7.93%) were sickle cell trait, 2 (1.22%) were sickle cell disease, 21 (12.80%) were HbE trait, 2 (1.22%) were homozygous HbE, 2 (1.22%) were HbD trait, 3 (1.83%) were Hereditary Persistence of Foetal Hb (HPFH)/delta beta-thalassaemia, 2 (1.22%) were HbJ trait, 1 (0.61%) was HbE beta-thalassaemia, 1 (0.61%) was sickle- beta-thalassaemia and 2 (1.22%) were alpha thalassaemia. Conclusion: From this study, beta-thalassaemia trait and beta- thalassaemia major were found to be the first and second most prevalent haemoglobinopathies in children below 12 years of age. This data suggests the importance of premarital and antenatal screening procedures that can help in reducing the possibility of such haemoglobinopathies in the future generation, suffering and burden of disease to the family and society.

Compound heterozygous delta beta thalassemia with IVS 1-5 (G>C) mutation presenting as thalassemia major phenotype

Delta beta thalassemia is an unusual variant of thalassemia with elevated level of fetal hemoglobin (HbF). Unlike beta thalassemia, delta beta thalassemia heterozygotes have milder phenotype and homozygotes present as thalassemia intermedia phenotype. We report a 11-month-old male child who presented with severe anemia, and hepatosplenomegaly, thalassemia major phenotype. On evaluation was diagnosed as compound heterozygous for δβ0/β thalassemia with IVS 1-5 (G>C) mutation. This case highlights the importance of genotyping of patients with δβ thalassemia and co-inheritance of δβ thalassemia deletion with point mutation for β-thalassemia results in severe clinical phenotype as thalassemia major.

English

BACKGROUND Sickle delta beta thalassemia is a rare genetic disorder, with varied symptoms, signs, requiring careful monitoring for potential complications. It is due to sickle mutation and thalassemia mutation occurring together, with sparse data available worldwide. The purpose of this study was to assess the clinical and laboratory profile of possible sickle delta beta thalassemia. METHODS The study design is retrospective analysis of clinical information of those selected patients done in our multi-specialty tertiary care referral hospital situated in Telangana state in south India. The case material was collected from December 2017 to December 2019 (2 years duration). All haemoglobin electrophoresis reports were collected with no prior blood transfusions in preceding 4 months. The information collected was analysed and presented. RESULTS Total 9 patients were diagnosed as possible sickle delta beta thalassemia, with male to female ratio of 5 : 4 and age ranging from 12 years to 45 years of age. The commonest symptoms were joint pain and jaundice in 5 patients and sign was splenomegaly in 2 patients. Ultrasonogram of abdomen showed that 3 patients had gall stones, 1 patient had gall bladder sludge, 1 patient had autosplenectomy and 3 patients had splenomegaly. Mild to moderate anaemia was seen with reticulocytosis, sickling test positive in all patients, with haemoglobin in the range of 5.5 g/dl to 12.7 g/dl. 3 patients had iron over load, 2 patients had hepatopathy, 5 patients had unconjugated hyperbilirubinemia, Acute chest syndrome, hepatic necrosis, and nephropathy was seen in 1 patient each. Haemoglobin electrophoresis showed Hb S was from 46.1 % to 76.4, Hb A from 5.3 % to 34.7 %, Hb F from 4.8 % to 22.7 %, Hb A2 from 1.5 % to 3.3 %. 2 patients were treated with hydroxyurea. 2 patients had mutation analysis elsewhere that was reported as compound heterozygous for β-globulin gene for Hb S (GAG-GTG) and IVS 1 - 5 (G-C). CONCLUSIONS Sickle delta beta thalassemia presents as mild to moderate anaemia haemolysis, splenomegaly with vaso-occlusive crises. Hydroxyurea may help in the treatment. Genetic analysis helps in diagnosis and future therapies. KEYWORDS Haemoglobin S, Haemoglobin A2, Haemoglobin A, Haemoglobin F

Thalassaemia genes in Baghdad, Iraq

To estimate the prevalence of thalassaemia genes in Baghdad, a study was made of 502 randomly selected pregnant women attending a major maternity care clinic in the city. A reduced mean corpuscular volume [MCV] of < 80 fl was used as a screening test for thalassaemia in this population, followed by a battery of tests, including haemoglobin analysis and iron studies, to confirm the diagnosis. Using this approach, 22 cases were diagnosed of beta-thalassaemia trait, five cases of alpha-thalassaemia trait and one case of delta beta-thalassaemia trait. Based on these figures the estimated prevalence rates of beta-, alpha- and delta beta-thalassaemia traits in Baghdad, would be 4.4%, 1.0% and 0.2%, respectively. The study also includes a discussion of the above findings in the context of those reported in other neighbouring countries

Analysis of δ-globin gene alleles in Tunisians: description of three new delta-thalassemia mutations.

Thalassemia is one of the most prevalent worldwide autosomal recessive disorders characterized by a great molecular and clinical expression heterogeneity. Alpha and beta-thalassemia are the main two types observed in case of mutations affecting alpha and beta-globin genes respectively. Delta-thalassemia is noted when mutations occur on the delta-globin gene. In Tunisia, β-thalassemia prevalence isestimated at 2.21% of carriers. However, few reports investigated the delta-globin gene. In this work, we aimed to perform a molecular study to help define the molecular spectrum of δ-thalassemia mutations in Tunisia. The study involved 7558 patients among whom we selected 179 individuals with abnormal HbA2 values or fractions. Hemoglobin analysis was performed using Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). DNA sequencing was performed on ABI prism 310 Genetic Analyzer Applied Biosystems. CUPSAT (Cologne University Protein Stability Analysis Tool) was used for the prediction of protein stability changes upon missense mutations and mutants were modeled via DeepView-SwissPdbViewer and POV-Ray softwares for molecular dynamics simulation studies. We identified four mutations: HbA2-Yialousa described for the first time in Tunisia ( in 72.72% of cases) and 3 mutations reported for the first time in the world: (i) c.442T > C Stop147Arg ext 15aa-stop observed in 18.18% of cases, (ii) c.187 G > C (Ala62Pro) noted in 4.54% of casesand (iii) c.93-1G > C found in 4.54% of cases. Our data provide genetic basis that would be especially useful in screening for beta-thalassemia trait during delta-beta thalassemia associations.

Characterization of β-Globin Gene Cluster Deletions Using Multiplex-Gap Polymerase Chain Reaction (PCR) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background : Deletions in the β-globin gene cluster are usually rare, problematic to detect, and subsequently possess a challenge in many diagnostic laboratories. They are normally related to the heterozygous of the delta beta (δβ) thalassemia, hereditary persistence of fetalhemoglobin (HPFH) and some of the hemoglobin variants. These disorders are typically presented by elevated levels of hemoglobin F (Hb F), but with low to normal hemoglobin A2 (Hb A2). However, despite their existence, there is still a limited number of studies focusing on this area, hence no definitive diagnosis could be conclusively established. Therefore, this pilot study was carried out to fill this knowledge gap.
 Methods: In this study, screening of the selected deletional mutations in the β-globin gene cluster among patients with Hb F (>1%) and Hb A2 (<4%) were performed using multiplex Gap-PCR and multiplex ligation-dependent probe amplification (MLPA).
 Results: The results showed that out of 54 samples tested using multiplex Gap-PCR against four target deletions; Thai (δβ)°-thalassemia, HPFH-6, Siriraj J and HbLepore, one sample was found positive with Thai (δβ)°-thalassemia. Further findings from the MLPA screening on 12 randomly selected samples revealed that another patient was positive with double deletions within the region of the β-globin gene cluster. These deletions occur at the gamma-globin gene 1 (HBG1) and gamma-globin gene 2 (HBG2) in exon 3.
 Conclusions: In conclusion, this study highlighted the importance of these deletions’ characterization using multiplex Gap-PCR and MLPA which helps in establishing a definitive diagnosis among this selected group of patients.
 Bangladesh Journal of Medical Science Vol.20(3) 2021 p.618-624

Incidental Detection of Hemoglobin Variants During Evaluation of HbA1c.

HemoglobinA1c (HbA1c) is used to diagnose Diabetes mellitus and monitor glycemic control over the previous eight to twelve weeks in diabetic patients. Detection of HbA1c by cation exchange-high performance liquid chromatography (CE-HPLC) gives a chromatogram by which abnormal hemoglobin variants are also picked up. Some of these may interfere with HbA1c values affecting clinical management. Due to increased inter-state migration as well as medical tourism, there is a high possibility of finding various hemoglobin variants in any part of India. We did a prospective analysis over 1.5years, of the hemoglobin variants detected during all the HbA1c runs. The HbA1c was tested on Bio-Rad D10 dual HbA2/F/A1c platform, which uses the CE-HPLC method. Every chromatogram was carefully studied to look for unknown peaks. The samples showing unknown peaks >6% were re-run in extended HbA2/F mode to categorize the hemoglobin variants. We had 9595 HbA1c samples, of which 70 cases showed a variant window. There were 40 males and 30 females, age ranging from 28 to 76years. The different hemoglobin variants detected were HbD, HbE and HbS in heterozygous state, high HbF (with a differential diagnosis of HPFH heterozygous and delta-beta thalassemia heterozygous), HbE homozygous, HbQ heterozygous and HbJ heterozygous. We conclude that in the process of monitoring glycemic control using HbA1c, we can also pick-up hemoglobin variants. Hence, it is essential to review HbA1c graphs, so that the diagnosis of hemoglobin variants is not missed and the HbA1c reported is reliable.

Contrasting co-inheritance of alpha and beta mutations in delta beta thalassemia and hereditary persistence of fetal hemoglobin: a study from India

ABSTRACT Background and objectives: Coinheritance of δβ thalassemia and HPFH with inherited factors is sparsely documented and may affect treatment modalities. So, we screened the presence of α deletion and β mutations in δβ thalassemia and HPFH disorders in 52 cases with high Hb F concentration. Material and methods: Fifty-two individuals with raised HbF levels were study subjects. CZE was done for quantitative assessment of hemoglobin variants. Asian Indian inversion deletion break point type A, B and HPFH-3 were done by GAP-PCR. Results: 18/52 cases of δβ Gγ (Aγδβ)0 thalassemia and 28/52 cases of HPFH-3 deletion were characterized. 6/52 patients with raised HbF levels were negative for δβ Gγ (Aγδβ)0 and HPFH-3 deletion. 9/18 (50%) were heterozygous for Gγ(Aγδβ)0 break point type A, 6/18 (33%) were heterozygous for break point type B and 3/18 (17%) were homozygous. Of the nine patients heterozygous for Gγ(Aγδβ)0 break point type A, three (33%) patients were double heterozygous with alpha 3.7 kb deletion and two (22%) patients showed compound heterozygosity with IVS 1–5(G–C) mutation. 4/9 (45%) patients were Gγ(Aγδβ)0 heterozygous. Discussion and conclusion: We found 5/18(27.β) δβ-thalassemia cases with co-inherited alpha 3.7 deletion and 3/18 (16β) cases with IVS 1–5(G–C) mutation. Patients showed features of thalassemia intermedia phenotype among which those with co-inherited IVS 1–5(G–C) mutation showed severe phenotype as compared to those with co-inherited alpha 3.7 deletion. So, we highlight importance of genotyping of patients with δβ thalassemia or HPFH and coinheritance with inherited factors which plays crucial role in clinicopathological profile and setting up prenatal diagnostic protocol.

Large Deletions Involving the Beta Globin Gene Complex: Genotype-Phenotype Correlation of 119 Cases

Background: Large deletions involving the beta globin complex are relatively rare. They can be categorized generally into five groups by deletion size and/or location: 1) beta zero thalassemia (BZT); 2) delta beta thalassemia (DBT); 3) hereditary persistence of fetal hemoglobin (HPFH); 4) gamma delta beta thalassemia (GDBT); and 5) epsilon gamma delta beta thalassemia (EGDBT). These deletions are not well understood but often have significant clinical impact, either when present alone or in combination with other hemoglobin mutations. In this study, we analyze phenotypic and molecular data on a large number of cases with deletions in the beta globin gene complex to better classify these five groups of deletions as they occur in isolation.Methods: A query of the routine clinical testing patient files from the Mayo Clinic Metabolic Hematology and Molecular Genetics Laboratories from 2010 to 2015 identified 179 patients with a deletion confirmed by a Multiplex Ligation-dependent Probe Amplification (MLPA) assay. Twenty-four probes sets were placed from the 5' locus control region (LCR) to the 3' hypersensitivity region, spanning the beta globin gene complex. Using a Luminex LX200 flow cytometer, a gene dosage ratio was calculated for each probe set using the median fluorescent intensity value collected. The size and location of the deletion and patient phenotype were compared.Results: Of the 179 total cases, the following large deletions were identified: beta gene (HBB) (n = 47), delta (HBD) through HBB (n = 105), A-gamma (HBG2) through HBB (n = 20), and locus control region (LCR) through HBB (n = 7). One case had a deletion involving the LCR epsilon with the rest of the complex left intact. A subset (n = 60) of cases had compound hemoglobin mutations that altered the phenotype. The BZT cases had relatively high Hb A2 levels and variable Hb F levels consistent with promotor region loss. The main differences between DBT and HPFH included Hb F and Hb A2 levels. GDBT cases presented with median Hb F levels higher than that observed in DBTs, normal Hb A2, and microcytic anemia. EGDBT cases had variable features according to age of the patient and Hb F level; severe microcytic anemia was observed in neonates, milder microcytic anemia in young children, and microcytosis without anemia in an adult case. The phenotypic features of 119 patients with isolated large deletions are compiled in table 1.Conclusion: In general, all five categories of large deletions in an isolated heterozygous state can present with microcytic anemia and are typically benign with the exception of transient severe microcytic anemia in neonatal EGDBT cases. Although phenotypes associated with large deletions involving the beta globin gene complex are frequently distinctive, significant phenotypic overlap can be seen in a subset of cases. These cases require molecular analysis due to their clinical importance when in combination with another beta globin gene complex mutation for an adequate diagnosis and treatment approach.Table 1Deletion typeAgenHbF (%)HbA2 (%)Hb (g/dL)MCV (fL)RBC (10^12/L)RDW (%)MCH (pg/cell)BZT206.3 (0.6-94.4)6.8 (3.4-11.6)11.1 (8.3-14.5)65.4 (60.8-77.2)5.4 (4.2-6.2)19.2 (16.6-21.2)20.9 (18.3-25.7)DBT5610.6 (2.7-22.4)2.7 (2.5-3.1)11.7 (8.6-14.4)68.9 (61.3-83.5)5.3 (4.1-7.3)21.4 (18.2-26.8)21.6 (19.9-39.2)HPFH2325.9 (17.6-39.7)2.0 (1.5-2.4)11.6 (8.1-16.7)78.4 (60.2-101.9)4.4 (3.0-6.3)17.5 (14.1-22.3)25.4 (17.6-29.7)GDBT1413.3 (8.2-19.0)2.6 (1.8-2.7)11.0 (8.6-14.1)72.5 (57.9-82.1)5.1 (3.5-6.2)20.6 (17.4-23.5)22 (17.9-25.1)EGDBT*28 Y10.3313.359.46.915.419.21-4 Y30.9 (0-1.6)3.2 (2.9-3.5)9.5 (8.8-13.3)57.8 (57.6-59.4)5.2 (4.9-6.9)16.6 (15.4-17.4)18.5 (18.1-19.2)<6 month221.4 (14.8-27.9)2.6 (2.2-2.9)6.3 (6.0-6.6)61.3 (59.9-62.6)3.4 (3.3-3.3)21.5 (21.2-21.7)18.4 (18.1-18.7)medians, (min, max); *stratified by age DisclosuresNo relevant conflicts of interest to declare.

Deletional HPFH Vs. Delta Beta Thalassemia: Closing in on a Possible Hb F Silencer Location

Background: The control of hemoglobin F (Hb F) expression has proven an elusive puzzle with several postulated but not mutually exclusive hypotheses. A silencing mechanism hypothesis, which predicts the existence of a regulatory element that suppresses HbF expression, has been supported by recent investigations into a 3.5 kb region upstream from the delta globin gene (HBD). To test this hypothesis, we have investigated our historical case files in a tertiary care high-throughput clinical laboratory and compared the patient phenotype (Hgb, MCV, MCH, RDW, Hb F %, and flow cytometry Hb F distribution) with the status of this 3.5 kb region.Methods: A query of clinical testing patient files from Mayo Clinic Metabolic Hematology Laboratory yielded 179 patients confirmed by Multiplex Ligation-dependent Probe Amplification (MLPA) to contain large deletions located within the epsilon through beta globin genes. Of these, 27 unrelated patients with breakpoints between the pseudobeta (HBBP1) and HBD genes were identified. Four additional MLPA probe pairs were placed in this region and the patient phenotypes were compared.Results: The interior two (of the four added) probes between HBBP1 and HBD stratified all 27 cases. The other two (flanking) probes were never discriminatory, thus refining the area of interest to 2.4 kb. Sixteen cases showed breakpoints within hg19 g.5260154-5259135 (5' region) and eleven cases showed breakpoints within hg19 g.5259136-5257692 (3' region) upstream of HBD. Patients who displayed a phenotype of HPFH (n = 9) contained breakpoints in the 5' region whereas those with a delta beta thalassemia (DBT) phenotype (n = 11) were associated with breakpoints in the 3' region. A subset (n = 7) with breakpoints in the 5' region had indefinite phenotypic features, but of these, all but one showed homocellular Hb F distribution.Conclusion: Our molecular and phenotypic correlation of 27 patients with large deletional breakpoints between HBBP1 and HBD supports the hypothesis of a silencing element located upstream of HBD and further narrows the area that segregates many HPFH and DBT patient phenotypes from 3.5 kb to 2.4 kb. Several potential silencing effectors with binding sites in this region include HDAC1, GATA1 and H3K27me3; interestingly, the BCL11A binding site may be outside of the regulatory area.Table 1Phenotypic data of patients with deletions between HBBP1 and HBD.CaseAge/SexHb FHb A2Hb XHgbRBCMCVMCHMCHCRDWHb F Flow(Y)(%)(%)(%)(g/dL)( 10^12/L)(fL)(pg/cell)(hg/cell)(%)3' Region147 M25.52.116.76.381.426.732.815.7H25 M28.32.412.54.974.625.43415.9H324 F30.31.711.64.2392.727.429.615.2H49 F36.32.2S = 6213.15.269.9NANA14.8524 F37.32.1C = 6112.95.172.925.53514.363 M39.72.112.44.488.728.131.716.3727 F40.23.4S = 5612.24.479.727.834.915816 M43.72.2S = 5415.96.871.523.532.914.197m M100.0*0.012.84.87626.534.916.83' Region1028 F20.9210.114.470.922.732.119H1164 M21.42.310.514.181.125.831.818.1122.5 M242.110.215.359.419.332.520.9H1358 F242.19.514.472.221.529.817.7H1414 F25.41.510.814.969.42231.817.6H1552 F26.91.88.423.67223.532.719.4T1634 M38.71.7S = 6011.44.771.6NANA21.65' Region1724 F2.72.912.36.162.22032.218.21832 F6.12.710.25.161.319.932.519.31927 F7.22.9115.172.921.729.819.92029 F7.82.710.24.772.221.73020.52115 M7.82.512.76.264.720.431.521.22242 M7.83.110.64.975.121.528.623.72360 M10.12.5114.574.824.73323.42441 M10.72.88.64.173.321.22926.82530 F11.52.712.15.567.421.932.521.82625 F13.22.812.15.368.422.833.321.6273 F18.32.611.95.7963.620.632.322.7TNormal values<12-3.30H = homocellular; T= heterocellular; I = indeterminate; *homozygous deletion -- = no deletion; + = deletion present (all alpha deletions were single -3.7 kb 'rightward' deletions) 1 = Suspected Fe deficiency; 2 = recent transfusion DisclosuresNo relevant conflicts of interest to declare.

Reticulocyte parameters of delta beta thalassaemia trait, beta thalassaemia trait and iron deficiency anaemia

AimsTo analyse the differences in reticulocyte indices between delta beta thalassaemia trait (δβ-TT), beta thalassaemia trait (β-TT) and iron deficiency anaemia (IDA), and to correlate those differences with the physiopathological...

Clinical relevance of erythrocyte ferritin in microcytic anemias

BackgroundErythrocyte ferritin (EF) reflects the balance between iron supply and its utilization for hemoglobin synthesis. This balance is altered in microcytosis. We aimed to evaluate the diagnostic value of both EF and the ratio (FRR) plasma ferritin (PF)/EF in these disorders. MethodsA total of 231 subjects participated in the study. Samples from 93 adult patients with different causes of microcytosis, 57 healthy subjects and 81 full-term newborns were analyzed to determine EF and PF concentrations and other hematological parameters. ResultsIn patients with iron deficiency, and in contrast to PF, EF decreased only in the presence of anemia and in direct correlation with the degree of microcytosis (Pearson's p<0.001). EF values for thalassemia patients were higher than those observed in controls (p<10e−5), while PF concentrations were similar between these groups. This EF increase was more marked in the delta-beta thalassemia group (p<0.05). Finally, FRR was much higher in patients with anemia of inflammation than in those with thalassemia (p<10e−5), thus helping to discriminate between these disorders. ConclusionsEF and FRR are tools that may be useful in the diagnosis of the main causes of microcytosis.

Persistent Pulmonary Hypertension of the Newborn Associated With Severe Congenital Anemia of Various Etiologies

Among the many associated features of persistent pulmonary hypertension of the neonate (PPHN), severe congenital anemia has been described only occasionally and is not included in the list of conditions that may cause PPHN in the neonate. We describe the clinical course of a group of 12 full-term neonates with PPHN and congenital anemia due to congenital dyserythropoietic anemia (7/12), α thalasemia (1/12), Diamond-Blackfan (1/12), and epsilon gamma delta beta thalassemia (3/12). The association of congenital anemia and PPHN is more common than previously thought; it can exist with various etiologies and severity of anemia. Congenital anemia has not been described until now as a cause or risk factor for PPHN; it should be considered as such alone or in combination with other known causes to be recognized early and treated appropriately to improve outcome. In families with known cases of congenital anemia due to the above-mentioned diagnosis, closer prenatal follow-up should be offered to anticipate possible fetal distress and/or fetal anemia and PPHN after birth.

Pattern of hemoglobinopathies and thalassemias in upper Assam region of North Eastern India: High performance liquid chromatography studies in 9000 patients

The hereditary hemoglobin (Hb) disorders are the most commonly encountered single gene disorders in India. Data pertaining to the pattern of hemoglobinopathies and thalassemias is scarce in North East India, and hence it was considered worthwhile to study these disorders using a large series of patients referred to a clinical diagnostic laboratory. A total of 9000 patients referred for Hb variant analysis were studied to identify hemoglobinopathies and thalassemias in Upper Assam region of North East India. This study was performed by high performance liquid chromatography (HPLC) using BIORAD variant Hb typing system. Out of 9000 patients studied, abnormal Hb fractions were seen in 5320 patients. The HbE gene was detected in 4315 patients of which HbE trait was seen in 2294 followed by HbE disease in 1892. There were 114 HbE beta thalassemia patients and 15 double heterozygotes of HbE with HbS or HbD. Beta thalassemia trait was seen in 313 patients and beta thalassemia homozygous in 32. HbS gene was detected in 460 patients comprising of HbS trait in 189, HbS disease in 203, S beta thalassemia in 53 and double heterozygotes of SD and ES in 15. The rest comprised of HbD trait in 6, delta beta thalassemia in 33, hereditary persistence of fetal hemoglobin trait in 5 and J chain hemoglobinopathy in 8 patients. Evidence of alpha thalassemia though suspected, could not be confirmed. A high incidence of hemoglobinopathies and thalassemias and their combinations is unique for this part of the country.

A novel epsilon gamma delta beta thalassemia presenting with pregnancy complications and severe neonatal anemia

The epsilon gamma delta beta (εγδβ)-thalassemias are rare sporadic disorders caused by deletion of the β-globin gene cluster. The main clinical feature is marked prenatal and neonatal anemia that resolves spontaneously within a few months. Reports originating mainly from Europe have so far identified 30 such deletions The aim of the present work was to describe a novel 1.78-Mb deletion, the longest ever reported, and to detail the clinical features in 12 members of an extended Bedouin family. The deletion was identified by globin gene multiplex ligation-dependent probe amplification (MLPA) of the β-globin cluster and further characterized by comparative genomic hybridization. Past and present clinical and laboratory data of ten symptomatic and two asymptomatic patients were collected. A 1.78-Mb εγδβ-deletion, the largest ever described, was identified in all patients. Although other genes were included in the deletion, no other symptoms were observed. Of the ten symptomatic fetuses and neonates, three died of the disease. The remainder required packed cell transfusions during the first months of life. Pregnancy complications included intrauterine growth restriction and oligohydramnios, as well as additional neonatal complications including prematurity and persistent pulmonary hypertension of the neonate. We suggest that εγδβ-thalassemia be added to the list of hemoglobinopathies that can cause neonatal anemia and that MLPA of the β-globin cluster be used to confirm its diagnosis. Careful surveillance during pregnancy is important to reduce neonatal mortality and morbidity, especially given the dramatic improvement that occurs later.