Search

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression. more...

Two types of alpha-globin variants were found in 0.2% of a large number of newborn from Malta. The two hemoglobins were identified from tryptic maps on a Vydac C18 column and by alpha-globin gene sequencing as Hb St. Luke's (isoelectric point = 7.18+/-0.017) and Hb Setif (isoelectric point = 7.26+/-0.010). Hb St. Luke's [alpha95(G2)Pro-->Arg] was found to result from a C-->G mutation at the second position of codon 95 on an alpha1-globin gene, and Hb Setif [alpha94(G1) Asp-->Tyr] resulted from a G-->T mutation at the first position of codon 94 on an alpha2-globin gene. Quantification of Hb St. Luke's (11.1+/-1.12%) and Hb Setif (14.7+/-2.22%) in peripheral blood hemolysates indicated that, in the absence of either an alpha- or a beta-thalassemia allele, the protein products of the alpha1- and alpha2-globin genes were nearly equal in quantity. more...

The product of the Polymerase Chain Reaction (PCR) may be generically suitable for four types of investigations: Discovery PCR, Analytical PCR, Modification by PCR, and Synthetic PCR. Despite the potential problem of contamination with extraneous DNA, PCR is relatively simple and inexpensive, and thereby offers opportunities for laboratories from smaller or developing countries to invest limited resources in competitive global research and development, or in applications of economic and social value in health, agriculture and other sectors. more...

In vitro DNA amplification and dot blot analysis with synthetic allele specific oligonucleotides (ASO) identified the beta + IVS, I-6 (T --> C) thalassaemia in 78% of 32 chromosomes from 16 beta-thalassaemia homozygotes in Malta. The preponderance of a single thalassaemia mutation in one population is unusual. The beta + IVS, I-6C thalassaemia mutation was also found in three carriers who had an associated beta globin heterozygosity, i.e. Hb Valletta (or alpha 2 beta 2 87PRO) or Hb S (or alpha 2 beta 2 6VAL). The proportion of Hb A in these cases (av. = 29.7%) provided objective documentation of the relatively mild effect of this mutation on in vivo globin gene expression. However, the expression of homozygous disease was more severe in developing children compared to adults. The beta + IVS, I-6C mutation complicates population testing because heterozygotes can have Hb A2 levels below those classically associated with beta thalassaemia. more...

We have identified a new stable abnormal hemoglobin called Hb Valletta, which is characterized by a Thr----Pro substitution at position 87 of the beta chain. This mutation was found to be linked to that of the gamma chain variant Hb F-Malta-I with a His----Arg mutation at position 117 of the G gamma chain. Both variants were detected in the blood samples of 34 Maltese and two Italian newborn babies with isoelectrofocusing and reversed phase high performance liquid chromatography. Similar analyses of cord blood from 388 additional Maltese newborns failed to identify either one of these two variants. Additional analyses of 353 Maltese adults (including 39 beta-thalassemia heterozygotes) resulted in the detection of two adult Hb Valletta heterozygotes. Dot-blot hybridization analyses of amplified DNA with a probe specific for the G gamma-F-Malta-I variant showed that both also carried that mutation. These results show close linkage of the mutant forms of the G gamma- and beta-globin genes, 27-28 kb apart, and a failure to identify chromosomes with either the Hb F-Malta-I mutation alone or with the Hb Valletta mutation alone, indicating a low recombination frequency. more...

The synthesis of alpha and non-alpha chains (beta, delta, G gamma, and A gamma) was studied in cultures of peripheral blood mononuclear cells from eleven beta-thalassemia heterozygotes, two HPFH heterozygotes, and one HPFH homozygote. The synthesis of Hb F in the thalassemia colonies (average value: 12.6%) was comparable to that in normal adult colonies (average value: 12.6%) was comparable to that in normal adult colonies (average value: 12.2%). The percent G gamma chain in the Hb F varied greatly but a relationship between the G gamma chain percentage in the Hb F from colonies and that from peripheral blood was established. The relative synthesis of Hb A2 in colonies of beta-thalassemia heterozygotes (average value: 5.8%) was 1.6 times as much as that in colonies of normal adults (average value: 3.6%). Hb A2 and Hb A were absent in the colonies of the HPFH homozygote. The alpha/non-alpha (i.e., beta, gamma, and delta) ratio of the hemoglobins in the cultured cells of the beta-thalassemia heterozygotes and the alpha/beta and alpha/beta ratios of isolated Hb A and Hb A2 were about one (range 0.74 to 1.38). The alpha/gamma ratio of the Hb F synthesized in BFUe-derived colonies of the HPFH homozygote, however, was 1.5. These results suggest a deficiency in the in vitro culture system resulting in decreased levels of alpha-mRNA or in a partial inhibition of initiation of protein synthesis which is known to reduce the synthesis of alpha chains more than that of the beta chains. more...

The hematology and pathophysiology of sickle cell disease during the postnatal development of younger hemoglobin (Hb) S homozygotes (SS) could be considerably affected by a variability of alpha globin gene numbers. We have documented longitudinal developmental changes of hematological values and hemoglobin composition on 147 patients with SS (alpha alpha/alpha alpha), 64 with SS (-alpha/alpha alpha), and 9 with SS (-alpha/-alpha) between the ages of 1 and 15 years. Non-steady-state data were excluded from these analyses. The number and organization of alpha globin genes was established by gene mapping. As anticipated, mean corpuscular volume and erythrocyte counts correlated with alpha globin gene numbers throughout the 15-year age interval. On the other hand, SS children with alpha alpha/alpha alpha, -alpha/alpha alpha, -alpha/-alpha had similar hemoglobin concentrations up to the ages of 5-10 years. Around the age of 7, the SS patients with -alpha/-alpha developed a higher Hb concentration than that of the SS (-alpha/alpha alpha), which in turn was higher than that of the SS (alpha alpha/alpha alpha). The emergence of this difference coincided with a developmental increase of the mean corpuscular hemoglobin concentration (MCHC) in patients with SS (alpha alpha/alpha alpha) and the decline of Hb F % under 15%. This newly observed developmental change of the MCHC could lead to increased hemolysis and anemia after the age of 5-10 years. It occurs to a smaller extent among SS (-alpha/alpha alpha) or not at all among SS (-alpha/-alpha) such that these two categories of patients have less severe hemolysis and higher hemoglobin levels at older ages. Although the proportion of Hb F was independent of alpha globin gene numbers, the absence of Hb Bart's suggested that alpha-thalassemia promotes the intracellular assembly of Hb F over Hb S tetramers. Thus, the interaction of alpha-thalassemia and Hb F in young SS patients may be more complex than revealed by Hb F levels in cell lysates. Among older SS children (greater than 7 years) alpha-thalassemia and Hb F levels exceeding 15% appear to have additive effects in diminishing the rate of hemolysis. more...

Seven Hb G-Philadelphia (Hb G) heterozygotes from three Caucasian families from Northern Italy and Sardegna were found to have proportions of Hb G averaging 23%. This value is considerably lower than the 34% or 48% found in Blacks from the Southeastern U.S.A. in whom the alpha G gene is in linkage with alpha-thalassemia-2, i.e. the alpha o alpha G/alpha alpha or alpha o alpha G/alpha o alpha genotypes. Gene mapping identified tandem organization of the alpha G gene in cis with a normal alpha A gene, i.e. the alpha alpha G/alpha alpha genotype, among the Hb G heterozygotes from Italy. The data on the Italian heterozygotes are similar to those obtained by Bruzdzinski et al (14) on a Black family. These results indicate alternate organization of the alpha G genes probably across racial or ethnic boundaries. Comparison of the mean cellular globin amount of alpha G/alpha G gene/cell among Hb G heterozygotes with 4, 3, 2 or 1 alpha globin genes (i.e. alpha A + alpha G) revealed considerable reactivation of individual alpha genes in conditions of mild to severe alpha globin deficiencies. more...

Four types of chromosomes with a deletion between the human embryonic zeta and psi zeta globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5' to the zeta globin gene [(X+) or (X-)]. The deletions are identifiable in Xba I digests of genomic DNA using an alpha or a zeta globin gene probe which yield fragments of 23 kb from (X+)-zeta alpha alpha chromosomes or 27 kb from (X-)-zeta alpha alpha chromosomes. Digestion with other enzymes and probing with both alpha and zeta probes gave fragments typical of the two zeta globin gene deletions previously identified in Polynesians. Among Black Americans, these zeta globin gene deletions have been found in combination with alpha globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the zeta globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult alpha globin genes. more...

Structural analysis of the alpha chain of the hemoglobin from a Caucasian female with a mild hemolytic anemia showed the presence of a variant with a Val----Met substitution at position alpha 62. The valine at this position forms one of the contacts with heme and its replacement by methionine will likely decrease heme binding and cause a distortion of the heme crevice and a decreased stability of the abnormal protein. Dot-blot analysis of amplified DNA with 32P-labeled synthetic oligonucleotide probes confirmed the suspected G----A mutation in the first position of codon 62, and also located the mutation in the alpha 2-globin gene. The mutation was found in the proposita and one of her daughters but was most probably absent in her parents. more...

Three delta beta-thalassemia homozygotes were found in a Mexican family. Both parents and two sibling had heterozygous delta beta-thalassemia with about 10% Hb F, mild microcytosis and mild hypochromia, while three siblings were normal. Hb F, which was the only Hb component in the homozygotes, had equal quantities of Ggamma and Agamma chains as in BgammaAgamma-delta beta-thalassemia. The homozygotes had comparable erythrocytic indices which were about the same as those of the heterozygotes. However, two were clinically and hematologically healthy but the third had a severe chronic hemolytic anemia and a more severe in vitro chain synthesis imbalance than her homozygous sisters. Comparison of these cases with other GgammaAgamma-delta beta-thalassemia homozygotes and with GgammaAgamma-HPFH homozygotes indicates the possibility that the proliferation of F-cell precursors may be defective in delta beta-thalassemia. more...

Restriction endonuclease mapping defined a partial deletion of about 1.35 kb in the beta-globin gene of a black American patient with hemoglobin S-beta zero-thalassemia and in his uncle with a beta zero-thalassemia trait. The 5' endpoint of the deletion is about 600 bases upstream from the cap site, and the 3' endpoint lies within about 500 bases from the 5' splice junction of the second intervening sequence. The deletion is different from that of a previously reported Indian beta zero-thalassemia allele, where 0.6 kb is deleted at the 3' end of the beta-globin gene. more...

Restriction endonuclease mapping with alpha and zeta-globin gene probes showed differences between the alpha-thalassemia-1 (alpha-thal-1) condition in two patients with HbH disease. One patient had the rare black type of alpha-thal-1 together with alpha-thal-2 and HbS heterozygosities. The second patient was a Laotian child with HbE, Hb Constant Spring (alpha-thal-2), and alpha-thal-1 heterozygosities. The diagnoses were based on clinical, hematologic, and biochemical data. Whereas DNA fragments hybridizing to a zeta-probe were obtained from the Laotian type of alpha-thal-1, neither alpha nor zeta-gene fragments could be identified deriving from the black type of alpha-thal-1. Therefore, the black type of alpha-thal-1 is associated with a deletion of the entire zeta 2-psi zeta-psi alpha-alpha 2-alpha 1 gene complex and can be considered a zeta alpha-thal-1. It is likely that homozygosity for such a condition will lead to embryonic wastage, explaining the absence of hydrops fetalis in blacks. more...

DNA from members of 10 Black families with conditions considered to be G gamma (delta beta)0-thalassaemia or G gamma (delta beta)0-HPFH were studied by using restriction enzyme analysis. One or more affected members from each family were shown to have the same deletion of 34 kbp of DNA in the human beta-globin gene cluster. A clone spanning the deletion was isolated from the DNA of one such person and studied in detail. The deletion removed part of the A gamma and all of the psi beta, delta and beta-globin genes and is different from the four previously identified deletions which caused a condition presently known as (A gamma delta beta)0-thalassaemia. more...

A microassay for erythropoietin (Ep) activity in serum using [3H]thymidine uptake by K562 cells is presented. The method is similar to that of Krystal except that cells of the K562 human pluripotent leukemia cell line replace spleen cells from phenylhydrazine-treated anemic mice. Response to the hormone by K562 cells and spleen cells was colinear. Using the Krystal bioassay, 14 young hemoglobin S homozygotes had Ep activity levels of 17.9-113.8 mU/ml serum, whereas the new method with K562 cells gave a range of 19.2-115.3 mU/ml. The correlation coefficient between the two sets of data (r) was 0.999 (p less than 0.001). With the modified technique we have assayed 34 sickle cell patients, whose sera ranged from 19.2 to 1400 mU of Ep/ml with corresponding hemoglobin concentrations of 10.7 g % to 3.0 g %. Values for normal subjects were 22.1 +/- 2.1 mU/ml (n = 7). The stimulation of [3H]thymidine uptake is significantly inhibited by an anti-Ep antiserum. The assay permits quantification of stimulatory activities in a large number of samples with relative ease and is also suitable to explore the interactions of erythropoietic factors with their appropriate receptors on stem cells. more...

The production of five alpha chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average sigma alpha/beta values of about 1.1. They were assigned a normal genotype (alpha alpha G/alpha alpha), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low alpha G/beta values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher alpha G/beta values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three alpha chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate alpha chain deficiency due to an alpha-thalassemia-2 heterozygosity (alpha alpha G/alpha O alpha or alpha O alpha G/alpha alpha) or a homozygosity (alpha O alpha G/alpha O alpha), respectively. more...

The in-vitro synthesis of hemoglobin (Hb) chains was studied among 60 Hb S heterozygotes (AS) having different quantities of Hb S, including five with an associated alpha-chain heterozygosity (ASAG). Hematologic values and hemoglobin composition were studied in these cases and in 15 other ASAG heterozygotes. The percentages of Hb S (which fell between 27% and 42%) and the mean corpuscular volume values correlated directly with the alpha/non-alpha values, confirming previous suggestions (Huisman, Hemoglobin 1:349, 1977) that the concomitant occurrence of an alpha-thalassemia-2 heterozygosity (alpha alpha(0)/alpha alpha; beta/beta(S)) or homozygosity (alpha(0) alpha/alpha(0) alpha; beta/beta(S)) resulted in intermediate or lower levels of Hb S compared with Hb S heterozygotes having four active alpha-chain genes (alpha alpha/alpha alpha; beta/beta(S)). Among ASAG heterozygotes, the occurrence of low (about 25%), intermediate (about 33%), or high (about 45%) proportions of an alpha-chain variant resulting from a variability in the number of active alpha-chain genes due to alpha-thal-2 coincided with high (39%), intermediate (34%), or low (28%) levels of Hb S, respectively. However, the overlap of biosynthetic data between Hb S heterozygotes with four, three, or two active alpha-chain genes prevents a reliable diagnosis in individual cases. more...

The synthesis of alpha and non-alpha chains of human hemoglobin (Hb) was studied in reticulocytes and in BFUe-derived cell colonies from patients with alpha chain or beta chain deficiencies. The subjects included normal adults (alpha alpha/alpha alpha) with or without a beta chain variant (Hb S, Hb Leslie) or an alpha chain variant (Hb G-Georgia); alpha-thalassemia-2 heterozygotes (alpha 0 alpha/alpha alpha) with an alpha chain variant (G-Georgia or G-Philadelphia); an alpha-thalassemia-1 heterozygote (alpha 0 alpha 0/alpha alpha); alpha-thalassemia-2 homozygotes (alpha 0 alpha 0/alpha 0 alpha) with a beta chain variant (Hb S), an alpha chain variant (G-Philadelphia), a Hb S homozygosity with Hb G-Philadelphia, or a Hb G-Philadelphia homozygosity; and three black beta +-thalassemia homozygotes. Data from reticulocyte in vitro synthesis analysis showed the expected deficiencies. However, similar analyses of the Hb synthesized in cell colonies (even from the black beta-thalassemia homozygotes) gave (nearly) balanced sigma alpha/sigma non-alpha ratios. It is speculated that this balanced synthesis is due to a most effective proteolysis in the immature erythroblast which rapidly removes free alpha or beta chains. The levels of Hb F and Hb A2 were considerably increased in these proerythroblasts; a two- to threefold increase in the synthesis of Hb A2 was observed over that seen in the reticulocytes. more...

Two homozygous delta 0 beta 0-thalassemia patients, one with the G gamma A gamma type and the other with the G gamma type, and their heterozygous parents are described. Red cell indices among the heterozygotes with the G gamma A gamma type of delta 0 beta 0-thalassemia were markedly different from those in heterozygotes with the G gamma type. However, the imbalance in in vitro hemoglobin synthesis was quite similar in the two heterozygous conditions. The same was observed for the homozygous patients; the in vitro chain synthesis was severely imbalanced as seen in beta-thalassemia major. The clinical and some of the hematological findings were milder in the G gamma-delta 0 beta 0-thalassemia homozygote than in the G gamma A gamma-delta 0 beta 0-thalassemia homozygote. The death of a sibling of the G gamma-delta 0 beta 0-thalassemia homozygote with a diagnosis of thalassemia major suggests that both types of delta 0 beta 0-thalassemia could follow a severe clinical and hematological course. The discovery of the G gamma type of delta 0 beta 0-thalassemia in a Turkish child shows that two types of delta 0 beta 0-thalassemia can be found in that country. Differentiation between the two types can only be made through structural analyses of Hb F. more...

Hb P-Nilotic which is produced by a hybrid of beta and delta genes was found in several members of a Sudanese family, three of whom had an associated beta-thalassemia. Chemical analyses confirmed the crossover between positions 22 and 50 of the beta delta P chain. The Hb p-Nilotic heterozygote had completely normal hematology, but the patients with the Hb P-Nilotic--beta-thalassemia condition had moderately severe clinical and hematological abnormalities which were considerably more pronounced than those in the father who had a beta-thalassemia heterozygosity. The absolute cellular contents of normal and abnormal non-alpha chains in these subjects and the results of in vitro chain synthesis analyses suggested that the thalassemia gene in this family is of the beta0 type and that the beta A gene which is present in cis to the beta delta P gene is incapable of being stimulated when the beta0-thalassemia determinant is present in trans. It is proposed that a number of recombination events produced a beta delta P hydrid gene with duplication of the beta A gene in cis as well as a change in an untranscribed strand of DNA which controls the expression of the beta A gene. more...