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Hemoglobin phenotypes of European mouflon and sheep were analyzed by isoelectric focusing; the results show that the sheep HbA migrated faster than that of other animals, while the mouflon HbA was more anodic than the sheep HbB. The oxygen dissociation curve of blood from mouflon is similar to that of sheep with HbA. The authors suggest that the mouflon should not be considered a direct ancestor of the Sardinian breed of sheep.

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.

A simple thin-layer isoelectric focusing technique was used to separate Hb F-Sardinia, containing the A gamma T-globin chain, from the Hb F containing the G gamma- and the A gamma I-globin chains. The identity of the slow-moving Hb F fraction as Hb F-Sardinia was verified by PAGE. A negative correlation (R2 = 0.747, p less than 0.001) was found between the percent Hb F-Sardinia and percent G gamma-chain in homozygotes for beta-thalassemia. Of 31 Sardinian beta-thalassemic patients studied, 21 were homozygous and eight heterozygous for the A gamma T polymorphism with a gene frequency of 0.823. The mean values of Hb F-Sardinia were 39.1 +/- 5.9% for the homozygotes and 17.1 +/- 3.6% for the heterozygotes. The percentage of Hb F-Sardinia found in beta o-thalassemic newborns was similar to that of corresponding normal newborns who also had the A gamma T polymorphism. No measurable differences in the percent Hb F-Sardinia level were observed among beta o-thal patients who were polytransfused, beta o-thal patients studied before transfusion, and beta o-thal patients exhibiting the intermediate form of the disease who had never been transfused.

As many as 7,717 babies born consecutively and 3,412 blood donors of Sardinian ancestry have been examined for the detection of the Hb J-Sardegna variant [alpha 50(CE8)His----Asp]; all subjects were from Northern Sardinia. Hemolysates were analyzed by isoelectricfocusing and the identification of the variant was made by reversed phase high performance liquid chromatography of the tryptic peptides. A total of 28 carriers (1:397) of Hb J-Sardegna were identified. The incidence of 0.25% makes this hemoglobin one of the most common alpha-globin structural mutants in humans. The distribution of the anomaly appears to be nonhomogeneous in the island. The quantity of the variant ranged from 19 to 36%; this wide range probably reflects the co-inheritance of an alpha-thalassemia anomaly.

11,129 individuals of the northern area of the island of Sardinia (7,717 newborns and 3,412 adults) were examined for the Hb G-Philadelphia variant [alpha 68(E17)Asn----Lys]. Hemolysates were analysed by isoelectric focusing, and the variant identified by reversed phase high performance liquid chromatography of tryptic peptides. A total of seven heterozygotes (1 in 1,589) were identified. This is probably the highest prevalence of this mutant so far described. Percentages of the variant (average 35.1 +/- 6.2%) were trimodally distributed with modes centering on 28, 36, and 42%, respectively. These values suggest that the G-Philadelphia allele occurs in Sardinians both on a single and on a double locus chromosome. The linkage with alpha-thalassemia may be the reason for the high frequency of the variant.

G gamma:A gamma composition of 54 newborns from Sardinia was determined by electrophoresis on polyacrylamide gels containing urea and Triton X-100. G gamma was present at a concentration of 67% with bimodal distribution (with mean values at 65 and 70% respectively). Concentration higher than 76% was not observed. When Hb F in peripheral blood was below 70%, G gamma:A gamma ratio correlates with Hb F%.

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.

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.

We considered the possible application of globin chain separation on cellulose acetate strips electrophoresis to thalassemia screening. The method shows good accuracy and reproducibility when compared with the chromatographic method on CM-cellulose. The electrophoretic method could be recommended as the simplest test of hemoglobin biosynthesis in countries where high incidence of thalassemic syndromes occurs.

In this study, we investigated the clinical and hematological features and carried out alpha- and beta-globin gene analyses in 11 Sardinian adult beta zero-thalassemia homozygotes from Northern Sardinia who were not transfusion-dependent. Oligonucleotide analysis revealed in nine out of 11 patients the nonsense mutation at codon 39, which was associated either with haplotype II or IX (14/16 and 2/16 chromosomes, respectively). Haplotype II was linked to the A gamma T mutation. The G gamma globin level ranged from 50 to 70%. Four out of nine patients (44%) were heterozygous and 3/9 (33%) homozygous for the rightward deletional type of alpha-thalassemia; two (22%) had the normal alpha-gene complement. Patients who were alpha-thalassemia homozygotes (-alpha/-alpha) showed a more balanced globin chain synthesis ratio. This study confirms that alpha-thalassemia may ameliorate the clinical picture of homozygous beta zero-thalassemia.

Forty-three patients with beta-thalassemia from Northern Sardinia (31 severe and polytransfused, six follow-up babies, five adults with mild thalassemia who were not transfusion dependent, and a young transfused patient was also affected by a disease of intermediate severity) were studied in order to establish the fetal hemoglobin composition, restriction fragment length polymorphism haplotypes at the beta-globin gene cluster, and the type(s) of mutation. Haplotype II was prevalent, [56/86 chromosomes (65%)], haplotype I was also fairly common, [22/86 chromosomes (25%)], while other types were relatively rare. The nonsense mutation at codon 39 was nearly exclusive, [76/80 chromosomes (95%)]. Other beta-thalassemia mutations occurred on chromosomes with haplotypes III, IX, X, and perhaps V, and a new type related to II. The mutated A gamma T gene was associated with type II, X, and the new type. Type IX was linked to a beta(0) gene and to an Xmn I site 5' to the G gamma gene, to a high G gamma globin level, and to a disease of mild severity. Type III was associated with a beta(+)-thalassemic gene. The (0)39 mutation linked to type II was associated with thalassemia intermedia in three patients.

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.

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.