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We have studied the relative levels of gamma-mRNA [%gamma/(gamma + beta)], Ggamma- and Agamma-mRNAs [%Ggamma/(Ggamma + Agamma)], hemoglobin (Hb) F, and the Ggamma and Agamma chains in some 50 patients with sickle cell anemia (SS) and with different haplotypes. As expected, the Hb F levels varied greatly and were high in patients with the Saudi Arabian-Indian haplotype. Similarly, the Ggamma values varied greatly (from 19.5 to 76.5%) and depended on the haplotypes. A rare haplotype, named Mor, was found in 3 SS patients, 1 of whom was a homozygote Mor/Mor; this haplotype is associated with the lowest Ggamma value (19.5% in the homozygote) and with a C-->T mutation at position -202 of the Agamma promoter. The levels of gamma-mRNA roughly parallel those of Hb F, but older patients have increased levels of mRNA, which appears not to be efficiently translated into Hb F. Similar observation have been reported for other hemoglobinopathies such as deltabeta-thalassemia heterozygotes and Hb Lepore heterozygotes. The relative quantity of Ggamma-mRNA was closely related to that of the Ggamma chain in the 15 patients who were studied; the Ggamma- to Agamma-mRNA ratio did not change with age.

We have determined the relative quantities of gamma- and beta-mRNAs and the alpha/beta-mRNA ratios in 37 patients with beta-thalassemia major with specific genotypes, namely 8 with a homozygosity for codon (CD) 39 (C-->T), 7 with a homozygosity for IVS-I-110 (G-->A), 5 with a homozygosity for IVS-I-6 (T-->C), for 15 patients with compound heterozygosities for 2 of these 3 mutations, and for 2 patients with the IVS-I-110 (G-->A)/-87 (C-->G) mutations. None had an alpha-thalassemia. Twelve patients had thalassemia intermedia and the remainder, transfusion-dependent severe conditions. Differences in phenotype were observed for compound heterozygotes involving the IVS-I-6 (T-->C) mutation in combination with either the IVS-I-110 (G-->A) or the CD 39 (C-->T) mutations: patients with thalassemia intermedia had a lower alpha/beta-mRNA ratio, about half of that of the patients with severe beta-thalassemia major. This might suggest a higher beta-mRNA synthesis in some patients than in others with the same genotype; mutations in promoter, enhancer, and/or locus control region sequences may be responsible for these differences. In vitro chain synthesis data were too incomplete to be helpful in this study. The RT-PCR procedure allowed the separation of abnormal (extended) mRNA from normal beta-RNA in subjects carrying the IVS-I-110 (G-->T) mutation. The relative quantities of this beta Th-mRNA (% of beta A + beta Th) were determined by scanning of the appropriate autoradiograms; they averaged 25% for homozygotes and about 4% for heterozygotes, indicating a considerable instability of the message.

We have treated 9 patients with sickle cell anemia (SS) with hydroxyurea (HU). All 9 patients carried 4 alpha-globin genes and the beta s-globin haplotypes 19/19 (Benin/Benin), except for 1 who had haplotype 19 together with type 3 (Benin/Senegal). Six patients received HU for 10 months and were again treated with the drug for 5 months after an interval of 1 year. One patient was given HU for 22 consecutive months. A record was kept of hematological and biochemical data, Hb F and G gamma levels, as well as possible clinical complications. Our data show that HU generally improves the hematological and biochemical values and the level of Hb F, and reduces painful crises in some patients. However, although the clinical symptoms improved in some patients during HU therapy, the older patients did not observe any changes in their general condition; the same is the case for the patient with haplotype 19/3. One patient also experienced life-threatening liver sequestration during treatment. We conclude that the selection of patients who may benefit from HU therapy needs further evaluation.

The author summarizes the Hb A2 levels in over 600 beta-thalassemia heterozygotes with 32 different base changes or frameshifts and in 22 heterozygotes for 1 of 5 large deletions. Three major groups are recognized: persons with beta zero-thalassemia or beta (+)-thalassemia (severe) have Hb A2 levels between 4.5 and 5.5%, those with mild beta (+)-thalassemia alleles have levels between 3.6 and 4.2%, with still lower values for those with silent mutations. High values were observed in subjects with the 2 mild beta + alleles with mutations in the beta-globin gene promoter (-88, C-->T and -29, A-->G); unusually high Hb A2 values were also present in several -88 and -29 homozygotes. Data for several members of 8 families in which the -88 (C-->T) or the -29 (A-->G) mutation, or the -1,393-bp deletion, is present in cis or in trans to a delta-globin gene mutation support earlier observations that an increase in delta-chain synthesis occurs in cis to either one of these 3 alleles. A review of these data confirms the suggestion that the increase in Hb A2 levels results from at least two mechanisms: in a posttranslational system, the formation of alpha delta-dimers is promoted when excess alpha-chains are available, while certain promoter mutations increase the transcription of the delta-globin gene in cis because of a change in the binding of transcription factors.

Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the alpha 2/alpha 1-, alpha/beta-, and gamma/beta-mRNA ratios in subjects with beta-thalassemia (beta-thal), hereditary persistence of fetal hemoglobin (HPFH), and normal adults. The alpha- and beta-globin gene mutations were characterized with gene mapping, PCR, and DNA sequencing. The average alpha 2/alpha 1-mRNA ratio was the same in normal adults and beta-thal heterozygotes with four alpha-globin genes (2.61-2.63) or with an alpha-thal-2 trait (1.48-1.55). The average alpha/beta-mRNA ratios were 4.47 and 3.84 in normal adults with four alpha-globin genes and with alpha-thal-2 trait (-alpha/alpha alpha), respectively. There was an increase of approximately 50% in beta-thal heterozygotes with transcriptional mutants [-88 (C-->T) and -29 (A-->G)] with lower values (approximately 25%) in those with alpha-thal-2 trait (-alpha/alpha alpha). High alpha/beta ratios were also observed for heterozygotes for nonsense or frameshift mutants located in exon 1 or exon 2. Increases of approximately 150-165% were seen in subjects with RNA processing defects; an exception was the IVS-1-110 (G-->A) mutation with a normal value in the heterozygote. The increases were also less pronounced in heterozygotes for the codon (CD) 121 (G-->T) mutation and the CDs 134-137 insertion/deletion. Normal alpha/(gamma + beta) values were seen in 3 heterozygotes each with a different deletion involving part of the beta-globin gene. The presence of the silent beta-thal allele, -101 (C-->T), in trans to a CD 8 (-AA) allele has a minor effect on the alpha/beta-mRNA ratio. The alpha/beta-mRNA ratio in HPFH heterozygotes was approximately 145% of normal, but with a gamma-mRNA level of 35.4-44.7% the calculated alpha/(gamma + beta) ratio became as in normal adults. The RT-PCR methodology appears useful in expression studies in beta-thal (and HPFH) and values of mRNA appear to correspond to the type of prevailing mutation(s) and concomitant alpha-thal.

The present study concerns the identification of four alpha-globin gene deficiencies, one alpha 1-globin gene mutation, and one beta-globin gene mutation in a Laotian couple and their newborn baby. The parents were Hb E heterozygotes and the baby was an Hb E homozygote. The father carried the 4.2-kb deletion on one chromosome and a TAA-->CAA mutation at the terminating codon of the alpha 2-gene (Hb Constant Spring or CS) on the other chromosome. Moreover, the remaining alpha 1-globin gene on the chromosome with the 4.2-kb deletion was mutated at codon 74 (GAC-->CAC; Asp-->His; Hb Q-Thailand). The mother had the 3.7-kb deletion on one chromosome and a TAA-->TAT mutation at the terminating codon of the alpha 2-globin gene (Hb Paksé) of the second chromosome. The baby was a compound heterozygote for the two termination codon mutations and, at birth, had a high level of Hb Bart's (16.6%) reflecting a mild form of Hb H disease.

We report the identification of an Hb S homozygosity in a 29-year-old multiple-transfused patient with severe kidney disease using a new reverse-transcription polymerase chain reaction (RT-PCR) technique with mRNA as starting template. The procedure requires considerably less time than sequencing of genomic DNA and is recommended for use in the diagnosis of Hb S and other hemoglobinopathies.

Using amplification, allele-specific oligonucleotide (ASO) hybridization and DNA sequencing we have documented the molecular basis of 64 alpha- and 123 beta-thalassemia (thal) chromosomes, and the haplotypes of 18 beta S chromosomes from patients followed in three hospitals in Kuwait. Of the 30 chromosomes from 15 patients with Hb H disease, 26 (86.7%) carried the polyadenylation (poly A) signal mutation (AATAAA-->AATAAG) in the alpha 2-globin gene, 3 (10%) had the -alpha (3.7 kb) deletion, and 1 (3.3%) had the pentanucleotide deletion in the 5' IVS-I splice junction (alpha-5nt alpha). As many as 12 different beta-thal mutations were identified; 6 Mediterranean alleles [IVS-II-1 (G-->A), IVS-I-6 (T-->C), codon (CD) 39 (C-->T), IVS-I-110 (G-->A), CD 8 (-AA), and IVS-I-1 (G-->A)] were present in 79 (64.2%) of the chromosomes tested. Four East Indian alleles [IVS-I-5 (G-->C), IVS-I 3' end -25 nt deletion, CDs 8/9 (+G), and 619-bp deletion] were found in 31 (25%), and the two Kurdish/Iranian alleles [CD 44 (-C) and CDs 36/37 (-T)] were found in 13 (10.6%) chromosomes. Fourteen beta S chromosomes carried haplotype No. 31 (Saudi Arabia/India); 3 had haplotype No. 19 (Benin), and 1 was a hybrid with haplotype No. 31-specific characteristics in the locus control region hypersensitive site-2 (LCR-HS-2), and haplotype No. 19-specific mutations in the 5' flanking region of the G gamma-promoter. The patient homozygous for haplotype No. 19 was a Jordanian, while the others were Kuwaiti Arabs. The latter appear to be fairly homogeneous in terms of the prevalent alpha-thal determinants and beta S-haplotypes, but there is considerable heterogeneity of their beta-thal alleles. This has implications for genetic counseling and prenatal diagnosis programs.

We have used the gel retardation assay to investigate the binding of nuclear proteins to the duplicated CACCC boxes in the beta-globin gene promoter region. The effect of beta-thalassemia mutations affecting both of these consensus sequences (the -88 C-->T and -101 C-->T mutations) were studied by using appropriate mutant oligonucleotides. Upon incubation with nuclear proteins from human erythroleukemia cells, the synthetic oligonucleotides containing single and/or duplicated CACCC box(es) generated five retarded bands. Bands B1 and B2 appeared to be highly specific complexes as determined by competition experiments whereas bands B3, B4 and B5 were nonspecific. The binding of the specific trans-acting factors was stable and could be competed out only by relatively high concentrations of competitor DNA. The proximal CACCC box (as in the -88N probe) appeared to be essential in the binding of nuclear proteins and a mutation at nt -88 (C-->T) abolished binding completely. In contrast, the distal CACCC box showed no binding activity either as normal (-101N) or as mutant (-101M). A full competition with the Sp1 oligonucleotide, even at low concentrations, suggested a high affinity binding to the Sp1 consensus sequence. The close similarities in the binding patterns of the labelled -88N, -88N/-101N and that of the labelled Sp1 probe add further credence to the possibility that bands B1 and B2 may be due to the Sp1 protein, a ubiquitously expressed trans-activator.

We have evaluated hematological data for numerous blood samples collected from 51 sickle cell anemia (SS) patients during their first 10 years of life. Haplotypes and alpha-globin gene status were determined in all patients. A total of 482 hematological evaluations and 317 fetal hemoglobin (Hb F) determinations are presented. The data show that the Hb F levels are the highest in patients with haplotypes 3/3 and the lowest in those with haplotypes 20/20. This is reflected in differences in total hemoglobin, PCV value, and RBC counts, which are the lowest in the 20/20 group and the highest in the 3/3 group. Female SS patients with haplotypes 19/19 have higher Hb F levels than their male counterparts; the same was observed for the patients with the 19/3 haplotype combination but not for those with the 20/3 haplotype combination. A concomitant alpha-thalassemia-2 heterozygosity had little or no effect on the hemoglobin level. The data confirm that SS children, aged 1-10 years, with a homozygosity for haplotype No. 20 are most severely affected by their disease.

We have identified three types of alpha-thalassemia in 28 members of an Indian family from Durban, South Africa. The rare South African (SA) type of alpha-thalassemia-1, which is characterized by an approximately 23-kb deletion involving the psi zeta, psi alpha 2, psi alpha 1, alpha 2, alpha 1, and theta 1 genes, was present in 13 members [6 simple heterozygotes, 5 with Hb H disease of the --(SA)/-alpha(-3.7 kb) type, and 2 with Hb H disease of the --(SA)/-alpha(-4.2 kb) type]. Seven others were heterozygotes for alpha-thalassemia-2 (-3.7 kb), 1 was homozygous for this deletion, and 1 was a compound heterozygote [-alpha(-3.7 kb/-alpha(-4.2 kb)]. Hematological and hemoglobin composition data indicated a moderate anemia in all 7 patients with Hb H disease with severe microcytosis and hypochromia, no elevation of gamma-chain synthesis, low levels of Hb A2 (0.3-0.7%), and low levels of Hb H. The most severe disease was present in 2 teenagers with the --(SA)/-alpha(-4.2 kb) combination.

We have identified 7 patients with Hb H disease as homozygotes for a mutation in the polyadenylation site (AATAAA-->AATAAG) and have compared their hematological data with those of Hb H patients having other types of alpha-thalassemia determinants. All 7 patients exhibited moderate anemia with microcytosis and hypochromia being similar to that observed in the other patients. Relatives with a heterozygosity for this mutation are borderline microcytic and hypochromic without a significant anemia but with a low in vitro alpha/beta chain synthesis ratio. Analyses of the hemoglobin components identified low levels of Hb A2 and Hb H that were comparable to those found in other patients with Hb H disease; the level of the zeta-chain was low (average 0.14%).

In this paper we describe the distribution of some specific sequence differences in the 5' flanking regions of the A gamma- and G gamma-globin genes from 100 Black adult and 57 newborn SS patients from the southeastern United States, from 76 individuals with AS, S-beta-thal, SC, AC, or A-beta-thal, and from 31 normal individuals. Haplotypes for all adult individuals have been previously determined using various restriction endonucleases. The DNA samples were amplified, dot blotted, and hybridized with 32P-labeled specific oligonucleotide probes. All 134 chromosomes with haplotype 19 were positive for the G----T substitution at position -657 (A gamma), while 132 were also positive for the C----G mutation at -369 (G gamma). The three specific changes for the chromosome with haplotype 20 were found on all 54 chromosomes with this haplotype. The C----T mutation at -158 5' to G gamma was present on all 41 chromosomes with haplotype 3, and on two chromosomes with a related atypical haplotype. Normal and beta-thal chromosomes with each of these substitutions had the same 5' subhaplotype as beta S haplotypes 19 or 20, respectively. The close relationship between the occurrence of specific mutations and the haplotype of beta S chromosomes makes the determination of these haplotypes with specific oligonucleotide probes attractive with respect to time and expense.

Hematological and hemoglobin composition data are presented for 14 members of a Surinam family (and for 1 unrelated subject) with either a beta-thalassemia heterozygosity [5 with the -29 (A----G) beta + mutation and 5 with the IVS II-849 (A----G) beta(0) mutation] or a compound heterozygosity (the 5 remaining patients). Identification of the mutation was by hybridization of amplified DNA with 32P-labelled synthetic oligonucleotides. The data indicate distinct differences between the two groups of heterozygotes, mainly in degree of microcytosis and hypochromia, in Hb A2 level, and in the level of G gamma (high in the -29 heterozygotes and low in the IVS II-849 heterozygotes). The 5 compound heterozygotes had a thalassemia intermedia with high Hb F levels (high G gamma), elevated Hb A2, and Hb A levels comparable to those seen in patients with a homozygosity for the -29 mutation or with the combination of this beta(+)-thalassemia and Hb S. An alpha-thalassemia-2 heterozygosity (-3.7 kb deletion) was present in 2 patients. Their hematological data were improved over those for the patients with four alpha globin genes; one was the mother of two sets of twins. The high G gamma value in the Hb F of the compound heterozygotes suggests that the high Hb F production in the condition is mainly directed by the chromosome with the -29 (A----G) mutation.

The level of adult hemoglobin (Hb A, Hb S, Hb C, Hb E) was determined in cord blood samples of 66 Black babies (41 with four alpha-globin genes; 25 with two alpha-globin genes) with Hb A, of 51 SS, 7 CC, and 6 EE babies, of 359 babies from Mediterranean countries, and 197 babies from Japan and China. Methodology involved high-performance liquid chromatographic procedures, which are considered most accurate because of a complete separation of Hb F (or gamma chains) and the adult hemoglobins (or beta A or beta X chains). The presence of an alpha 2-thalassemia homozygosity (-alpha/ -alpha versus alpha alpha/alpha alpha) did not affect the average Hb A level. The levels of Hb S and Hb E in homozygous Hb S or Hb E babies were about one-third lower than the Hb A of the normal baby, while that of Hb C in homozygous Hb C babies was not decreased. The average level of Hb A in Chinese (50 babies) and Japanese (147 babies) was about 16.0%, which was significantly lower than the average level of 19.0% in newborns of Italian (221 babies), Yugoslavian (68 babies), and Turkish (70 babies) origin.

This short review summarizes data obtained with two different high-performance liquid chromatography (HPLC) methods, useful in the study of numerous haemoglobinopathies. The first concerns the use of a cation exchange HPLC column which, when developed with specific gradient elution systems, will allow the separation of many normal and abnormal haemoglobins in blood from adults and newborn babies. The method has great diagnostic value. The second procedure uses a reverse-phase HPLC system and acetonitrile-H2O-trifluoroacetic acid developers at low pH for the separation of globin chains (alpha, zeta, beta, delta, G gamma, A gamma, epsilon). The method can be applied as an analytical as well as a preparative tool; it is ideally suited to separate several normal and abnormal chains with neutral amino acid substitutions, including the three gamma chains (G gamma = gamma 75Ile; 136Gly, A gamma = gamma 75Ile; 136Ala, A gamma T = gamma 75Thr; 136Ala).

Clinical, haematological and biochemical aspects of a new family with heterozygous haemoglobin Presbyterian [beta 108 (G 10) Asn----Lys] are described. It could be readily separated by isoelectric focusing, DEAE-cellulose chromatography and by cation exchange high-performance liquid chromatography (HPLC). The characterization of this mildly unstable haemoglobin was made by reverse-phase HPLC. This is the fourth family with Hb Presbyterian so far described.

Detailed hematological and fetal hemoglobin data as well as results from gene mapping analyses are reported for nine members of an Indonesian-Dutch family. An alpha-thalassemia-1 heterozygosity (Southeast Asian type) was present in five subjects and a gamma-globin gene triplication of the type-G gamma-G gamma-A gamma in three; the propositus and one of her daughters had both conditions. Comparison of gene mapping data with quantitative results of the fetal hemoglobin analyses provided an explanation for the slight increases in fetal hemoglobin levels and for the extremely high G gamma levels of this fetal hemoglobin.

We report data on a fetus with hydrops fetalis due to a homozygosity for alpha-thalassemia-1, type -(alpha)-20.5 kb; this is the first reported case in a Turkish family. Characterization of the abnormality was based on data from family studies and from alpha-globin gene mapping of the DNA from the parents.

The electrophoretically fast-moving hemoglobin variant Hb Camden [beta131 (H9) gln leads to glu] has been found in a middle-aged female suffering from pulmonary disease, and Hb Hope [beta136 (H14) gly leads to asp] which migrates just ahead and very close to Hb A was present in a young obstetrical patient with a mild hemolytic anemia.

A total of 38 patients with beta-thalassemia intermedia from 30 families were were studied. Twelve of the thirty unrelated patients had beta zero-thalassemia which was due to a homozygosity for one of two different thalassemia defects, namely the frameshift at codon 8, and the IVS-II-1 G----A mutation. Another mild variation, a beta +-thalassemia, was a homozygosity for the mutation of T----C at position 6 of IVS-1 (10 patients). Compound heterozygosities for mild thalassemic determinants or for one mild and one severe beta-thalassemic determinant were also found in some patients with beta-thalassemia intermedia. The mutations at beta-39 and IVS-I-110 were the most commonly occurring thalassemic determinants in these patients. Correlations between genotype and phenotype indicated significant differences in some of the hematological parameters among patients with the IVS-I-6 and the frameshift at codon 8, IVS-I-6 and IVS-II-1, and the frameshift at codon 8 and IVS-II-1 mutations.

Detailed gene mapping data are provided for members of a Yugoslavian and Canadian family with a thalassemia heterozygosity characterized by mild anemia with severe microcytosis and hypochromia, normal levels of Hb A2 and slightly raised Hb F levels. The condition in both families results from large deletions (minimally approximately 148 kb in the Yugoslavian family and minimally approximately 185 kb in the Canadian family), which include all functional and psi genes of the beta globin gene cluster. The Canadian propositus was a newborn baby who has been followed for nearly 2 years; severe anemia developed some 30-40 days after birth when the Hb F level was still 70%; recovery was evident at the age of 90 days when the Hb F level had decreased to 40%.

This paper summarizes various anomalies involving the duplicated gamma-globin genes which are normally arranged as -G gamma-A gamma-. Variations include G gamma----A gamma and A gamma----G gamma replacements; newborn heterozygotes for these anomalies have low G gamma (A gamma A gamma/G gamma A gamma) or high G gamma values (G gamma G gamma/G gamma A gamma). Additional abnormalities include the deletion of one gamma-globin gene (-A gamma-; gamma-thalassemia), gamma-globin gene triplications (G gamma-AG gamma-A gamma; G gamma-G gamma-A gamma) and gamma-globin gene quadruplications (G gamma-G gamma-G gamma-A gamma-); several of these conditions are found in different populations but at low frequencies. In adults, the G gamma to A gamma ratio in the Hb F and often also the Hb F level, are directly related to specific structural characteristics of the chromosome; specific mutations in the promoter sequences 5' to G gamma or to A gamma, for instance, may result in increased G gamma or A gamma chain production with an increase in Hb F levels (nondeletional G gamma- or A gamma-HPFH) or with only modest changes in the total Hb F (normal adults; Swiss-HPFH). Similar variations have been observed in SS patients.