Search

The adult beta-globin gene of Ammotragus lervia (Barbary sheep or aoudad) has been sequenced completely, including 185 base pairs of 5' flanking region and 168 base pairs 3' to the stop codon, and compared with that of other caprines such as goat (Capra hircus), wild Corsico-Sardinian (Ovis aries musimon) and Cypriot (O. a. ophion) mouflons, and domestic sheep (Ovis aries). The gene was identified as being located on a triplicated four-gene set cluster containing the HBBA locus (A-haplotype) as is the case of goat, sheep of the Hb A type and Corsico-Sardinian mouflon. Phylogenetic analyses support the evidence that caprines share a common ancestor that probably carried the A-haplotype and that a more recent deletion of a gene set gave rise to the duplicated cluster containing the HBBB locus (B-haplotype) found in sheep of the Hb B type and Cypriot mouflon, which evolved independently. Data also suggests that the Ammotragus beta-globin gene is older than genes of the examined caprines and indicates it followed an independent evolution after separating from species having the same HBBA locus. Similarly, phylogenetic analyses of beta-globin chain sequences suggest a different evolution for globins coded by the HBBA locus with respect to the HBBB. Ammotragus beta-globin chain shows all the amino acids responsible for the low oxygen affinity of ruminant Hbs. Investigations on the oxygen transport properties indicate that the intrinsic oxygen affinity of aoudad Hb is higher than the Hb B of the domestic sheep and, at the same time, more similar to that of other A type Hbs, whereas in the presence of the Cl(-) effector the oxygen affinity is approximately the same as that of the other species.

A clinical, haematological, biochemical and molecular study was carried out in 17 patients affected with thalassaemia intermedia, who were compound heterozygotes for the beta-thalassaemia mutation beta-87 C-->G to determine the genetic basis of their clinical heterogeneity. The beta-87 was found associated with haplotype VIII (beta-87/VIII) or V (beta-87/V). The 10 patients with the beta-87/VIII showed milder clinical conditions, with significantly higher levels of haemoglobin (Hb) (9.8 +/- 1.1 g/dl vs. 8.5 +/- 1.3 g/dl) and fetal haemoglobin (Hb F) (6.2 +/- 1.5 g/dl vs. 2.6 +/- 1.5 g/dl; P = 0.0034) and higher synthesis of (G)gamma ((G)gamma/(Total)gamma 69.4 +/- 2.6% vs. 42.8 +/- 16.2%; P = 0.0042) than the seven patients with the beta-87/V. The beta-87/VIII showed a configuration of rare polymorphisms in the 5' sub-haplotype, which have been reported to exert an increasing effect on Hb F. They were "T"-158 (G)gamma-globin gene, T-A-G in pre-(G)gamma framework, (TG)(11)(CG)(3) in the (G)gamma-IVS2, (AT)(9)N(12)(AT)(10) in LCR-HS2; in contrast, the haplotype V had, respectively, "C", T-G-A (TG)(19)(CG)(2)CACG in the (G)gamma-IVS2, and (AT)(10)N(12)(AT)(11). In all patients the beta-87 was associated with the (AT)(9)T(5) motif 5' beta-globin gene with increased affinity for the BP-1 protein, and with the (TG)(13) in the (A)gamma-IVS2. The high increase of the Hb F, mostly of the (G)gamma-type, strongly suggests the hypothesis that the 'T'-158 (G)gamma plays a principal role and that the other polymorphisms could exert a cooperative role in the modulation of Hb F in patients with erythropoietic stress.

Sequencing of alpha-globin genes of 18 Sardinian heterozygotes for the Hb G-Philadelphia [alpha 68(E17)Asn-->Lys] variant, with four active alpha genes and circulating level of the variant of about 27%, showed the AAC-->AAG change at codon 68 of the alpha 2-globin gene (alpha(G)alpha/alpha alpha). Two heterozygotes with level of about 37% were the carriers of the same mutation on the same alpha 2 gene, and of the alpha 2 alpha 1 hybrid gene, because of the 3.7-kb deletion, in trans (alpha(G)alpha/-alpha(3.7)). In Black people, the same C-->G mutation occurs on the hybrid gene (-alpha(G)3.7), whereas in Caucasians the Lys for Asn change is because of the C-->A transversion occurring on the alpha 2 gene of a normal alpha alpha arrangement. The identification of the C-->G mutation on the normal alpha alpha chromosome points to an undescribed genotype for this rather common variant, which is probably because of the high rate of recombination between the duplicated alpha-globin genes.

The abnormal Hb F-Porto Torres [Agamma75(E19)Ile-->Thr, 136(H14)Ala-->Ser] was observed during a cord blood survey for hemoglobinopathies in North Sardinia. This silent variant showed the same mobility as Hb F-Sardinia in isoelectric focusing (IEF) of the tetramers, whereas the abnormal globin chain was clearly separated by acid-urea-Triton polyacrylamide gel electrophoresis (AUT-PAGE) from the normal Ggamma- and Agamma-globin chains. Separation of the globin chains by reversed phase high performance liquid chromatography (HPLC) indicated the following percentages: Ggamma 68.4, Agamma 14.0, Xgamma 17.6, that strongly suggested the abnormal chain as being a variant of the Agamma-globin. Sequencing of the gamma-globin genes indicated that the mutated gene was in fact an Agamma with two nucleotide replacements, one being the ATA-->ACA (Ile-->Thr) at codon 75 (the so-called AgammaT of the rather common Hb F-Sardinia) and the second the GCA-->TCA (Ala-->Ser) at codon 136. This new variant is the seventh having the sequence of the AgammaT chain with an additional mutation so far described and the third characterized by gene sequencing.

Four novel alleles of the adult beta-globin gene of Capra hircus were observed in an extended study on hemoglobin polymorphism in goat breeds living in the island of Sardinia Nucleotide sequencing showed that one of these alleles is due to a 2 bp substitution at codon 125 (CTG --> GAG, Leu --> Glu). Two substitutions, the silent CTT --> CTC for Leu at codon 78 and the conservative AAG --> AGG (Lys --> Arg) at codon 104, are shared by the other three alleles, two of them having additional mutations, which suggests a common origin. The allele we provisionally called the beta(Y) shares four out of five amino acid substitutions, together with the same polymorphisms in the IVSII, we observed previously in the rather common beta(E) gene. This evidence allowed the origin of the beta(E) gene to be better characterized. The data increase to seven the number of alleles at the goat beta(A)-globin locus characterized thus far at the molecular level. A simplified nomenclaturefor the increasing number of goat beta-globin alleles is presented.

The cord blood sample of a Caucasian newborn contained about 40% of an abnormal fetal hemoglobin. The mutated gamma chain was isolated using reversed phase high performance liquid chromatography and characterized by means of electrospray and fast atom bombardment mass spectrometric techniques as a G gamma-globin variant with an Ile-->Thr substitution at position gamma 75. The variant chain shows the same structure as the previously described Hb F-Charlotte that was demonstrated to be an A gamma variant with an Ile-->Thr substitution at position gamma 75 and an additional Ala-->Gly substitution at gamma 136.

The beta zero-thalassemia codon 39 nonsense mutation predominant in Sardinia is severe, and homozygotes are transfusion dependent. Two-thirds of beta zero 39 alleles are linked to A gamma T (haplotype II). One-fourth are linked to A gamma I (haplotypes I and IX), as is the mild beta +-thalassemia -87 C-->G mutation (haplotype VIII). beta +/beta zero-Thalassemia VIII/II compound heterozygotes have significantly higher A gamma I:A gamma T (23:7) than beta zero-thalassemia I/II (24:20) or IX/II (16:17) cases. This suggests that the beta + -87 mutation is associated with elevated gamma expression in cis, which may contribute to the lack of transfusion-dependence in beta +/beta zero cases.

1. In sheep alpha alpha 113His and alpha D alpha alpha 113His globin gene haplotypes, the percentage gene efficiencies, from the 5' to the 3' end, are about 32-18 and 30-14-6, respectively. 2. We previously found that in a alpha alpha alpha alpha homozygote there may be a 1-2% alpha 113His chains, which however were difficult to resolve from alpha 113Leu chains by CMC chromatography. 3. We report here the experimental conditions which allowed a neat resolution of the 1% alpha 113His chain peak by RP-HPLC. 4. This finding strongly suggests the occurrence of the alpha D alpha alpha alpha 113His haplotype where the percentage gene efficiencies are 30-(14-5)-1, supporting the existence of a gradient of decreasing expression in multiple alpha-globin gene haplotypes.

1. The amino acid substitution which characterizes the haemoglobin I variant from sheep has been ascertained using a combination of Fast Atom Bombardment mass spectrometry and protein sequencing. 2. A Ser for Gly substitution at position 13 (10 of the A helix) was found in a polypeptide with the overall sequence of the beta B globin. 3. On the basis of the nucleotide sequence of the beta B-globin gene, a C to T transition occurring on a CpG doublet is considered to be responsible for the amino acid substitution. 4. This represents the first observation of a variant sheep Hb due to a mutation which is rather common in the human genome. 5. Amongst ruminants, serine is normally present at position 13 of goat and sheep epsilon II and gamma chains and of bovine gamma chain which had an independent and more ancient evolutionary origin than the beta chains.

Isoelectric focusing in the ultranarrow immobilized (7.1-7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different II alpha-globin chains were separated from two different I alpha-chains by HPLC. Two alpha-chains (I alpha 1 and II alpha 3), and Hbs with similar mobilities (Hb1 and Hb3), were associated with the AA Hb phenotype: two alpha-chains (I alpha 2 and II alpha 4), and Hbs with different mobilities (Hb2 and Hb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two alpha loci. An allele at the beta locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.

Haemoglobin (Hb) phenotypes have been studied in 100 wild European mouflons living on the island of Sardinia by means of isoelectric focusing (pH 6.7-7.7 range) of the native tetramers, acid-urea-Triton gel-electrophoresis, and reversed-phase HPLC of globin chains. The result indicates the presence of two beta-globin alleles one of which, corresponding to the beta B, being the most common (f = 0.94). None were carriers of the earlier described Hb A. The new Hb was provisionally named Hb M. Severely anaemic mouflons were able to synthesize Hb C at expense of the Hb B alone, thus suggesting structural and physiological homologies between mouflon beta B and sheep beta A globin genes, and between the newly observed beta M allele and the beta B of the domestic Sardinian sheep.

1. Hemoglobin (Hb) of the river buffalo (Bubalus bubalis L.) was studied by employing isoelectric focusing (IEF) in the 6.7-7.7 pH range and by IEF in ultra-narrow immobilized pH gradient (IPG) 7.1-7.5. 2. Three Hb BB phenotypes were identified which were characterized by sets of two or four Hbs with different isoelectric points. 3. These type were called BB, BsBs and BBs, the Bs phenotype showing Hbs with slightly slower mobility. 4. Analysis of constituent globin chains by acid urea polyacrylamide gel electrophoresis in the presence of Triton X-100, provided clear evidence of a novel polymorphism at the beta-globin level. 5. Titration curves of beta-globins from heterozygous Hb BBs indicated a single curve thus suggesting the absence of a net charge in all the pH field. 6. A neutral-to-neutral amino acid replacement probably differentiates the two beta-globins.

We report here the identification of a 4 bp deletion in the A gamma T globin gene promoter by means of Fnu4HI digestion of DNA amplified by polymerase chain reaction (PCR). This deletion has been previously associated with haplotype II beta-thalassemia in Sardinia. This simple, non-radioactive procedure should facilitate the screening of various populations of normal and beta-thalassemic subjects for this specific genetic alteration.

The successful extension of the reversed-phase HPLC methodology to the study of non-human globin chains is shown. A large-pore C4 column, and simple acetonitrile in aqueous TFA gradients, were used to completely separate and quantitate globin chains from caprines, swines, rodents, and birds. Results indicate a large hemoglobin polymorphism in some species.

This article summarizes experience and data obtained using a previously developed reverse-phase high-performance liquid chromatography method (J.B. Shelton, J.R. Shelton and W.A. Schroeder, J. Liq. Chromatogr. 7 (1984) 1969) in the study of a number of hemoglobinopathies in the Sardinian population. The occurrence and incidence of several abnormal hemoglobins are described, as well as aspects of the expression of abnormal gamma-globin gene arrangements and thalassemic genes.

The hemoglobin of 2048 newborn babies from Sardinia was analyzed by isoelectric focusing and polyacrylamide gel electrophoresis in order to determine the level of Hb F-Sardinia (with A gamma T) and the G gamma chain. Hb F-Sardinia values of 15.5 +/- 2.6% were present in the A gamma T heterozygote whereas 30.7 +/- 5.2% were present in the homozygote. The A gamma T gene frequency was 0.17. Most of the babies tested showed the normal G gamma level either in the absence of the A gamma T anomaly (69.6 +/- 4.1%), or in the presence of the anomaly in both the heterozygous state (70.9 +/- 4.8%) and the homozygous state (71.1 +/- 3.4%). Similar values were shown in nine homozygotes for beta-thalassemia discovered during the screening. Nine newborn babies (0.44%) showed particularly low G gamma levels (38.3 +/- 6.8%) whereas 18 newborn babies showed high G gamma levels (83.9 +/- 2.6%). The frequencies of the anomalies (0.0022 for the low G gamma and 0.0044 for the high G gamma) were the lowest observed in Caucasian and other ethnic groups. Data suggest the presence of abnormal gamma globin gene arrangements in the Sardinian population.

An extended survey of 8,103 Sardinian newborns has been conducted for the study of abnormal gamma-globin gene arrangements. Fetal hemoglobin analysis and globin gene mapping have identified five different arrangements in 24 babies: five (21%) were carriers of the -GA gamma- hybrid (thalassemic) gene, directing the synthesis of the A gamma chain and resulting in low (41%) G gamma chain and decreased Hb F levels; two (8%) carried the -A gamma-A gamma T- duplication, characterized by even lower (37%) G gamma levels; five (21%) carried the -G gamma-AG gamma-A gamma- triplication; one (4%) carried the -G gamma-G gamma-A gamma- triplication, and 11 (46%) the -G gamma-G gamma- duplication, all resulting in high G gamma levels (85, 83, and 88%, respectively). Thirty-six additional babies could be phenotypically classified as carriers of the same mutations. Haplotyping and gamma-chain composition showed that the crossover event generating the -GA gamma- hybrid gene and the corresponding -G gamma-AG gamma-A gamma- triplication has occurred on at least three different chromosomes. The -G gamma-G gamma- duplication was associated with the chromosome having haplotype I, and the -A gamma-A gamma T- with the chromosome of haplotype II. As many as 2,260 babies (28%) were heterozygous, and 254 (3%) homozygous for the A gamma T mutation. The incidence and relative distribution of these anomalies in Sardinia are different when compared with those of other ethnic groups.

12 thalassaemic patients from Northern Sardinia showing the beta + phenotype were examined by isoelectric focusing and high-performance liquid chromatography techniques for the determination of the variant A gamma T globin chain of the foetal haemoglobin. Two patients (16.7%) were homozygotes for the A gamma T gene variant, 2 (16.7%) were heterozygotes and 8 (66.7%) were homozygotes for the normal A gamma I allele. The A gamma T gene frequency was 0.183, much lower than the observed 0.823 in beta zero homozygosity. These data suggest the presence of at least 2 beta +-thalassaemic chromosomes in Sardinians, one associated with the variant A gamma T allele and one associated with the normal A gamma I. The latter is prevalent among adult patients showing the intermediate form of the thalassaemic disease, which is not transfusion-dependent.

Fetal hemoglobin analysis and globin gene mapping have identified one type of beta(0)-thalassemia and four different gamma globin gene arrangements among newborn babies from the northern part of Sardinia. The beta(0)-thalassemia with a nonsense mutation at codon 39 was found on two chromosomes, each with a distinct pattern of polymorphic restriction sites; one had the A gamma T (A gamma 75 Ile----Thr) mutation, while the second did not. Four closely related haplotypes were identified for chromosomes with the A gamma T mutation. The gamma-thalassemia heterozygosity with the -GA gamma- hybrid gene fell into two categories. One apparently originated through crossing-over between mismatched chromosomes characterized by the most common haplotype, while the other had polymorphisms resembling those of a less frequently occurring chromosome. Chromosomes with the -G gamma-AG gamma-A gamma- triplication had polymorphic sites to be expected for this condition, being complimentary to the -GA gamma- thalassemias. Of the two additional gamma globin gene variations the -G gamma- G gamma- arrangement was associated with the chromosome with the most commonly occurring haplotype, while the chromosome with the -A gamma-A gamma- arrangement had a haplotype characteristic for that with the A gamma T mutation, which identified an -A gamma-A gamma T- arrangement. The incidental discovery of a silent beta-chain mutant, Hb Hamilton, with the Val----Ile substitution at position beta 11, in five newborns was also reported.

Globin gene mapping analyses of DNA from numerous Black babies, and from newborns from Sardinia, Sicily, Turkey, and Spain have identified the following: A high incidence of alpha-thalassemia-2 heterozygotes among Black babies with less than 1% Hb Bart's at birth and a high incidence of alpha-thalassemia-2 among Sardinians, but not among Sicilian, Turkish, and Spanish babies. A relatively high incidence of zeta-thalassemia was present among Black babies only, while triplicated zeta was seen in four of the five populations. Two Black babies were each found to have a different theta 1 deletion; two Sardinian babies had a newly discovered approximately 2.5 kb deletion between zeta and psi zeta; four babies had the rare Bgl II polymorphism between psi zeta and psi alpha; and one Black baby lacked the Eco RI site 3' to zeta. Quantitation of the zeta chain by reversed phase high performance liquid chromatography showed that two-thirds of the babies with four alpha genes (alpha alpha/alpha alpha) had levels between 0.1 and 1.0%, while nearly 90% of the babies with -alpha/alpha alpha had similar levels (averaging 0.2% for alpha alpha/alpha alpha; 0.35% for -alpha/alpha alpha; 0.75% for -alpha/-alpha). Additional data indicated that the occurrence and level of zeta are related to the level of beta, i.e. the gestational age. The presence of a zeta triplication did not affect the level of zeta in cord blood. The extensive search for gamma-globin gene anomalies resulted in the discovery of a chromosome with five gamma genes. gamma-Thalassemia was rare in all populations, while the -G gamma-G gamma- gene arrangement was mainly observed among Black babies; this arrangement is primarily responsible for high G gamma levels in cord blood samples. The strong correlation between the presence or absence of a C----T mutation at position -158 (measured in Xmn I digests) and the level of G gamma was confirmed for adult blood samples. A search for possible anomalies in the -delta-beta- region through gene mapping with Eco RV gave negative results except for the discovery of a polymorphic site 5' to delta in one of the 371 Black babies tested.