Analysis of mRNA from red cells of patients with thalassemia and hemoglobin variants

During the past decade new procedures have been developed for the isolation of RNA from a few mL of freshly collected blood. This material is reverse transcribed and the resulting cDNA can be used for the determination of the ratios between different types of globin mRNA, namely alpha 2/alpha 1, alpha/zeta, alpha/beta, gamma/beta, beta A/beta X, delta beta Lep/beta, and G gamma/A gamma. Details about these polymerase chain reaction-based methods are reviewed, and information about their usefulness in studying alpha-thalassemia, beta-thalassemia, sickle cell anemia and other beta-globin gene abnormalities, Hb Lepore heterozygosity, and heterozygosity for alpha 2- or alpha 1-globin gene mutations will be provided. The methods are also most useful in characterizing the mRNA types in single, in vitro cultured, BFU-E colonies; in colonies derived from cells of a Hb S heterozygote; for instance, the beta A- and beta(S)-mRNAs were present in all colonies and in about equal quantities, while many of those cells from a subject with a somatic cell mutant (Hb Costa Rica) contained beta A-mRNA and no beta-Costa Rica mRNA, and only a few had both types. The techniques described have considerable diagnostic value and offer a rather simple approach to the study of some of the listed diseases.     

Prevalence of alpha-thalassemias in northern Thailand

The population of northern Thailand has one of the highest frequencies of alpha-thalassemia in the world. However, the available distributional data are controversial. In addition to deletional types of alpha-thalassemia Hb, type Constant Spring should also be taken into consideration in alpha-thalassemia population studies, because it causes clinical alpha-thalassemia in the homozygous state or when present with both alpha-globin genes deleted in trans. We have examined a sample of 215 healthy subjects from four rural districts of Chiang Mai province. Out of these, 77 exhibited anomalies of the alpha-globin genes (alpha alpha/-alpha 3.7 in 36; -alpha 3.7/-alpha 3.7 in 3; -SEA in 30; alpha alpha/alpha CS alpha in 5; alpha alpha alpha anti 3.7 in 3). Therefore, no fewer than 2% of the children in northern Thailand are expected to be born with HbH disease or thalassemic hydrops fetalis. The considerable public health problem of hemoglobinopaties and the increasing acceptance of family planning necessitates facilities for the pre- and postnatal diagnosis of these disorders at the DNA level.     

High incidence of the CD8/9 (+G) beta 0-thalassemia mutation in Spain

BACKGROUND AND OBJECTIVE: In Spain, as in other Mediterranean regions the most common beta-thalassemia mutations are due to point mutations in gene regions that are critical for production of mRNA, such as [IVS-I-nt1 (G-->A), IVS-I-nt6 (T-->C), IVS-I-nt110 (G-->A)] which interrupt normal RNA processing or nonsense mutations [CD39 (C-->T)] which interrupt the translation of mRNA. The frameshift mutation CD8/9 (+G) is a very common allele in Asian Indians but is rare in the Mediterranean regions in which isolated alleles with this mutation have been found in Israel, Greece, Portugal and Turkey. DESIGN AND METHODS: We performed a molecular analysis of 175 chromosomes corresponding to 233 beta-thalassemia patients (221 heterozygous, 10 homozygous and 2 compound heterozygous) who belong to 169 Spanish families. The study of beta-thalassemia was made by PCR-ARMS, the alpha genes by Southern blot, the phenotype of Hb Lepore by enzymatic amplification and the presence of -158 gamma G C-->T mutation by PCR and digestion with the restriction enzyme XmnL. RESULTS: Twenty of these 233 patients showed the beta-thalassemia mutation CD8/9 (+G) (17 were heterozygous, 2 homozygous and in one patient the mutation was associated with a structural variant Hb Lepore Boston). INTERPRETATION AND CONCLUSIONS: These data reveal the heterogeneity of beta-thalassemia in Spain and the relatively high frequency (8.6%) of the frameshift mutation CD8/9 (+G). It is surprising that homozygotes for beta zero-thalassemia due to this mutation with very high Hb F values (around 90%) present a phenotype of intermediate thalassemia.     

Eosinophils and serum eosinophilic cationic proteins in interleukin-2-based immunotherapy for cancer

BACKGROUND: Extramedullary hematopoiesis (EMH) occurs in many disorders, including thalassemias and other hemoglobinopathies, and commonly presents in the spleen and liver. We present a case of spinal cord compression in a patient with beta-thalassemia intermedia, and review the literature and available treatment options. PATIENT AND METHODS: A 35-year-old black female with beta-thalassemia intermedia presented with a 3-week history of back pain and lower extremity weakness. Neurologic examination was consistent with spinal cord compression, and gadolinium enhanced magnetic resonance imaging (MRI) confirmed this diagnosis. She was given intravenous steroids and radiotherapy was begun in 200 cGy fractions to a total dose of 2000 cGy. RESULTS: At the completion of radiotherapy the patient was ambulatory with mild residual weakness. MRI scans 16 months later showed smaller, but persistent masses, and she remains asymptomatic 5 years from her diagnosis. CONCLUSION: Recognition of spinal cord EMH requires prompt physical examination and MRI for accurate diagnosis. EMH can be managed with radiation, surgery, transfusions, or a combination of these therapies. Radiation in conservative doses of (750-3500 cGy) is non-invasive, avoids the surgical risks of potentially severe hemorrhage and incomplete resection, and has a high complete remission rate in the majority of patients. Relapse rates are moderate (37.5%), but retreatment provides excellent chance for second remission.     

The beta-thalassemia frameshift at codon 8 (-AA) observed in four Chinese of the Uygur nationality

During a survey for beta-thalassemia in Kashi City, Xinjiang Province, P.R. China, four unrelated Uygur students were identified as heterozygotes for the beta-thalassemia frameshift at codon 8 (-AA). Detection was with the polymerase chain reaction combined with allele-specific oligonucleotide hybridization. This beta-thalassemia allele has not been observed in the Chinese population before and might be rather specific for the Uygur nationality.     

Heme synthesis in hereditary hemolytic anemias: decreased delta-aminolevulinic acid synthetase in hemoglobin K?ln disease

The activities of delta-aminolevulinic acid (ALA) synthetase and ALA dehydratase in cord blood erythrocytes of newborn infants and peripheral blood red cells of patients with beta-thalassemia major, beta-thalassemia intermedia, hemoglobin K?ln (Hb K?ln) disease, sickle cell anemia, and pyruvate kinase deficiency were studied. The activity of ALA dehydratase did not vary appreciably with the number of immature RBC (reticulocytes and nucleated red blood cells) or the severity of the hemolytic anemia except in pyruvate kinase deficiency. The activity of ALA synthetase was linearly correlated with the number of immature RBC (r=0.974, p is less than 0.001). The ALA synthetase activity was significantly decreased in the RBC of Hb K?ln (p is less than 0.01) when compared with the activity in immature RBC of newborns and of patients with pyruvate kinase deficiency, sickle cell anemia, and thalassemia intermedia.     

Oxidative damage and erythrocyte membrane transport abnormalities in thalassemias

Oxidative damage induced by free globin chains has been implicated in the pathogenesis of the membrane abnormalities observed in alpha and beta thalassemia. We have evaluated transport of Na+ and K+ in erythrocytes of patients with thalassemias as well as in two experimental models that use normal human red blood cells, one for alpha thalassemia (methylhydrazine treatment, alpha thalassemia like) and one for beta thalassemia (phenylhydrazine treatment, beta thalassemia like). With the exception of the Na-K pump, similar alterations in membrane transport were observed in thalassemia and thalassemia-like erythrocytes. These were: increased K-Cl cotransport, Na-Li countertransport and reduced Na-K-Cl cotransport. The Na-K pump was reduced in thalassemia-like cells, whereas it was increased in severe alpha thalassemia and in beta thalassemia cells. The increased K-Cl cotransport activity could be observed in light and dense fractions of beta-thalassemic cells. K-Cl cotransport in thalassemic and thalassemia-like erythrocytes was partially inhibited by [(dihydro-indenyl) oxy] alkanoic acid and completely abolished by dithiothreitol. Thus, oxidative damage represents an important factor in the increased activity of the K-Cl cotransport observed in thalassemias, and of the K+ loss observed in beta-thalassemia erythrocytes.     

Genetic basis of human complement C8 alpha-gamma deficiency

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.     

The molecular basis of beta thalassaemia in Punjabi and Maharashtran Indians includes a multilocus aetiology involving triplicated alpha-globin loci

We have analysed 201 beta-thalassaemia (beta-thal) genes from natives of the Punjab (156) and Maharashtra states of India and found the causative mutation in 200 of them. The most common beta-globin gene mutations differed significantly between these two groups and between these groups and Indian immigrants in the U.S.A. and the U.K. In the Punjabi Indians the IVS-1, nt 1 (G-T) mutation accounted for nearly one-quarter of beta-thal genes, whereas it was 5% or less in the other groups. Likewise, the cap + 1 mutation was much more prevalent in the Punjabis, whereas the nonsense codon 15 allele had a higher frequency in the Maharashtrans of the Bombay region. The common IVS-1, nt5 allele had a frequency of 60% of beta-thal genes in the Maharastrans, 35% in North American immigrants, and only 23% in the Punjabis. Two-thirds of all beta-thal genes in Punjab were found in the merchant caste (Khatri-Arora), whereas the menial caste (Shudra) was highly represented among those with beta-thal genes in Maharashtra. Two novel beta-globin alleles were each found once; a frameshift codon 55 (+A) in Maharashtrans and a frameshift codons 47-48 (+ATCT) in Punjabis. Of three Punjabi patients with beta-thal intermedia in whom only a single severe beta-globin gene mutation was found, two had six alpha-globin genes (homozygosity for a triplicated alpha-globin locus) instead of the normal alpha-globin gene number of four. Thus, these two individuals had a multilocus aetiology of beta-thal and their parents have the unusual recurrence risk of 1 in 8 for conceiving a third with beta-thal intermedia. Since 15% of 126 alpha-globin clusters studies in Punjabis contained either single (10%) or triplicated (5%) alpha-globin genes, the alpha-globin gene number is a frequent modifier of the phenotype of beta-thal in this ethnic group.     

Studies on fetal hemoglobin and gamma globin gene triplication in newborns in Jordan

Quantitation of fetal hemoglobin (Hb F) and quantitation of it's gamma chain composition is important for the identification of different hemoglobinopathies. This is the first study done on the Jordanian newborns to test the hematological data and the gamma globin chain variants. A total of 52 randomly selected healthy Jordanian newborns were examined. The quantitation of the G gamma and A gamma chains combined with gene mapping using XmnI digestion, were used in the identification of one case of G gamma triplication among the studied samples. A family study of this case showed that adults carrying one copy of this G gamma triplication (13Kb XmnI fragment) had normal levels of HbF (< 1%) and high levels of G gamma (> 80%) while no homozygotes were detected. The remaining 51 newborns had normal frequency values of G gamma and A gamma chains. The frequency of the A gamma T chain among the 52 samples was 0.22. No abnormal alpha or beta chain variants were detected except for one case of HbS.     

Prevalence and genotypes of alpha- and beta-thalassemia carriers in Hong Kong -- implications for population screening

BACKGROUND: The thalassemias are common in southern China. We determined the prevalence of heterozygous carriers of these genetic disorders in Hong Kong and assessed the feasibility of a community-based screening program. METHODS: An educational and screening program for the thalassemias was carried out in three high schools with a total of 2420 students. Seventy-five percent of the students agreed to undergo screening, which consisted of blood counts, hemoglobin electrophoresis, serum ferritin measurements, and DNA analyses. RESULTS: Of the 1800 blood samples tested, 150 (8.3 percent) had microcytosis (mean corpuscular volume, <80 microm3). Ninety students (5.0 percent) were carriers of alpha-thalassemia, of whom 81 (4.5 percent) were carriers of the Southeast Asian type of deletion, in which both alpha-globin genes on the same chromosome 16 are deleted. Sixty-one students (3.4 percent) were carriers of either beta-thalassemia or the mutation coding for hemoglobin E. Six students were carriers of both alpha- and beta-thalassemias. On the basis of these figures, the estimated numbers of pregnancies in Hong Kong in which the fetus is at risk for homozygous alpha-thalassemia and beta-thalassemia major or intermedia are 145 and 80 per year, respectively. In Hong Kong the actual numbers of women referred for prenatal diagnoses of these disorders are approximately 95 and 40 per year, respectively. CONCLUSIONS: Despite the availability of hospital-based screening and prenatal diagnosis for many years in Hong Kong, many women carrying fetuses at risk for thalassemia are not referred for genetic counseling. A community-based program of education, screening, and counseling is needed in Hong Kong and southern China.     

The expression of beta-glucuronidase during preimplantation development of mouse embryos

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.     

Genetic control of the development by retinoic acid

Two families of nuclear receptors for retinoic acid (RA) have been characterized. Members of the RAR family (types alpha, beta and gamma and their isoforms alpha 1, alpha 2, beta 1 to beta 4, and gamma 1 and gamma 2) are activated by most physiologically occurring retinoids (all-trans RA, 9-cis RA, 4oxo RA and 3,4 dihyroRA). In contrast, members of the RXR family (types alpha, beta and gamma and their isoforms) are activated by 9cis-RA only. In addition to the multiplicity of receptors, the complexity of retinoid signalling is further increased by the fact that, at least in vitro, RARs bind to their cognate response elements as heterodimers with RXRs. Moreover, RXRs can also bind, in vitro, to some DNA elements as homodimers, and are heterodimeric partners for other nuclear receptors, including TRs, VDR, PPARs and a number of orphan nuclear receptors. To evaluate the functions of the different RARs and RXRs types and isoforms, we have generated null mutant mice by targeted gene disruption in ES cells. As to the functions of RARs, we found that RAR alpha 1 and RAR gamma 2 null mutant mice are apparently normal. Mice deficient in RAR alpha or RAR gamma (i.e., all alpha or gamma isoforms disrupted) show aspects of the post-natal vitamin A deficiency (VAD) syndrome which can be cured or prevented by RA, including post-natal lethality, poor weight gain and male sterility. RAR beta 2 (and RAR beta) null mutants display a retrolenticular membrane which represents the most frequent defect of the fetal VAD syndrome. That these abnormalities were restricted to a small subset of the tissues normally expressing these receptors suggested that some degree of functional redundancy should exist in the RAR family. To test this hypothesis we then generated RAR double null mutants. RAR alpha beta, RAR alpha gamma and RAR beta gamma compound mutants exhibit all the malformations of the fetal VAD syndrome, thus demonstrating that RA is the vitamin A derivative which plays a crucial role at many different stages and in different structures during organogenesis. Interestingly, almost all the structures derived from mesenchymal neural crests cells (NCC) are affected in RAR compound mutants. As to the functions of RXRs, RXR gamma null mutants are viable, fertile and morphologically normal. In contrast, RXR alpha null fetuses display a thin ventricular wall and die in utero from cardiac failure. A myocardial hypoplasia has also been observed in some RAR compound mutants as well as in VAD fetuses. Thus, RXR alpha seems to act as an inhibitor of ventricular cardiocyte differentiation and/or as a positive regulator of their proliferation, and these functions might involve heterodimerization with RARs and activation by RA. RXR beta null mutants are viable but the males are sterile, most probably because of an abnormal lipid metabolism in the Sertoli cells. New abnormalities, absent in RXR alpha mutants, are generated in RXR alpha/RAR (alpha, beta or gamma) compound mutants. All these abnormalities are also seen in RAR double mutants as well as in VAD fetuses. In contrast, such manifestations of synergism are not observed between the RXR beta or RXR gamma and the RAR (alpha, beta or gamma) null mutations. These data strongly support the conclusion that RXR alpha/RAR heterodimers represent the main functional units of the RA signalling pathway during embryonic development. Moreover, since RXR gamma-/-/RXR beta-/-/RXR alpha +/-mutants are viable, a single allele of RXR alpha can perform most of the developmental RXR functions.     

Comparison of expression of human globin genes transferred into mouse erythroleukemia cells and in transgenic mice

To examine whether transfer of gamma globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (microLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the muLCR(-201)Agamma and muLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.     

PMR-relaxation and steric computations give unequivocal nucleoside conformations

The configuration and the conformation of alpha and beta anomers of pyrazomycin, cytidine and pseudouridine in aqueous solution have been investigated by 1H-NMR at 250 MHz. T1 proton relaxation measurements are an excellent method to determine the conformation of the base around the glycosidic linkage. Frequently, steric hindrance considerations can help to decide which conformations are possible in nucleoside anomer pairs. The proton-proton coupling constants indicate that the N conformer is largely predominant in the alpha anomers while the S conformer is particularly abundant in beta-pyrazomycin. The steric hindrance is much larger for alpha than for beta-nucleosides and change of a C-C to a C-N glycosidic bond reduces considerably the rotational possibilities of the base. The relaxation data show that alpha-cytidine adopts the anti conformation with gamma = 200 degrees in good agreement with the crystal structure and with the sterical computations. In the other case, when the syn and anti conformations are sterically accessible, the orientation of the base may be completely different from one nucleoside to the other. It can be predicted neither from the crystal structure nor from comparisons with similar compounds. For alpha-pseudo-uridine the predominant orientation of the base (gamma = 120 degrees) is in the boundary of the syn-anti regions; for beta-cytidine the syn (gamma = 65 degrees) and anti (gamma = 215 degrees) conformations are equiprobable at room temperature while beta-pseudouridine shows the syn conformation with gamma = 40 degrees, the smallest angle observed until now. There is no correlation between the N/S and syn-anti ratios.     

Protein interactions during assembly of the enamel organic extracellular matrix

This paper describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the detection of human globin chains in blood and bloodstains. The method involves direct injection of the filtered samples of dilute hemolysates or bloodstain extracts onto a microbore C4 reversed-phase column (2.1 mm I.D.) with UV detection at 220 nm. Microbore HPLC offers a significant improvement in sensitivity with little loss of the resolution of globin chains and only small variations in the determination of gamma chain composition. The detection limit of hemoglobin (Hb) was 0.1 microgram, which is equivalent to about 1 nl of fresh whole blood. Umbilical cord blood could be differentiated from adult blood in stains that were up to twenty weeks old, by the presence of gamma globin chains. The present method will be useful for detection of abnormal Hbs and for the determination of gamma chain composition in clinical laboratories, as well as in the practice of forensic science for the analysis of minute amounts of blood and bloodstains.