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We developed a series of interrelated locus-specific databases to store all published and unpublished genetic variation related to hemoglobinopathies and thalassemia and implemented microattribution to encourage submission of unpublished observations of genetic variation to these public repositories. A total of 1,941 unique genetic variants in 37 genes, encoding globins and other erythroid proteins, are currently documented in these databases, with reciprocal attribution of microcitations to data contributors. Our project provides the first example of implementing microattribution to incentivise submission of all known genetic variation in a defined system. It has demonstrably increased the reporting of human variants, leading to a comprehensive online resource for systematically describing human genetic variation in the globin genes and other genes contributing to hemoglobinopathies and thalassemias. The principles established here will serve as a model for other systems and for the analysis of other common and/or complex human genetic diseases. © 2011 Nature America, Inc. All rights reserved.

Hemoglobin (Hb) disorders are common, potentially lethal monogenic diseases, posing a global health challenge. With worldwide migration and intermixing of carriers, demanding flexible health planning and patient care, hemoglobinopathies may serve as a paradigm for the use of electronic infrastructure tools in the collection of data, the dissemination of knowledge, the harmonization of treatment, and the coordination of research and preventive programs. ITHANET, a network covering thalassemias and other hemoglobinopathies, comprises 26 organizations from 16 countries, including non-European countries of origin for these diseases (Egypt, Israel, Lebanon, Tunisia and Turkey). Using electronic infrastructure tools, ITHANET aims to strengthen cross-border communication and data transfer, cooperative research and treatment of thalassemia, and to improve support and information of those affected by hemoglobinopathies. Moreover, the consortium has established the ITHANET Portal, a novel web-based instrument for the dissemination of information on hemoglobinopathies to researchers, clinicians and patients. The ITHANET Portal is a growing public resource, providing forums for discussion and research coordination, and giving access to courses and databases organized by ITHANET partners. Already a popular repository for diagnostic protocols and news related to hemoglobinopathies, the ITHANET Portal also provides a searchable, extendable database of thalassemia mutations and associated background information. The experience of ITHANET is exemplary for a consortium bringing together disparate organizations from heterogeneous partner countries to face a common health challenge. The ITHANET Portal as a web-based tool born out of this experience amends some of the problems encountered and facilitates education and international exchange of data and expertise for hemoglobinopathies. Copyright © Informa Healthcare USA, Inc.

We report an update of the α-globin gene point mutations resulting in structural modification associated with an α-thalassemia (α-thal) phenotype. These variants, barely symptomatic in the heterozygous state, are either unstable due to folding defects and/or defects in binding to α-hemoglobin stabilizing protein (AHSP). This is predicted to result in precipitation of the unstable α chains or Hb variant, a concomitant decrease in the overall quantity of normal α-globin in the red cells and a potential degree of anemia and possibly, hemolysis. Genotype/phenotype correlation and potential genetic risk in combination with common or less common α-thal defects are discussed. Copyright © Informa Healthcare USA, Inc.

BACKGROUND: Hemoglobin (Hb) Groene Hart [alpha119 (H2)Pro-->Ser (alpha1)], also known as Hb Bernalda, is a nondeletional alpha-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The alpha-thalassemic behavior of this variant has been explained as an impaired interaction between the alpha-globin chain and the alpha-Hb-stabilizing protein. METHODS: To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile-water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC-ESI-MS/MS. RESULTS: RPLC detected an abnormal peak with a retention time substantially greater than that of the wild-type alpha(A)-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the -alpha(3.7) deletion). These patients possessed abnormal hematologic features suggesting an alpha-thalassemia phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue. CONCLUSIONS: This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity.BACKGROUND: Hemoglobin (Hb) Groene Hart [alpha119 (H2)Pro-->Ser (alpha1)], also known as Hb Bernalda, is a nondeletional alpha-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The alpha-thalassemic behavior of this variant has been explained as an impaired interaction between the alpha-globin chain and the alpha-Hb-stabilizing protein. METHODS: To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile-water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC-ESI-MS/MS. RESULTS: RPLC detected an abnormal peak with a retention time substantially greater than that of the wild-type alpha(A)-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the -alpha(3.7) deletion). These patients possessed abnormal hematologic features suggesting an alpha-thalassemia phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue. CONCLUSIONS: This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity.

We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion.We report a new hemoglobin (Hb) variant found in a 6-year-old girl of Moroccan origin, living in the Dutch city of Gouda. The child was referred because of microcytic and hypochromic parameters. A normal zinc protoporphyirin (ZPP) value excluded iron deficiency and gap-polymerase chain reaction (gap-PCR) revealed a heterozygosity for the common -alpha(3.7) thalassemia deletion, partially justifying the hematological picture. The Hb pattern on alkaline electrophoresis and capillary electrophoresis was normal, while a fraction of 9% preceding the Hb A peak, remained visible on different high performance liquid chromatography (HPLC) devices. This fraction, located in front of the Hb A peak, is usually considered as a Hb A derivate that becomes more expressed in older samples. However, the sample was freshly collected and the peak unusually evident. Therefore, direct sequencing of the alpha-globin genes was performed revealing a GTG-->CTG transversion at codon 1 of the alpha1-globin gene or of the hybrid gene. This point mutation induces a single amino acid substitution from valine to leucine. Electrospray-mass spectrometry (ES-MS) analysis revealed, in addition to this substitution, that the N-terminal methionine was retained and that about 20% of the variant was acetylated. As expected for an association with a -alpha(3.7)-thalassemia (thal) deletion, the non acetylated and acetylated abnormal alpha chain amounted to 32% of the total alpha chains. Family studies revealed that the mutated codon was located in cis of the deletion.

Hb Crete, an electrophoretically neutral, unstable, high oxygen affinity variant, was characterized by protein and DNA analyses in the homozygous state in a 32-year-old woman from Crete, with erythrocytosis and microcytosis. The proband and members of her family over 3 generations, including 5 carriers of Hb Crete, were subject to clinical, hematological and biochemical investigations, and DNA, RNA and protein studies were carried out. The proband demonstrated features associated with disturbed hemoglobin (Hb) structure and function, including erythrocytosis and additionally a state of functional anemia, the latter reflected by increased erythropoetin levels and cardiac output. In addition, all the carriers surprisingly had hematological and biosynthetic findings more usually associated with thalassemia trait. The structural change in Hb Crete only partly explains all the pathological manifestations of this variant, and other mechanisms are discussed. (c) 2005 Elsevier Inc. All rights reserved.

Hb Mont Saint-Aignan [beta 128(H6)Ala –> Pro] is a mildly unstable variant, associated with hemolytic anemia, marked microcytosis and increased alp biosynthetic ratio (1.55 versus 1.1 +/-0.1 in the control). The abnormal chain was isolated by selective precipitation with isopropanol and the structural modification determined by protein chemistry methods (reversed phase high performance liquid chromatography and mass spectrometry). Possible mechanisms underlying the beta (+)-thalassemia-like expression of this variant are discussed.

Hb Sitia [beta 128(H6)Ala –> Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Wb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT –> GTT mutation at codon 128, which is predicted to encode an Ala –> Val substitution. This was confirmed by mass spectrometry analyses of the beta -globin chain. Since alanine at beta 128(H6) interacts with several amino acids of the alpha1 beta1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.

We report here the functional and structural characterization of Hb Acharnes [beta 53(D4) Ala –> Thr], an unstable and electrophoretically silent variant, that was found associated in trans with a beta(0)-thalassemic mutation (IVSI-1 G –> A), in a patient with thalassemia intermedia syndrome. This case is discussed in comparison with other sporadic cases that we have previously investigated, resulting from the co-inheritance of a beta(0)-thalassemic mutation (CD39 C –> T) with two other types of unstable hemoglobins, Hb Koln [beta 98(FG5) Val –> Met], and Hb Arta [beta 45(CD4) Phe –> Cys]. it may be concluded that, in these associated forms, both the degree of instability of the variant and the altered oxygen binding properties (affecting the degree of tissue hypoxia) are major determinants of their clinical expression. (C) 1999 Wiley-Liss, Inc.

We report here the functional and structural characterization of Hb Acharnes [β53(D4) Ala → Thr], an unstable and electrophoretically silent variant, that was found associated in trans with a β°-thalassemic mutation (IVSI-1 G → A), in a patient with thalassemia intermedia syndrome. This case Is discussed in comparison with other sporadic cases that we have previously investigated, resulting from the co-inheritance of a β°-thalassemic mutation (CD39 C → T) with two other types of unstable hemoglobins, Hb Koln [β98(FG5) Val → Met], and Hb Arta [β45(CD4) Phe → Cys]. It may be concluded that, in these associated forms, both the degree of instability of the variant and the altered oxygen binding properties (affecting the degree of tissue hypoxia) are major determinants of their clinical expression.

The interaction of rare Hb variants with beta degrees-thalassaemia results in a quasihomozygous state where the erythrocytes contain the variant as the only major adult Hb component, Such a situation is a unique model that enables functional studies even in the case of a neutral variant that could not be isolated from Hb A, We report here an unusual patient carrying Hb Arta, a novel Hb variant [beta 45 (CD4) Phe –> Cys], in trans with beta degrees-thalassaemia gene (beta degrees 39). The aminoacid substitution at the critical CD corner of this Hb molecule renders the molecule unstable. In addition, haem is displaced in a position that favours the deoxy (T) conformation of the variant, but less than in Hb Cheverly [beta 45 (CD4) Phe –> Ser], and results in a p50 of 43 mmHg (pH 7.4, 37 degrees C) in the red cells with preservation of cooperativity. Solution studies of the almost pure Hb Arta show a 50% decrease in oxygen affinity and normal cooperativity; the Bohr effect and the interaction with organic phosphates are similar to those of Hb A. Hb Arta retains both normal homo- and heterotropic effects allowing a well-preserved oxygen transport in vivo despite a mild anaemia.

Hemoglobins A1c and A1a+b were measured by an automated chromatographic method in 11 control subjects, 100 diabetics and 30 subjects with renal failure not induced by diabetes. Hb A1c was higher in diabetics than in controls (8.26 +/- 0.31 versus 5.24 +/- 0.28, p less than 0.01) and strongly correlated with blood glucose values for the preceding 4 months. In poorly controlled diabetics, submitted to an intensive therapeutic program, Hb A1c decreased rapidly. Hb A1c was slightly but significantly elevated in the uremic, non-diabetic patients who were not submitted to periodic hemodialysis: 6.42 +/- 0.32, p less than 0.05. It returned, in hemodialyzed patients, to a level not significantly different from the control value. Hb A1a+b was elevated in diabetic subjects (2.43 +/- 0.04 vs. 1.55 +/- 0.01 in controls, p less than 0.001). It was also higher in the uremic patients (2.71 +/- 0.14, p less than 0.001). No decrease occurred in the hemodialyzed patients (3.27 +/- 0.31). Glycosylated hemoglobin values, as routinely estimated and expressed under the name of "Hb A1", should be interpreted with caution in patients with renal failure. Methods discriminating Hb A1a+b have to be used.

Three brothers with congenital recessive methaemoglobinaemia without mental retardation were found to be homozygous for NADH diaphorase deficiency. Twelve family members were heterozygous. One of the