Lepore Boston Research Articles

First cases of Hb Lepore in the Russian Federation

Hb Lepore, as the result of fusion of the b- and d-globin genes, leads to decreased amount of non-a-globin chains availability for hemoglobin formation. Hb Lepore, up to now, was not identified among Russian patients. We provide clinical and laboratory information on Hb Lepore Boston–Washington in two cases, one of them familial. A small amount of abnormal Hb was detected by capillary electrophoresis, an abnormal globin chain was shown by HPLC, and the final diagnosis of Hb Lepore Boston– Washington was made by molecular biological analysis of globin genes. Peripheral blood for all affected people revealed RBC’s hypochromia microcytosis and normal Hb concentration. The parents of the patients agreed to use the information, including photos of children, in scientific research and publications.

Accurate mass measurement and tandem mass spectrometry of intact globin chains identify the low proportion variant hemoglobin Lepore–Boston–Washington from the blood of a heterozygote

Within a mixture of proteins, minor polymorphic components are difficult to identify using a conventional proteomic approach. Their identification generally requires multi-dimensional separation steps, before or after proteolytic cleavage, followed by sequence analysis of the proteolytic products. In this study, we investigated the potential of tandem mass spectrometry for protein characterization by identifying the delta-beta hybrid human hemoglobin variant Lepore-Boston-Washington using electrospray ionization tandem mass spectrometry. Hemoglobin Lepore-Boston-Washington occurs mainly in heterozygotes, where it comprises approximately 10% of the total non-alpha-chains, the dominant non-alpha-chain being the normal beta (approximately 90%). Furthermore, Hemoglobin Lepore-Boston-Washington has an average molecular mass (15,865.23 Da) that is only 2 Da lower than that of the normal beta-chain (15,867.24 Da). Consequently, it cannot be resolved from the normal beta-chain by mass spectrometry. Here we show how Hemoglobin Lepore-Boston-Washington was identified directly from the diluted blood of a heterozygote by analyzing the product ions from the Lepore-Boston-Washington and normal beta-chain ions without prior separation of the individual chains. This study shows the potential of the tandem mass spectrometry for identifying a minor component in an unseparated mixture of proteins.

The emerging understanding of sickle cell disease.

The emerging understanding of sickle cell disease.

Characterization of Hemoglobin Lepore Variants by Advanced Mass-Spectrometric Procedures

We describe an analytical protocol for characterizing the molecular structure of hemoglobin (Hb) Lepore variants by using two different mass-spectrometric approaches. The first method consists of direct examination of the chromatographically separated hybrid globins by electro-spray mass spectrometry; the variant Lepore globin is identified through the accurate determination of its molecular mass. Alternatively, the anomalous globins are digested with trypsin and their structures are determined by fast atom bombardment mass-spectrometric analysis of the peptide mixture. The application of this procedure to the identification of Hb Lepore Boston and Hb Lepore Baltimore is described.

Diagnóstico da hemoglobinopatia de Lepore por análise do DNA.

We illustrate the application of recombinant DNA methods (namely Southern blotting) for the genotype diagnosis of haemoglobin Lepore Boston: the use of the restriction endonucleases PstI and XbaI along with a beta globin gene specific probe make it possible to detect a deletion of approximately 7kb which typically characterizes the delta beta Boston gene. The impact of using such methods in the prenatal diagnosis of major haemoglobinopathies is discussed.

Molecular Characterization of Italian Chromosomes Carrying the Lepore Boston Gene

Hemoglobin Lepore Boston is characterized by abnormal non-alpha-chains that are the product of a fusion delta beta gene originated from an unequal crossing over between misaligned delta and beta genes. The investigation of the restriction fragment length polymorphisms (RFLP) of the beta-globin gene cluster in 18 Italian Lepore Boston chromosomes indicates that the hybrid gene is linked to two RFLP patterns. The majority of chromosomes show a pattern which corresponds to haplotype V and a minority to haplotype I according to Orkin's classification. A single Hb Lepore Boston homozygote was homozygous for haplotype V. The two haplotypes differ only for a single site 3' to the beta cluster. Our data allow the speculation that in Italy the Lepore Boston gene might be the result of multiple recombination events.

The delta beta crossover region in Lepore boston hemoglobinopathy is restricted to a 59 base pairs region around the 5' splice junction of the large globin gene intervening sequence

Genomic DNA from a hemoglobin (Hb) Lepore Boston (delta 87 Gln beta 116 His) homozygote of Southern Italian origin has been studied in order to map the fusion point between the delta and beta genes. An Ava II restriction endonuclease recognition sequence, located 12 base pairs (bp) downstream from the 5' end of the beta gene large intervening sequence, has been taken as marker of the beta-like portion of the fusion gene. This site was present even in the delta beta gene, allowing the localization of the crossover area to a 59-bp region extending from the first nucleotide of the Leu codon in position 88 to the 11th nucleotide of the large intervening sequence. The analysis of the DNA restriction polymorphisms in the gamma delta beta globin gene region provides evidence that a single mutational event originated the Lepore delta beta genes, at least in the Italian population.

Sickle Cell-Hb LeporeBoston Syndrome

Two siblings assumed on the basis of clinical and hematological evidence to have homozygous sickle cell (SS) disease were found to have a mother without sickle hemoglobin. Subsequent investigation and hemoglobin structural studies indicated the diagnosis to be sickle cell-Hb Lepore Boston syndrome. This syndrome generally manifests clinically significant sickle cell disease, and this genotype should be borne in mind in apparent SS disease where a parent without sickle hemoglobin in discovered.

Localization of the site of recombination in formation of the Lepore Boston globin gene.

The site of crossover between the delta- and beta-globin gene sequences resulting in Lepore Boston globin gene formation has been localized at the DNA Level. Probes specific for detecting the intervening sequences (IVS) of the delta- and beta-globin genes were used to map the origin of cellular DNA fragments of a patient homozygous for hemoglobin Lepore Boston. Restriction endonuclease analysis and hybridization of this DNA to IVS specific probes show that most, if not all, of the large intervening sequences (IVS 2) in Lepore DNA are derived from the beta-globin gene IVS 2. In addition, the crossover occurs in a region of DNA in which the delta- and beta-genes have almost complete nucleotide homology, and might be expected to pair most tightly during meiosis.

Comparative studies of Hb Lepore Boston, Hb A2, and Hb A.

Several functional tests were performed to compare Hb A, Hb A2, and Hb Lepore Boston, which has a delta-beta crossover in the region of residues 87 to 116. Oxygen equilibrium curves determined by an automatic apparatus in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees showed that the p50 was 5.8 mm Hg for Hb Lepore Boston, in contrast to 8.1 and 10.3 mm Hg for Hb A2 and Hb A, respectively. The n values (Hill coefficinets) of Hb Lepore Boston and Hb A2 were slightly smaller than that of Hb A. The effect of 2,3-diphosphoglycerate and inositolhexophosphate on the p50 of Hb Lepore Boston and Hb A2 was less than that on the p50 of Hb A. The molecular stability to mechanical shaking of Hb Lepore Boston and Hb A2 showed that the oxy forms of Hb Lepore Boston and Hb A2 denatured at a rate 3 times faster than that of Hb A. MetHb Lepore Boston was more unstable than MetHb A2 to mechanical shaking. These results indicate that, although the molecular stability of Hb Lepore Boston is more similar to that of Hb A2 than that of Hb A, the oxygen-binding properties of Hb Lepore differ from both Hb A and Hb A2.

A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1

Abstract Lepore hemoglobins result from crossovers between normal beta and delta chain genes. Structural investigation of two newly discovered examples of Lepore hemoglobins revealed one of them to be structurally identical to hemoglobin Lepore Hollandia α 2 A δ 22 -x- β 50 , a rarely occurring Lepore variant, while the second had the structure of hemoglobin Lepore Boston α 2 A δ 87 -x- β 116 . Studies of the equilibrium and kinetic properties of the liganding reactions of these two Lepore hemoglobins, which differ only in three amino acid residues, and comparison of these with the known properties of hemoglobin A 1 ( α 2 β 2 ) and hemoglobin A 2 ( α 2 δ 2 ) have been carried out. A high value of n , the Hill coefficient, indicating normal heme-heme interaction, was observed in each hemoglobin along with a normal Bohr effect. However, a slight but definite increase in oxygen affinity was observed for each Lepore hemoglobin. Furthermore, kinetic studies indicated a slight but consistently increased rate of ligand combination and a somewhat decreased rate of oxygen dissociation for hemoglobins Lepore Hollandia and Lepore Boston at pH 7 and 20 °C. Apparently, the higher oxygen affinity of these Lepore hemoglobins over those of the normal hemoglobins A 1 and A 2 reflects changes of sequence that are common to both types of hemoglobin Lepore.

Haemoglobin Lepore Boston and elliptocytosis in a family of Indonesian-German ancestry.

A family is presented in which Hb Lepore Boston was found in six individuals over three generations. The gene must have had its origin either in Java (Indonesia) or in what is now the Federal Republic of Germany. The haemoglobin was characterized by amino-acid analysis of the six tryptic peptides that have a different composition in the beta- and the delta-chain. The ratio of glycine to alanine in position 136 of the fetal haemoglobin, which was somewhat raised in the Hb Lepore carriers, averaged 31:39. In addition an elliptocytosis gene was found, which was inherited independently from Hb Lepore; the simultaneous presence of elliptocytosis in three family members did not seem to aggravate the mild anaemia caused by Hb Lepore.

Synthesis of haemoglobin Lepore.

HAEMOGLOBIN Lepore is a class of haemoglobin variants composed of α-chains and hybrids of δ and β-chains, which can be designated as α2(δβ)2. The N-terminal amino-acid sequence of the hybrid chains corresponds to that of δ-chains, while the C-terminal sequence corresponds to that of β-chains. A misplaced synapse of the δ and β gene loci is thought to have occurred during meiosis, followed by a non-homologous cross-over1. Three types are known which differ in the position of the cross-over: Hb Lepore Hollandia2,3, Hb Lepore Baltimore4, and Hb Lepore Boston (Washington)5,6.

An unstable haemoglobin with reduced oxygen affinity: haemoglobin Peterborough, 3 (GI3) Valine lead to Phenylalanine, its interaction with normal haemoglobin and with haemoglobin Lepore.

Summary: A new unstable haemoglobin, Hb Peterborough, is described in father and son. In the former it was found with Hb A but in the latter with Hb Lepore, the mother being a heterozygote for Hb A and Hb Lepore Boston (or Washington). In this respect it differs from other unstable haemoglobins so far described which have been observed in heterozygotes together with normal adult haemoglobin. It was therefore possible to compare the clinical and biochemical interactions of Hbs A and Peterborough, A and Lepore, and Peterborough and Lepore.Hb Peterborough has been identified as βiii (G‐13) Val → Phe. It has a low oxygen affinity, but the Bohr effect and haem‐haem interaction appear to be normal.

Human abnormal hemoglobins. 11. A chemical study of hemoglobin Lepore from a homozygote individual.

The hemoglobins of a child homozygous for the Lepore Boston gene have been analyzed. Only hemoglobin F and Lepore were observed. Hemoglobin Lepore was isolated and the peptide chains of this hemoglobin were separated. The amino acid composition of the Lepore chain and of the tryptic peptides obtained from this chain was determined. The peptides isolated accounted for the amino acid composition of the Lepore chain and indicated that this chain is 146 amino acid long. The switch region from the δ-like sequence of the Lepore chain to the β-like sequence has been localized between residue 87 and residue 116.

Abnormal human hemoglobins X. A study of hemoglobin lepore Boston

An abnormal hemoglobin has been isolated from two related individuals of Italian extraction. This abnormal hemoglobin has been identified with hemoglobin Lepore Boston on the basis of the fingerprinting pattern. Chemical studies have been performed on the “core” of this hemoglobin Lepore. Tryptic digestion after carboxy-methylation has been found to be the most suitable procedure for analyzing the “core” of the non-α chains. The fingerprinting analysis of the carboxymethylated hemoglobin has given sufficient information on the structure of this hemoglobin to pinpoint accurately the region along the amino acid sequence of the abnormal peptide chains, where the δ-like portion of these chains is joined to the β-like portion. Evidence has been obtained to show that the abnormal peptide chains of hemoglobin Lepore are equal in length to either β- or δ-chains (146 amino acids).