Clinically relevant updates of the HbVar database of human hemoglobin variants and thalassemia mutations.

HbVar (http://globin.bx.psu.edu/hbvar) is a widely-used locus-specific database (LSDB) launched 20 years ago by a multi-center academic effort to provide timely information on the numerous genomic variants leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Here, we report several advances for the database. We made clinically relevant updates of HbVar, implemented as additional querying options in the HbVar query page, allowing the user to explore the clinical phenotype of compound heterozygous patients. We also made significant improvements to the HbVar front page, making comparative data querying, analysis and output more user-friendly. We continued to expand and enrich the regular data content, involving 1820 variants, 230 of which are new entries. We also increased the querying potential and expanded the usefulness of HbVar database in the clinical setting. These several additions, expansions and updates should improve the utility of HbVar both for the globin research community and in a clinical setting.

Label-free detection of thalassemia and other ROS impairing diseases.

Pathogenesis of different diseases showed that some of them, especially thalassemia (T) and rheumatoid arthritis (RA) have an implicit association with oxidative stress and altered levels of reactive oxygen species (ROS). Introducing ROS level and the balance between ROS and antioxidants as essential metrics, an attempt was made to classify T and RA from normal individuals (treated as controls)(C) using synchronous fluorescence spectroscopy (SFS) and Raman line intensity of water. This non-invasive and label-free approach was backed up by a categorization algorithm that helped in the prediction of disease types from serum samples. The predictive system constituted principal component analysis (PCA) with four parameters, namely spectral intensity ratios of reduced nicotinamide adenine dinucleotide (NADH) to tryptophan (Trp) (NADH/Trp), kynurenine (Kyn) to tryptophan (Kyn/Trp), kynurenine to NADH (Kyn/NADH), and logarithmic changes in Raman line intensity of water (Rline), with the index headers containing the disease types. Rline has a positive correlation with both Kyn/Trp and Kyn/NADH and a negative correlation with NADH/Trp ratio, implying its direct or indirect association with oxidative stress. In addition to the classification of T, RA, and C a sub-classification of T into beta major and E-beta in their post and pre-splenectomized surgical stages could also be realized. Furthermore, receiver operating characteristic (ROC) analysis was deployed to ascertain that the misclassification error (ME) was negligible for the disease types. Graphical Abstract A schematic representation of the workflow converging into the categorical classification of disease classes.

Molecular characteristics of thalassemia and hemoglobin variants in prenatal diagnosis program in northern Thailand.

Molecular analysis of globin genes is an essential process for prenatal diagnosis (PND) of severe thalassemia. This study aimed to describe the molecular characteristics of thalassemia and hemoglobin (Hb) variants in PND program in northern Thailand. The type and frequency of globin gene mutations from 1290 couples at risk of fetal severe thalassemia diseases that were tested at Thalassemia Laboratory at Chiang Mai University from 2012 to 2017 were retrospectively reviewed. The PND program detected 444 (34.4%), 196 (15.2%) and 642 (49.8%) couples at risk of fetal Hb Bart's hydrops fetalis, beta-thalassemia major (BTM) and beta-thalassemia/Hb E disease, respectively. Coinheritance of more than one type of thalassemia was common and eight (0.6%) couples were at risk of two types of severe thalassemia. There were two types of alpha0-thalassemia; 893 (99.7%) Southeast Asian and 3 (0.3%) Thai deletions. Twenty beta-globin gene mutations were found with 94.3% of beta0-thalassemia. The codon 41/42 (- TTCT), codon 17 (A>T), IVS-I-1 (G>T) and codon 71/72 (+ A) comprised 90% of beta-thalassemia mutations. The study shows a high percentage of couples at risk of fetal Hb Bart's hydrops fetalis and BTM. The percentage of beta0-thalassemia is higher than those seen in other regions of Thailand.

Establishing and evaluating an auto-verification system of thalassemia gene detection results.

The manual verification of gene tests is time-consuming and error prone. In this study, we try to explore a high-efficiency, clinically useful auto-verification system for gene detection of thalassemia. A series of verification elements were rooted in the auto-verification system. Consistency check was applied initially as one of the essential elements in our study. One hundred twenty-four archived cases were used to choose the consistency-check rules' indices from routine blood examination and hemoglobin electrophoresis by the receiver operating characteristic curves. Rule 1 and rule 2 established by the chosen indices were compared by their passing rate, consistency with manual validation, and error rate. Finally, 748 cases were used for verifying the system's feasibility by evaluating the passing rate, turn-around time (TAT), and error rate. The rule 2 had a higher passing rate (67.7% vs. 50.8%) and consistency (0.623 vs. 0.364) than the rule 1 with an error rate of zero. In a "live" valuation, the auto-verification system can reduce the TAT and error rate of verification by 51.5% and 0.13%, respectively, with a high passing rate of 82.8%. The auto-verification system for gene detection of thalassemia in this study can shorten the validation time, reduce errors, and enhance efficiency.

A two-layered classifier based on the radial basis function for the screening of thalassaemia.

The thalassaemias are blood disorders with hereditary transmission. Their distribution is global, with particular incidence in areas affected by malaria. Their diagnosis is mainly based on haematologic and genetic analyses. The aim of this study was to differentiate between persons with the thalassaemia trait and normal subjects by inspecting characteristics of haemochromocytometric data. The paper proposes an original method that is useful in screening activity for thalassaemia classification. A complete working system with a friendly graphical user interface is presented. A unique feature of the presented work is the adoption of a two-layered classification system based on Radial basis function, which improves the performance of the system.

Classification of the disorders of hemoglobin.

Over the years, study of the disorders of hemoglobin has served as a paradigm for gaining insights into the cellular and molecular biology, as well as the pathophysiology, of inherited genetic disorders. To date, more than 1000 disorders of hemoglobin synthesis and/or structure have been identified and characterized. Study of these disorders has established the principle of how a mutant genotype can alter the function of the encoded protein, which in turn can lead to a distinct clinical phenotype. Genotype/phenotype correlations have provided important understanding of pathophysiological mechanisms of disease. Before presenting a brief overview of these disorders, we provide a summary of the structure and function of hemoglobin, along with the mechanism of assembly of its subunits, as background for the rationale and basis of the different categories of disorders in the classification.

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

OBJECTIVE: The epsilon gamma delta beta (εγδß)-thalassemias are rare sporadic disorders caused by deletion of the ß-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. METHODS: The deletion was identified by globin gene multiplex ligation-dependent probe amplification (MLPA) of the ß-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. RESULTS: A 1.78-Mb εγδß-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. CONCLUSIONS: We suggest that εγδß-thalassemia be added to the list of hemoglobinopathies that can cause neonatal anemia and that MLPA of the ß-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.

One step forward in health promotion.

Thalassemias are inherited blood disorders. In 2011, there was no clear situation regarding the number of thalassemia or other hemoglobinopathy patients in Romania. The luck of information has led to an increased number of patients registered by Fundeni Hospital, Bucharest in the last years. The main goal of this article is to underline the importance of implementing a screening program in Romania for thalassemia and other hemoglobinopathies, parallel with an awareness program. The importance of this objective is strongly supported by the following example: in Sardinia, thalassemia major was present in one out of 250 births, and has declined to one out of 4000 births after they implemented the screening program. The article is written by Monica Rosu, a participant to the World Wide Opportunities for Woman course, held in Canada, in 1999 and states one of the first results of thalassemia awareness in Romania with a positive impact on the patient's life, registering the following results: 2000: A website in Romanian language regarding thalassemias and addressed to patients was created. 2005: The Association of People with thalassemia major was founded and become member of Thalassemia International Federation. 2011: Thalassemia major patients from Romania have access to free treatment and the latest medical oral chelator, Exjade. The association participated in various meetings addressed to patients and doctors as well, created their own website, held workshops, fought together for an improved quality of life for themselves, for healthy people (possible thalassemia carriers) and for all Romanian children.

The definition and epidemiology of non-transfusion-dependent thalassemia.

Inherited hemoglobin-related disorders, which include the structural variants (hemoglobin S, C, and E) and the alpha (α)- and beta (ß)-thalassemias, affect more than 300,000 children annually, particularly in malaria-endemic regions stretching from sub-Saharan Africa and the Mediterranean to Southeast Asia. Screening for carriers of these traits is important to provide prenatal genetic counseling and to accurately estimate the true prevalence and public health burden of these disorders. The clinical course of thalassemias, which affect nearly 70,000 children annually, is highly variable depending on the mixture of inherited alleles. The primary forms of non-transfusion-dependent thalassemia include ß-thalassemia intermedia, hemoglobin E ß-thalassemia, and hemoglobin H disease. Early clinical recognition of these disorders is essential to prevent affected children from being mistakenly placed on life-long transfusion therapy.

Craniofacial deformities in transfusion-dependent thalassemia patients in Malaysia: prevalence and effect of treatment.

This comparative cross-sectional study was conducted in the pediatric daycare unit, Hospital Universiti Sains Malaysia to determine the prevalence of craniofacial deformities (CFD) and the association between these deformities and different clinical presentations among thalassemia patients. Patients were classified as either craniofacial deformity positive (CFD+) or craniofacial deformity negative (CFD-) by two examiners based on the presence or absence of deformity of the cheeks, frontal and/or maxillary bones. Fifteen clinical parameters were compared between the groups. Nineteen out of 43 patients (44.2%; confidence interval, 30.2-58.2%) had craniofacial deformities (CFD+). Both groups were comparable among the clinical parameters studied. Patients in the CFD+ group did not start their blood transfusions significantly earlier than the CFD- group (p = 0.50) and had a nonsignificantly lower mean pretransfusion hemoglobin level than the CFD- group (p = 0.71). Patients receiving regular monthly blood transfusions had a nonsignificantly smaller percentage of CFD than those transfused less often (p = 0.495). CFD+ patients had a splenectomy at a nonsignificantly younger age than CFD- patients (p = 0.36). HbE/beta thalassemia patients were not significantly less likely to develop CFD than other varieties (p = 0.50) and males had a nonsignificantly higher percentage of CFD than females (p = 0.29). This study shows CFD in thalassemia patients are still prevalent but no significant associated factors were found; however, a nonsignificantly higher prevalence of CFD was observed in patients with signs of severe disease and less efficient treatment.

Thalassemic mothers and their babies.

Thirty-two mothers with thalassemia, more than half of whom had hemoglobin H disease with or without other forms of thalassemia or hemoglobinopathies along with their 46 children were evaluated. The mean maternal hematocrit was 22.2% and the mean maternal body mass index was 18.7. The majority of women were not transfused during pregnancy. Eight point seven percent developed toxemia of pregnancy. All had spontaneous labor, 69.5% had a normal vaginal delivery and 23.9% had a cesarean section. The mean infant birth weight was 2,542.2 grams. Low birth weight was found in 34.7%; no extremely low birth weights occurred.

Molecular mechanisms underlying thalassemia intermedia in Iran.

To improve the differentiation of thalassemia intermedia from other hemoglobinopathies in Iran, four known genetic mechanisms-XmnI (G)gamma polymorphism, inheritance of mild and silent beta-thalassemia alleles, delta beta deletion, and coinheritance of alpha- and beta-thalassemia-were investigated in 52 Iranian individuals suspected to have thalassemia intermedia based on clinical and hematological characteristics. Beta-globin mutations were studied using a reverse-hybridization assay and sequencing of the total beta-globin gene. The XmnI (G)gamma polymorphism, the Sicilian delta beta deletion, and four alpha-globin mutations (-a(3.7), -a(4.2), -(MED), aaa(anti-3.7)) were studied using PCR-based techniques. The inheritance of the XmnI (G)gamma polymorphism with severe beta-thalassemia alleles in the homozygous or compound heterozygous state was the predominant mechanism observed in 27 individuals (55.3%). In five cases, this status overlapped with the -a(3.7)/aa genotype. The second most frequent cause for thalassemia intermedia (14.8%) was the inheritance of mild beta-thalassemia alleles, including IVS-I-6 (T > C), -88 (C > A), and + 113 (A > G). In three subjects (4.3%) the Sicilian delta beta deletion was identified. HbS in association with beta-zero-thalassemia was found in three patients with thalassemia intermedia phenotype. In 11 cases (21.3%) no causative genetic alteration could be identified. Our results reflect the diversity underlying thalassemia intermedia, and the limitations of the applied clinical, hematological, and molecular approaches for correct diagnosis. Some of the unresolved cases will offer an opportunity to discover additional molecular mechanisms leading to thalassemia intermedia.

[The use of hydroxyurea in severe forms of sickle cell disease: study of 47 Tunisian paediatric cases]. / L'utilisation de l'hydroxyurée dans les formes sévères de la drépanocytose: étude de 47 cas pédiatriques tunisiens.

INTRODUCTION: The Sickle Cell Disease (SCD) is a serious illness considering its complications. For the children seriously affected, three therapeutic options are currently validated: transfusion therapy, hydroxyurea and bone-marrow transplantation. OBJECTIVES: To see the contribution of hydroxyurea therapy on severe forms of SCD in affected Tunisian children. MATERIAL AND METHODS: This investigative study lasted over 6 years and 9 months, (September 2000-May 2007), enrolling 47 patients including 27 homozygous SCD and 20 double heterozygote SCD-S/beta thalassemia. The median age was 12 years and a half. The average dosage were 20mg/kg/d (14-30 mg/kg/d). The average duration of treatment was 52 months (18-81 months). RESULTS: The main indication for hydroxyurea treatment was prevention of recurrence of an acute chest syndrome in seven cases; episodic vaso-occlusive crises exceeding three events per year in 38 cases and prevention of deterioration of cerebral vascular accident in two cases. We observed a fast and sustained improvement of the clinical expression of the disease with a significant decrease of the number of days of hospitalization per patient and per annum from 29.3 d (10-84 d) to 3.2 d/(p<0.01). Treatment was well tolerated. We observed a significant increase of haemoglobin fetus (HbF) rates from 3 to 30% (p<0.01), hemoglobin from 7.8 to 9.6g/dl (p<0.05), average blood cells volume from 79.1 to 100.3 fl (p<0.05) and a significant fall of the white blood cells rates from 14,914 to 8464 per millimetre cube (p<0.05), polynuclear neutrophils from 6799 to 3486 per millimetre cube (p<0.05) and platelets from 508,666 to 293,500 per millimetre cube (p<0.05). CONCLUSIONS: Hydroxyurea represents a privileged choice of treatment in the severe forms of SCD in children, for homozygous SCD-SS as well as for double heterozygote SCD-S/beta thalassemia. Used carefully, with frequent monitoring, it appeared as a safe treatment in short and medium term, but studies of long-term tolerance should be undertaken.

A novel epsilon gamma delta beta thalassemia of 1.4 Mb deletion found in a Japanese patient.

A novel large deletion, causing epsilon gamma delta beta thalassemia (here called, epsilon gamma delta beta thalassemia Jpn-I) was discovered in a 6-year-old Japanese boy. He was born uneventfully, but revealed thalassemia minor after birth. The mutation was inherited from his mother. The deletion, caused by an illegitimate recombination extended from 750 kb upstream to 660 kb downstream of e-globin gene, and removed about 1.4 Mb of DNA, the largest in epsilon gamma delta beta thalassemias. A 19-nucleotide orphan sequence and direct repeats were present at the junction. The deletion lost several functional genes, but no relevant symptoms manifested. The breakpoints were determined by relatively simple methods.

Reduced intensity stem cell transplantation for treatment of class 3 Lucarelli severe thalassemia patients.

Bone marrow transplantation is the only therapeutic option that can potentially eliminate thalassemic disease. Early results indicated that children in Class 3 Lucarelli had a much worse outcome because of high nonrejection mortality and high rejection rate. In the present study, reduced intensity stem cell transplantation (RIT) was performed in eight Class 3 Lucarelli patients conditioned by busulfan, fludarabine, and antilymphpcute globulin. One of the eight patients additionally received thiotepa, and total lymphoid irradiation (TLI), while one only received TLI. All patients received hydroxyurea 20 mg/kg/day daily >/=3 months before RIT. Peripheral blood stem cell (PBSCs) were given to a target number of CD34(+) cells more than 5 x 10(6) cells/kg of recipient weight. Seven patients received T cell nondepleted PBSCs from matched siblings while one patient received purified CD34(+) cells from two HLA antigen mismatched maternal PBSCs. The graft-versus-host disease (GvHD) prophylaxis included cyclosporine or tacrolimus and mycophenolate mofetil. Initially, an engraftment of donor cells was observed in all eight patients, but subsequently only six of eight patients had stable full donor engraftment. There were no deaths or Grade 3-4 acute GvHD in our patients. The present study lends support that the regimens described here produced minimal toxicity and resulted in stable full donor engraftment in the majority of the severe Class 3 Lucarelli thalassemia patients.