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During a screening for hemoglobinopathies, we found a carrier of the Sardinian δβ-thalassemia condition. The proband's hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α2Aγ2 and α2Gγ2 instead of nearly 100% α2Aγ2. In order to explain the unusual γ-chain ratio, sequencing of the Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the Gγ promoter. Results indicated that the observed in vivo Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

Here we describe Hb F-Avellino [(G)γ41(C7)Phe → Leu; HBG2: c.124 T > C], a new hemoglobin (Hb) variant observed in a healthy newborn. The proband's hemolysate was found to be mildly unstable by the isopropanol test. The occurrence of the variant was assessed by both chromatographic and electrophoretic methods. DNA sequencing analysis of the (G)γ gene showed a T to C transition at codon 41 (TTC > CTC) corresponding to the Phe → Leu substitution. Normal functional properties have been hypothesized.

The molecular basis of β-thalassemia (β-thal) mutations in North and in South Vietnam have been described during the past 15 years, whereas limited data were available concerning the central area of the country. In this study, we describe the molecular characterization and frequency of β-globin gene mutations in the Thua Thien Hue Province of Central Vietnam as the result of a first survey conducted in 22 transfusion-dependent patients, and four unrelated heterozygotes. Nine different known mutations were identified (seven of the β

A new variant of the fetal hemoglobin (Hb) was observed in a newborn baby subjected to phototherapy due to jaundice, by means of electrophoretic and chromatographic techniques. The variant Hb resulted unstable by the isopropanol stability test. After HBG2 gene sequencing, the G to A transversion at codon 64, position eight of the E helix, was found, which corresponds to the Asp for Gly amino acid substitution. The new variant was called Hb F-Turritana [(G) γ64(E8)Gly→Asp, HBG2:c.194G>A]. Incoming aspartic acid residue, bulky and negatively charged, may be responsible for alteration of the heme pocket steric configuration and for instability. The new abnormal HBG2 gene was found to be associated in cis with the mutated HBG1 gene, which characterizes the Hb F-Sardinia [(A) γ (E19)Ile→Thr, HBG1:c.227T>C] variant.

We report a new hemoglobin (Hb) variant on the HBA2 gene, Hb Zara [α91(FG3)Leu→Ile (α2); HBA2: c.274C > A], which was found in a Caucasian man from Croatia. It was observed by routine cation exchange chromatography as an abnormal 21.8% fraction overlapping Hb A2, and associated with normal hematology. It was slightly unstable by the standard isopropanol precipitation test. DNA analysis revealed the CTT > ATT mutation at codon 91 on an α2 gene of a normal α-globin gene arrangement. This new variant represents the sixth described mutation at codon α91 and fourth on the α2 locus. As a result of the slight instability due to the significant role of the α91 residue in the α1β2 contact, the level of the Hb Zara variant was lower than levels observed for several stable variants codified by the α2 locus.

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation.

Two healthy newborns, heterozygous for two different γ-globin chain mutations, were observed during an electrophoretic screening for hemoglobinopathies in Sassari, North Sardinia (Italy). The variants were characterized by reversed phase high performance liquid chromatography (HPLC) and sequencing of amplified γ-globin genes. One of the two abnormalities was a novel Aγchain variant and the tetramer was named Hb F-Osilo [Aγ119(GH2)Gly→Ser]. The other was a Gγ chain variant, Hb F-Paulinia [Gγ80(EF4)Asp→Tyr], already described in a Brazilian baby of African ancestry. No functional studies could be performed.

In order to investigate the polymorphism of ?-globin chain of hemoglobin amongst caprines, the linked (I)? and (II)? globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5? and 3? untranslated regions. European and Cyprus mouflons, which do not show polymorphic ? globin chains, had almost identical ? globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different ? genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the (I)?-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.

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.

In this report we describe the complete sequence of the mtDNA genome of Ammotragus lervia (Barbary sheep or aoudad) as obtained by PCR and sequencing with primer walking using flanking sequences. The molecule is 16,530 base pairs in length, resulting similar to those of goat and sheep. The genome organization matches to those of other mammalian mitochondrial genomes. The phylogenetic position of the Ammotragus relative to twelve other mammalian species was assessed based on protein-coding sequences. A goat-aoudad split of 9.9–8.9 MYBP has been estimated, whereas the divergence ancestor/caprines was dated at 12.1–10.8 MYBP.

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (alpha(2)(G)gamma(2) and alpha(2)(A)gamma(2)) that differ only at position 136 reflecting the products of two nonallelic gamma-globin genes. At the time of birth, HbF accounts for approximately 70% of the total Hb. The (G)gamma:(A)gamma globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a gamma- to beta-globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to (G)gamma and (A)gamma chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as gamma-thalassemias, duplications, triplications, and even sextuplications of the gamma-globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the beta-globin gene cluster, such as beta-thalassemia, sickle cell disease, deltabeta-thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of gamma-globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching.

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.

Four novel alleles of the adult β-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 ( $$\underline {CT} \to \underline {GA} G,$$ G, "Leu→Glu). Two substitutions, the silent CT $$\underline T \to CT\underline C $$ for Leu at codon 78 and the conservative A $$\underline A G \to A\underline G $$ G (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 β Y shares four out of five amino acid substitutions, together with the same polymorphisms in the IVSII, we observed previously in the rather common β E gene. This evidence allowed the origin of the β E gene to be better characterized. The data increase to seven the number of alleles at the goat β A -globin locus characterized thus far at the molecular level. A simplified nomenclature for the increasing number of goat β-globin alleles is presented.

Owing to the presence of several variants at the adult β -locus and at both α-globinloci, the Hb polymorphism in goat is remarkably complex. We recently confirmed this high polymorphism in a survey on some 400 goats from Sardinia, Egypt and Cyprus. Eight alleles at the β-adultlocus and two allelic genes at both α-globinloci have been detected by electrophoretic and Chromatographic techniques. Five of the β genes identified have been completely sequenced. Four of them produce globin chains already described in previous studies at the protein level, whereas the globin chain encoded by the remaining one was undescribed. Results at the coding and non-coding regions level provide a possible basis for further interpretations on the origin of these alleles.

In order to obtain molecular and biochemical data, which might help to elucidate aspects of evolution of Caprinae globin genes, we characterized the Hb phenotype ofAmmotragus lervia (Barbary sheep) and compared it withO. aries (of the A and B-haplotypes),O. g. musimon (of the B and M-haplotypes),O. g. ophion (Cyprus mouflon), andCapra hircus. DNA sequencing analysis on PCR amplified segments was used to define the adult β-globin gene of Cyprus mouflon and Hb B type sheep. The structure of the adult β-globin gene of Barbary sheep was partially determined for the first time indicating closer relationships with goat than with sheep.

Primary structure analysis of the four river buffalo alpha-globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two alpha-globin chains, Ialpha1 and IIalpha3, which differ at positions 129 and 131: Ialpha1 has 64 Ala, 129 Phe, 131 Asn; IIalpha3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two alpha-globin chains, Ialpha2 and IIalpha4, which differ at positions 10 and 11: Ialpha2 has 10 I1e, 11 Gln, 64 Asn; IIalpha4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic alpha-globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo alpha-globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.

The occurrence of several abnormal hemoglobins (Hbs) in the population of the island of Sardinia has been documented [[1]]. Because of continuing screening programs for hemoglobinopathies among new...

Haemoglobin (Hb) J-Sardegna [α50(CE8)His → Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 °C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 °C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the α1-β1 interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [α50(CE8)His → Arg] at the same position.

(2000). Characterization by DNA Sequencing of Hb F-Columbus-GA [Gγ94(FG1)Asp→Asn] Observed in Sardinian Newborn. Hemoglobin: Vol. 24, No. 1, pp. 53-57.

Hb F-Calabria [Ggamma118(GH1)Phe-->Leu] is a new fetal hemoglobin variant that was found during routine screening for abnormal hemoglobins in a newborn of Calabrian (Southern Italy) ancestry. The variant chain was identified (acid urea gel electrophoresis of dissociated globin chains in the presence of Triton X-100, and by reversed phase high performance liquid chromatography) as a slightly hydrophilic Ggamma chain. Sequencing of the polymerase chain reaction-amplified exon 3 of the Ggamma-globin gene demonstrated the TTC-->CTC mutation at codon 118 leading to the Phe-->Leu conservative substitution at position GH1. A molecular modeling study supports that the variant might not have clinical implications. This is the 40th example of a Ggamma chain variant.

(1998). Sheep Hb I Variant, or βB13(A10)Gly→Ser, in Breeds From Corsica: Detection by Gene Sequencing. Hemoglobin: Vol. 22, No. 5-6, pp. 525-528.

Different alleles at the two linked α-globin loci are present in sheep: Iα113Leu and IIα113Leu, Iα8Ala,113Leu, Iα15Asp,113Leu, and IIα113His. Triplicated α-gene haplotypes are also common. Six different β-globins (A, B, E, G, H, and I) contribute to the Hb polymorphism. A comparative study on the biochemical polymorphism of α- and β-globins in pure sheep breeds of the Mediterranean islands of Chios, Cyprus, and Sardinia is described. Cyprus and Sardinia breeds have the lower frequency of α-globin variants. Chios sheep possess the high 0.40 frequency of the Iα8Ala,113Leu-IIα113His arrangement. No α-gene triplications were observed amongst Chios and Cyprus breeds. None showed the Iα15Asp,113Leu variant. The βB allele largely predominates; the frequency of the βA was lower (f = 0.03) amongst Sarda sheep that also possess the βI variant. A novel βB-globin variant was observed in the Chios breed. Both natural and artificial selection may be the cause for these differences.

The -117(G-->A)A gamma hereditary persistence of fetal hemoglobin (Greek HPFH) and beta zero 39-thal mutations are rather frequent in Sardinia so that their interaction is to be expected. Characterization of eight compound heterozygotes for these defects indicated that HPFH was linked to haplotype VII and beta zero 39-thal to haplotype II. Haplotype II beta zero 39-thal chromosome carries the A gamma T gene which is a useful marker of gamma-gene expression. Since the Hb F level in these compound heterozygotes was significantly higher than in 46 -117 HPFH carriers, the A gamma I, A gamma T, and G gamma globin level was determined. A gamma T was underexpressed while G gamma was significantly increased, which suggest that in -117 A gamma HPFH/beta zero 39-thal healthy subjects the increase in Hb F production is determined only by the -117 mutated A gamma gene and the adjacent G gamma gene.

Sheep are thought to have been one of the first livestock to be domesticated in the Near East, thus playing an important role in human history. The current whole mitochondrial genome phylogeny for the genus Ovis is based on: the five main domestic haplogroups occurring among sheep (O. aries), along with molecular data from two wild European mouflons, three urials, and one argali. With the aim to shed some further light on the phylogenetic relationship within this genus, the first complete mitochondrial genome sequence of a Cypriot mouflon (O. gmelini ophion) is here reported. Phylogenetic analyses were performed using a dataset of whole Ovis mitogenomes as well as D-loop sequences. The concatenated sequence of 28 mitochondrial genes of one Cypriot mouflon, and the D-loop sequence of three Cypriot mouflons were compared to sequences obtained from samples representatives of the five domestic sheep haplogroups along with samples of the extant wild and feral sheep. The sample included also individuals from the Mediterranean islands of Sardinia and Corsica hosting remnants of the first wave of domestication that likely went then back to feral life. The divergence time between branches in the phylogenetic tree has been calculated using seven different calibration points by means of Bayesian and Maximum Likelihood inferences. Results suggest that urial (O. vignei) and argali (O. ammon) diverged from domestic sheep about 0.89 and 1.11 million years ago (MYA), respectively; and dates the earliest radiation of domestic sheep common ancestor at around 0.3 MYA. Additionally, our data suggest that the rise of the modern sheep haplogroups happened in the span of time between six and 32 thousand years ago (KYA). A close phylogenetic relationship between the Cypriot and the Anatolian mouflon carrying the X haplotype was detected. The genetic distance between this group and the other ovine haplogroups supports the hypothesis that it may be a new haplogroup never described before. Furthermore, the updated phylogenetic tree presented in this study determines a finer classification of ovine species and may help to classify more accurately new mitogenomes within the established haplogroups so far identified.

The cord blood sample of a Caucasian newborn contained about 40% of an abnormal fetal hemoglobin. The mutated γ 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 Gγ-globin variant with an Ile->Thr substitution at position -γ75. The variant chain shows the same structure as the previously described Hb F-Charlotte that was demonstrated to be an Aγ variant with an Ile->Thr substitution at position γ75 and an additional Ala->Gly substitution at γ136.

A new silent hemoglobin variant, Hb Ozieri (alpha 71(E20)Ala-->Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the alpha-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the alpha-globin gene. This observation brings the number of variants due to a mutation in the alpha-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.

Background HbF-Monserrato-Sassari is a newly discovered abnormal fetal hemoglobin observed in an apparently normal newborn baby during a hemoglobinopathies survey at birth in North Sardinian population. Methods Electrophoretic analysis of the cord blood lysate evidenced for an abnormal tetramer due to a mutated fetal globin chain. Electrospray ionisation-mass spectrometry and gene sequencing were used to identify the mutation. Oxygen binding ability of the variant Hb was determined. Results Sequencing of the γ globin genes revealed the TGT → CGT transition at codon 93 in one of the two Gγ genes, which leads to the Arg for Cys amino acid replacement at position 9 of the F α-helix. The amino acid substitution was confirmed by mass spectrometric analysis of the globin chains. Since modifications or substitutions at position β93 are known to affect the arrangement of a salt bridge at the α1β2 sliding contacts that are crucial for subunit cooperativity, the functional properties of the variant were studied to evaluate the effect of the replacement at the same position in the γ globin chain. With respect to normal HbF, the variant showed a significant increase in oxygen affinity and a slight decrease of both Bohr effect and cooperativity. General significance Result indicates a key role of the Cys γ93 residue for subunit cooperativity in the T → R transition of the HbF tetramer. Substitutions at the F9 position of the Gγ globin may result in stabilization of the high affinity R-state of the Hb tetramer. Because of the loss of Cys γ93 residue, this variant is considered to be potentially compromised in nitric oxide transport.

1. 1. The hemoglobin (Hb) polymorphism of the Italian catfish (Ictalurus sp.) was investigated by means of isoelectric focusing of Hb and of electrophoresis of globin chains. 2. 2. Multiple chains, due to allelism and non-allelism at the level of both α- and β-globin loci, were observed in the AA and BB Hb phenotypes. 3. 3. Isolated Hb tetramers consisted of the usual doublets of α- and β-globin chains or of stable combinations of different α- and different β-chains (asymmetric tetramers). This evidence explains the notable Hb multiplicity of this species. 4. 4. Hypotheses are advanced on the possible number, and chromosomal organization, of globin genes of the A and B Hb haplotypes.

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.

1. Hemoglobin (Hb) switching in the perinatal life of wild mouflon (Ovis musimon) was characterized by the replacement of Hb F by 60% levels of Hb C, and subsequently of Hb C by Hb B. 2. The recently discovered Hb M variant was not replaced by Hb C; thus, Hb BM heterozygote newborns synthesized 30% Hb C at the expense of Hb B. 3. Hybrid B mouflon x B sheep synthesized only 5% Hb C at birth but were able to produce 30% Hb C in adult life following induced anemia. 4. Adult BB and BM mouflons, after the same extent of induced anemia, synthesized HB C levels similar to those produced at birth. The results indicate a mouflon beta-globin gene cluster arrangement similar to those of sheep and goat, the beta C gene having an intermediate expression. Results also suggest a selective disadvantage in hybrid animals.

A procedure for the determination of the gamma chain heterogeneity of adult human blood samples with low Hb F has been developed. It consists of the isoelectrofocusing of lysates at a rather high hemoglobin concentration, the isolation of the focused Hb F by elution from the gel, and the analysis of the isolated hemoglobin by reversed phase high performance liquid chromatography for the separation and quantitation of globin chains. Depending on the isoelectrofocusing apparatus, many samples with as little as 1% Hb F may be focused in a single run and the gamma chain composition of the recovered hemoglobin (enriched from 15% to 95% in Hb F) easily analyzed by high performance liquid chromatography. The same procedure might be used for the isolation of low level hemoglobin variants for structural studies.

The propositus was a newborn baby (#398) from the area of Sassari, North Sardinia. The Hb Lepore was detected by isoelectrofocusing (IEF) (14); the same abnormality was present in the mother (Fig. 1) but not in the father. Its initial identification was confirmed by cation exchange high performance liquid chromatography (HPLC) (15) with a PolyCat column.

A novel hemoglobin variant was observed in pure sheep ( Ovis aries ) breeds of the island of Chios (Greece), Egypt and Hungary. This silent variant was identified by gel electrophoresis and RP-HPLC of dissociated globin chains. Two Arg for Lys substitutions were detected, by means of MALDI TOF electrospray mass spectrometric analysis for the intact globins, at positions β66(E10) and β144(HC1) of a globin chain having the sequence of the β B chain. Sequencing of the β-globin gene confirmed the variant gene as being an allele of the HBBB locus having the AAG → AGG and the AAA → AGA mutations at codons 66 and 144, respectively, both corresponding to the Lys → Arg substitution. The intrinsic oxygen affinity of the variant Hb (log P 50 = 0.79 at pH 7.0) was found to be intermediate between that of the sheep Hb B (log P 50 = 0.92) and that of Cypriot mouflon ( O. a. ophion ) Hb (log P 50 = 0.53), the latter having only the Lys → Arg change at β144, whereas nearly no differences were observed in the presence of the Cl − physiological effector. Result supports the indication that Arg at β144 enhances the role of the ligand in decreasing oxygen affinity, this effect being partially counteracted when Arg is at β66. Data also shows that the Lys → Arg change at β66 is responsible for 1.49 fold reduction in the intrinsic oxygen affinity. This hitherto undescribed variant increases to seven the number of alleles at the sheep HBBB locus . Following the nomenclature used for human Hb variants, the new allele was termed as the Hb Chios or [β B 66(E10) Lys → Arg, 144(HC1)Lys → Arg], whereas the proposed genetic nomenclature of the locus is HBBK.

Hb F-Emirates [Ggamma59(E3)Lys --Glu] was first described in a newborn from the United Arab Emirates. Here we describe the occurrence of this variant in a family of Sardinian ancestry. Direct DNA sequencing analysis of the selectively amplified Ggamma gene shows that the AAA --GAA transition, corresponding to a Lys --Glu substitution, is responsible for this abnormal hemoglobin (Hb). Our observation indicates a multiple origin of the mutation. In order to facilitate future studies at the level of population genetics, the structure of the entire Ggamma gene that carries the mutation was assessed and compared with that of normal Ggamma genes.

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.

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.

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.

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr--Ser) and A2 (Pro--Gly) substitutions observed in the beta chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb.

The 13-globin gene cluster of the domestic sheep (Ovis aries) shows two common haplotypes: the A haplotype, which bears the adult sA allele (HBBA), and the B haplotype, which bears the adult sB allele (HBBB) [1, 2]. The chromosomal organization of the A haplotype was found to be similar to that present in goat (Capra hircus) since it shows the same triplication (5’ Er_Ea_NJsI sc_E[u_Erv_Jsrr_sn_£v_Evr_ NJs,rr-sF-3’) of an ancestral four gene set (E-E-NJs-s) [3]. This set is characterized by the presence of two embryonic genes (e), one pseudogene (NJs), and one adult gene (s). The expression of the adult gene varies during ontogenic development and under different physiological conditions. The sc, sA, and sF genes are, in fact, expressed during juvenile, adult and fetal life, respectively, and the sc gene expression is reactivable, at the expense of sA gene, under particular physiological or experimental conditions such as anemia and hypoxia or the administration of erythropoietin [4-6]. The B haplotype is considered to have diverged from the A haplotype, as the result of a recent deletion from a triplicated locus. In fact, due to the lack of the whole juvenile four-gene set containing the sc gene, it is duplicated (5’ er srr NJsr sB Errr-EIV yrsrI sF-3’) and sheep which are homozygous for the sB allele do not exhibit the sBia>scswitching

As in most vertebrates, the goat α-globin gene locus is duplicated though producing Iα and IIα globin chains that differ in three amino acid residues [[1]–[3]]. As the result of an unusual evolutionary history, however, a goat (β-globin gene cluster consists of 12 genes organized as a triplicated four-gene set located on chromosome 7: 5’-eI-eII-ψβX-βC-eIII-eIV-ψβY-βA-eV-eVI-ψβZ-βF-3’[[4]-[6]]. The βA-globin gene is expressed in adult animals, whereas βF and βC genes are expressed in the fetus and in juveniles up to 6 months of age, respectively [[7]]. During the first year of postnatal life, the synthesis of pre-adult βC-globin is supplanted by the synthesis of adult βA. The βC→βA switch is reversible and the reactivation of the βC synthesis, at the expense of βA, can be induced by anemia, hypoxia, or the administration of erythropoietin [[1],[8]]. Due to this unusual feature, which is also common in sheep hemoglobin (Hb) type A and in Sardinian mouflon [[5], [7], [9], [10]], the organization of globin genes, the nucleotide sequence, and the evolution of goat and sheep globin genes have been extensively studied [[1]–[2], [11]–[13]].

Biogeographia - vol. XXI - 2000 (Pubblicato il 30 giugno 2000) Biogeografia deI|’Anatolia The origin and extinction of the large endemic Pleistocene mammals of Cyprus ELEFTHERIOS I-IAD]ISTERKOTIS*, BRUNO MASALA**, DAVID S. REESE *** *Gzzme and Fzzunzl Service, Ministry of Interior — Nicosia (Cyprus) “Department of Physiological Bioc/aemiczzl and Cell Sciences, Faculty of Biological Sciences, University of S[l55ll7’i - Sizssrzri (Italy) ***Depimment of/lntbropology, Field Museum 0fNzztzmzl Histmy — Chicago (USA) Key words: Fossils, hippopotamus, elephant, Cyprus, Pleistocene, extinction, Mediterranean SUMMARY All fossil terrestrial mammal sites on the island of Cyprus are dated from the Late Pleistocene period and consist almost exclusively of the remains of two terrestrial mammals, pygmy hippopotamus (P/J/mmzrios nzizzz/ms) and pygmy elephant (Elep/ms cypriater). Two theories exist on the arrival of these species on Cyprus. The first is that they arrived by a land bridge. This took place during the Pliocene about five to six million years ago when the Mediterranean sea was sealed at both ends clue to tectonic movements and its water evaporated creating a land bridge. However, there are no fossils dating from the Pliocene The second theory is that they arrived under circumstances described by the Island Sweepstakes model. The latter theory refers to cases in which animals may venture far from the coast, reach an isolated island from which they cannot return and are Forced to settle there. We reject the possibility that the Late Pleistocene mammals of Cyprus arrived by a land bridge, because during the Pleistocene such a bridge never existed. The cause of extinction of the earlier Cypriot endemic large mammals remain unclear. The discovery at the site ofAltrotiri /letokrenmos of the above pygmy mammal species together with man—made artefacts has been interpreted by some researchers as proof of a human role in the extinction of these mammals. Others rejected this view. We review the facts about this discovery and believe that humans did play a role in the final extinction of these species. THE ORIGIN OF THE LARGE PLEISTOCENE MAMMALS OF CYPRUS In Cyprus, Late Pleistocene fossil and subfossil sites consist almost exclusively of pygmy hippopotamus (P/annourios minzitus) (Forsyth Major, 1902; Bate 1906; Boekschoten and Sondaar, 1972; Houtekamer and Sondaar, 1979; Faure et al., 1983, Hadjisterkotis and Masala, 1995; Reese, 1975, 1989, 1992, 1995, 1996; Simmons, 1988a, b, 1989, 1991a,b,c, 1992, 1996; Swiny, 1988, 1995; Simmons and Reese, 1993; Simmons and Wigand, 1994) and pygmy elephant (Elepnczs Q)/priotes) (Bate, 19033, 1904a,b,c; Hadjisterkotis and Masala, 1995; Reese, 1995, 1996b; Simmons, 1988, 1989, 1991a,b,c, 1992, 1996). Both species were considerably smaller than their mainland counterparts, and most researchers believe that the dvvarfism was an evolutionary response both to the lack of predators and to the limited resources available (Sondaar, 1977; Azzaroli, 1982).

In Sardinia, the beta-39 nonsense mutation is the primary cause of beta 0-thalassaemia. This mutation is found mainly on beta-globin gene cluster haplotypes I and II, which differ in their A gamma globin types (A gamma I and A gamma T, respectively). This report presents data on G gamma, A gamma I and A gamma T levels, and the presence or absence of a 4 base pair (bp) deletion at -225 to -222 of the A gamma globin promoter, in 55 poly-transfused beta 0-thalassaemia major patients. Six patients were homozygotes for the normal (N) A gamma promoter lacking the 4 bp deletion, had no A gamma T globin, and their mean G gamma:A gamma I: A gamma T ratio was 52.9:47.1:0. Twenty-five patients were homozygotes for the mutant (M) A gamma promoter with the 4 bp deletion, had no A gamma I globin, and the mean G gamma:A gamma I: A gamma T ratio was 62.1:0:37.9. For M/M compared to N/N, the lower A gamma T than A gamma I was significant by the t-test (P less than 0.001). Twenty-four N/M cases had mean G gamma:A gamma I:A gamma T of 56:24.4:19.6, and the lower A gamma T than A gamma I was also significant (P less than 0.001). Partial haplotype analysis on these and 17 other beta 0-thalassaemia patients suggested that the 4 bp deletion was strongly associated with haplotype II. Of 33 M/M, 32 were haplotype II/II and one was II/5a; of 31 N/M, 29 were I/II and two were II/IX; of eight N/N, seven were haplotype I/I and one was I/IX. These data show a strong association of the 4 bp promoter deletion with decreased expression of the A gamma T globin gene on haplotype II.

Several factors influence the severity of homozygous β-thalassemia: β-thalassemia intermedia (non-transfusion dependent) patients often have at least one β + -thalassemia allele with only moderate impairment of β-globin production. β-thalassemia is also ameliorated by coinheritance of α-thalassemia or by γ-globin promoter mutations, such as T at position -158, which increase fetal hemoglobin production. In Sardinia, the codon 39 nonsense mutation (β39) is the primary cause of Β 0 -thalassemia. Two-thirds of northern Sardinian patients have this mutation on haplotype II, while one-fourth have it on haplotype I. Haplotype II, with ++ at the Hin d III sites in the G γ and A γ genes, is strongly associated with the variant A γ T globin, while haplotype I is associated with the normal A γ I . We have previously demonstrated that a β 0 -thalassemia haplotype found in a black family, with ++ at the Hin d III sites, had a 4-base-pair (bp) deletion at positions -225 to -222 of the A γ gene, in association with reduced A γ (elevated S γ ) levels. We have now extended this study to northern Sardinia, providing statistical data for the association of the 4-bp deletion with decreased A γ T expression.

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 functional properties of Hb B of the wild European mouflon (Ovis gmelini musimon), Hb B of domestic sheep (Ovis aries), and Hb C isolated from anemic mouflon were investigated. Mouflon and sheep Hbs appear to be very similar in their response to organic anions and protons, whereas sheep Hb B displays an oxygen affinity lower than that of mouflon Hb B and sheep Hb A. Mouflon Hb B and Hb C, like sheep Hb A and Hb C, have similar efficiencies in transporting oxygen to the tissues. As in other ruminant Hbs, the effect of temperature on the oxygen affinity is slight. Data suggest that mouflon Hb B is not only structurally, but even functionally, more similar to sheep Hb A than to sheep Hb B.

Publisher Summary This chapter discusses the principal for the detection of globin chains by reversed-phase high-performance liquid chromatography (HPLC). Reversed-phase high-performance liquid chromatography (RP-HPLC) has greatly influenced and stimulated research in detection, quantitation, and identification of normal and abnormal globin chains in human newborn and adult red blood cells. In fact, RP-HPLC methodology has a number of advantages: it is fast and accurate, minute amounts of material are sufficient for each determination, complete automation is possible, and, most importantly, identification of electrophoretically silent mutant hemoglobins caused by neutral-to-neutral amino acid substitutions is feasible and reliable. It also greatly facilitates the separation and isolation of even microquantities of a mutant globin chain, and of globin proteolytic fragments, for subsequent characterization of the substitution within the abnormal peptide. This is possible by means of HPLC techniques for the determination of the amino acid composition at the picomole or subnanomole level, and microsequencing techniques, fast atom bombardment mass spectrometry (FAB mapping), electrospray mass spectrometry, and capillary electrophoresis. The chapter describes several different HPLC procedures. Some use cation-exchange systems whereas others use the reversed phase with different columns, such as Waters μBondapack C18, Merck LiChrospher 100 CH-8/2, Aquapore RP-300, and Partisil C18.

Summary A rather common haemoglobin variant was detected in the Sardinian and Altamurana sheep breeds. The mutated globin chain appears to be produced under the control of an allele at the HBB locus and due to a neutral amino acid substitution. The variant will be provisionally referred to as the Hb I.