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.     

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.     

Molecular diagnosis of beta-thalassemia intermedia

OBJECTIVES: To analyze the molecular abnormalities of beta-thalassemia intermedia and contribute to the knowledge of the molecular diagnosis and prenatal diagnosis of this disorder. METHODS: In 14 patients with beta-thalassemia intermedia, we analyzed the hematologies, alpha, beta and gamma globin gene organization and structure as well as globin gene biosynthesis by Southern blot hybridization, multiplex allale specific PCR (MAS-PCR), DNA sequencing and micro-globin chain biosynthetic assay. Moreover, alpha globin gene organization was studied in 250 cord blood specimens. RESULTS: Of the 14 patients, 4 were found to be beta-thalassemia heterozygotes combined with rightward cross-over or/and leftward cross-over triplicated haplotype of alpha-globin gene loci (alpha alpha alpha anti3.7 or/and alpha alpha alpha anti4.2), 3 were compound heterozygotes for beta-thalassemia combined with alpha-thalassemia 1 or 2, one was identified to be a compound heterozygote for beta-thalassemia combined with G gamma promotor-158 (C-->T) mutation. The data of the alpha globin gene organization in 250 cord blood specimens showed that 8 of the 500 tested chromosomes (1.6%) were abnormal: 3 were alpha alpha alpha anti3.7, 4 were alpha -3.7, and one was --SEA. CONCLUSION: In addition to beta-thalassemia homozygote or compound heterozygotes with alpha thalassemia, the conjunctive abnormalities of beta-thalassemia heterozygote with alpha-globin gene triplication was another major cause of beta-thalassemia intermedia.     

A new method of nonradioactive labelling of oligonucleotides and their use as allele-specific probes for detecting mutations causing beta-thalassemia

We have developed simple and efficient methods for synthesis of biotin and horseradish peroxidase (HRP)-labeled oligonucleotides. Biotinylated oligonucleotides were obtained in quantitative yields, and oligonucleotide conjugates with HRP in 60-80% yields. Allele-specific oligonucleotide probes for the diagnostics of IVS 1-110 mutation in the beta-globin gene causing beta-thalassemia were thus obtained. Temperature conditions for the non-radioactive ASO hybridization with the amplified segment of the human beta-globin gene and wash conditions were selected. HRP-labelled probes were used in hybridization without preliminary separation after synthesis. To decrease nonspecific enzyme binding we have elaborated special conditions for membrane blocking. Detection of the biotinylated probe was carried out with the help of a streptavidin--HRP conjugate. O-Dianisidine was used as a chromogenic substrate. We have demonstrated the usefulness of this method in the analysis of amplified samples of DNA obtained from blood of patients homozygous in the mutant gene, and heterozygous carriers.     

Codon 4 ACT-->ACA, codon 5 CCT-->TCT, and codon 6 GAG-->TAG mutations in cis position: a form of thalassemia trait

A female of Uttar Pradesh, of Indian origin, who had a transfusion-dependent child, carried codon 4 ACT-->ACA, codon 5 CCT-->TCT, and codon 6 GAG-->TAG mutations at the cis position. The mutation was detected through sequencing of the amplified beta-globin gene. Heterozygosity is expressed as a thalassemia trait with moderate anemia, low MCV (57 fl), raised HbA2 (6.7%), and normal fetal hemoglobin (1.4%).     

Association of alpha and beta thalassemia with alpha gene triplication in one family

We describe the haematological data and molecular results of a native family from Cádiz in that one is produced the a within heterozygous beta 0 thalassaemia (IVS-1, nt 1-G-->A), heterozygous alpha+ thalassaemia (-alpha 3.7) and alpha gene triplication (alpha alpha alpha 3.7). PATIENTS AND METHODS) We are studied 7 members to a family composed by father (I1), mother (I2) and five children (II1, II2, II3, II4, II5). The molecular biology study of the alpha gene was realized by Southern blot method using the restriction enzymes Bam HI, Bgl II and Eco RI and hybridized with alpha probe of the plasmid PRB 1 (fragment of 1.5 Kb digested with the enzyme Pst I). The genes were studied by the technique of the polymerase chain reaction (PCR), modified according to designated method "Amplification Refractory Mutation System" (ARMS). RESULTS: The father (I1) presents an interaction of therozygous beta 0 thalassaemia with heterozygous alpha + thalassaemia (beta 0/beta 1;alpha alpha/-alpha 3). The mother (I2) shows an alpha gene triplication (beta A/beta A: alpha alpha alpha 3.7/alpha alpha). Finally the children are expressed 5 possibilities: II4 he is normal (beta A/beta A; alpha alpha/alpha alpha), II2 he has alpha gene triplication (beta A/beta A; alpha alpha/alpha alpha alpha 3.7), II3 he has heterozygous beta 0 thalassaemia (beta 0/beta A; alpha alpha/alpha alpha), II5 he has interaction between heterozygous beta 0 thalassaemia and heterozygous alpha gene triplication (beta 0/beta A; alpha alpha alpha 3.7/alpha alpha) and II1 presents an interaction between a heterozygous beta 0 thalassaemia and together with the lost of one alpha gene in one chromosome he also presents a alpha gene triplication in other one (beta o/beta A; alpha alpha/alpha alpha). The hematological data of II5 corresponds to a intermediate thalassemia with not transfusion dependent feature an opposite to II1 that presents a heterozygous thalassemic trait features with 4 alpha genes. DISCUSSION: The phenotypical expression of the different interactions of these mutations in this family, points out, the relevant role that the unbalance globins chains plays in the pathogenesis and development of the clinical manifestations of the patients with the thalassaemia syndromes.     

Update on occupational natural rubber latex allergy

Three beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) deficiency is a form of congenital adrenal hyperplasia characterized by severe impairment of steroid biosynthesis in the adrenals and gonads. To better understand the molecular basis of the phenotypic heterogeneity found in 3 beta HSD deficiency, we analyzed the structure of type I and II 3 beta HSD genes in a female patient with nonsalt-losing 3 beta HSD deficiency diagnosed at puberty. We directly sequenced DNA fragments generated by polymerase chain reaction amplification of the four exons, the exon-intron boundaries, and the 5'-flanking regions of each gene. No mutation was detected in the type I 3 beta HSD gene, which is the predominant species expressed in the placenta and peripheral tissues. We detected a novel missense mutation, Y254D, in one allele of the patient's type II 3 beta HSD gene, which is the almost exclusive type expressed in the adrenals and gonads. The influence of the Y254D mutation on enzymatic activity was assessed by analyzing the recombinant mutant enzyme generated by site-directed mutagenesis after its transient expression in COS-1 monkey kidney cells. Recombinant mutant type II 3 beta HSD enzyme carrying the Y254D substitution exhibits no detectable activity with C21 delta 5-steroid pregnenolone or C19 delta 5-steroid dehydroepiandrosterone used as substrate. The absence of restriction fragment length polymorphism by Southern blot analysis and the finding that all of the amplified DNA fragments possess the expected length suggest the absence of deletions, duplications, or re-arrangements in the other allele. A putative second mutation could be located farther than 1427 basepairs upstream of the initiation codon, thus potentially affecting the normal expression of this gene or within intronic regions, generating an alternative aberrant splicing site. These are possibilities that remain to be elucidated. The present findings, which describe the novel missense mutation Y254D in the human type II 3 beta HSD gene, provide useful information on the structure-activity relationships of the 3 beta HSD superfamily.     

4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-3-deoxy-D-manno-2-octulosonic acid, a constituent of lipopolysaccharides of the genus Pectinatus

A thyroglobulin (Tg) synthesis defect in Dutch goats causes congenital goiter and hypothyroidism. The disease is inherited in an autosomal recessive way and is linked to restriction fragment length polymorphisms (RFLPs) in the Tg gene. Previous studies showed that Tg mRNA isolated from the goiters was of normal size (8.4 kilobases). Translation of high mol wt polysomal Tg mRNA isolated from goiter in a cell-free rabbit reticulocyte lysate resulted in a single 35,000 mol wt Tg polypeptide. Tg antigens analyzed in T4-arrested goiters were glycosylated and had mol wt of 40,000 and 32,000. The aim of this study was to identify the molecular lesion responsible for this disease. Polysomal Tg mRNA, therefore, was isolated, and cDNA was made using oligonucleotides as primers. This cDNA was multiplied by the polymerase chain reaction and cloned. In comparing the normal and abnormal sequences, we found a C-->G point mutation in exon 8 causing a change from TAC (Tyr)-->TAG (termination signal) at amino acid position 296. This mutation resulted in the appearance of a KpnI restriction site in the goiter DNA. The sequence of Tg mRNA preceding the stop codon was equal for normal and goitrous goats, except for one C-->T mutation in exon 5 which gave a Ser-->Leu transition. The KpnI site introduced by the C-->G point mutation was present in chromosomal DNA of the goitrous goats, making it possible to distinguish goats heterozygous for the defect from normal and goitrous animals. We calculated that the stop codon in exon 8 would result in a Tg polypeptide chain with a mol wt of 39,000, in good agreement with the mol wt of the in vitro and in vivo translation products. In conclusion, the C-->G mutation causing a stop codon in exon 8 is responsible for the Tg synthesis defect in Dutch goats.     

Escherichia coli MutY protein has a guanine-DNA glycosylase that acts on 7,8-dihydro-8-oxoguanine:guanine mispair to prevent spontaneous G:C-->C:G transversions

Low rates of spontaneous G:C-->C:G transversions would be achieved not only by the correction of base mismatches during DNA replication but also by the prevention and removal of oxidative base damage in DNA. Escherichia coli must have several pathways to repair such mismatches and DNA modifications. In this study, we attempted to identify mutator loci leading to G:C-->C:G transversions in E.coli. The strain CC103 carrying a specific mutation in lacZ was mutagenized by random miniTn 10 insertion mutagenesis. In this strain, only the G:C-->C:G change can revert the glutamic acid at codon 461, which is essential for sufficient beta-galactosidase activity to allow growth on lactose. Mutator strains were detected as colonies with significantly increased rates of papillae formation on glucose minimal plates containing P-Gal and X-Gal. We screened approximately 40 000 colonies and selected several mutator strains. The strain GC39 showed the highest mutation rate to Lac+. The gene responsible for the mutator phenotypes, mut39 , was mapped at around 67 min on the E.coli chromosome. The sequencing of the miniTn 10 -flanking DNA region revealed that the mut39 was identical to the mutY gene of E.coli. The plasmid carrying the mutY + gene reduced spontaneous G:C-->T:A and G:C-->C:G mutations in both mutY and mut39 strains. Purified MutY protein bound to the oligonucleotides containing 7,8-dihydro-8-oxo-guanine (8-oxoG):G and 8-oxoG:A. Furthermore, we found that the MutY protein had a DNA glycosylase activity which removes unmodified guanine from the 8-oxoG:G mispair. These results demonstrate that the MutY protein prevents the generation of G:C-->C:G transversions by removing guanine from the 8-oxoG:G mispair in E.coli.     

Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.     

Epidural steroid-based technique for cervicogenic headache diagnosis

We present a case of a two year old infant with jaundice in the neonatal period, anemia and splenohepatomegaly. Hemoglobin electrophoresis of the child and siblings revealed double heterozygosity of Lepore/beta thalassemia in the child and father, and heterozygous beta-thalassemia in the uncle. Structural variants giving rise to thalassemia phenotype are discussed.     

Endoscopic stapling diverticulotomy of pharyngeal pouch

PURPOSE: Homozygosity for Hb D-Punjab (or Hb D-Los Angeles; codon 121; GAA-->CAA) is rare among Arabs. The co-inheritance of Hb D with beta(0)-thalassemia trait is even rarer, with only 10 previous cases reported worldwide. PATIENTS AND METHODS: We present clinical and hematological data for two Hb D homozygotes and three compound heterozygotes for Hb D-Punjab and beta(0)-thalassemia (IVS-II-1; G-->A). All the individuals belong to a consanguineous Kuwaiti Arab family. The hemoglobin variant and the beta-thalassemia allele were characterized by sequencing, allele-specific amplification, and oligonucleotide hybridization. RESULTS: The hematology was unremarkable except for a moderate elevation of Hb F (3-4%) and significant hypochromia and microcytosis in the subject with Hb D/beta(0)-thalassemia. CONCLUSION: This report confirms the benign nature of homozygosity for Hb D.     

Correlation between soluble transferrin receptor and serum ferritin levels following bone marrow transplantation for thalassemia

This study analyzes the serum transferrin receptor (sTfR) levels in a series of 230 ex-thalassemics with a follow-up of 1 to 9 years after bone marrow transplantation (BMT) for homozygous beta thalassemia. Ex-thalassemics are individuals, cured of homozygous beta thalassemia by BMT, who maintain different degrees of iron overload acquired during the pretransplant period. Both in experimental and clinical conditions, sTfR concentrations have been shown to be a quantitative measure of body iron status. This study was carried out to assess whether the level of sTfR may be of help in determining the extent of iron overload in ex-thalassemics. Patients who received the marrow from their HLA-identical sibling donor heterozygous for beta thalassemia, namely heterozygous ex-thalassemics, displayed significantly higher levels of sTfR than patients transplanted from their normal sibling donors (normal ex-thalassemics). This finding suggests that increased erythropoiesis, albeit in part ineffective in heterozygous ex-thalassemics, is responsible for the sTfR increment. Both heterozygous and normal ex-thalassemics had significant lower sTfR levels than their heterozygous (p < 0.003) or normal (p < 0.0001) donors, respectively. These differences may be ascribed to the presence of iron overload in ex-thalassemics in comparison to their normal or heterozygous donors who did not present excess of iron in the body. A significant inverse correlation between sTfR and serum ferritin levels (r = -0.54, p < 0.0001) was found when normal ex-thalassemics were considered. In heterozygous ex-thalassemics, the lack of correlation between these two parameters may be explained by the enhanced erythropoietic activity of individuals with thalassemic trait. These results suggest that the level of sTfR may be a useful indicator of iron overload in normal ex-thalassemics.     

Identification of Hb C [beta 6(A3)Glu-->Lys] in a Thai male

The propositus was a 29-year-old Thai male, whose electrophoretic pattern showed Hb A (58%) plus an abnormal hemoglobin (42%) with mobility identical to Hb A2 and Hb E. Protein sequencer analysis and tryptic peptide mapping of the beta chain indicated that the abnormal hemoglobin was Hb C [beta 6(A3)Glu-->Lys], rather than Hb E which is more commonly found in South East Asia. This conclusion was confirmed by direct sequence analysis of the propositus' DNA, which showed AAG as well as GAG at codon 6 of the beta gene, in agreement with heterozygosity for Hb C and Hb A. Furthermore, the beta gene framework (Ava II-, Bam HI+) of the propositus suggested that the beta C gene may have arisen from an independent mutation. Since Hb C and Hb E have the same mutation (Glu-->Lys) in the beta chain, although at different positions, and behave similarly in electrophoresis, cases of Hb C and Hb E may sometimes have been mistakenly identified for each other, based on whichever variant is most prevalent in the particular population.     

Mutation in the thyroid hormone receptor beta gene (A317T) in a Thai subject with resistance to thyroid hormone

Analysis of the thyroid hormone receptor beta (TRbeta) gene of a Thai female with the syndrome of resistance to thyroid hormone (RTH) revealed a missense mutation at codon 317, changing the guanine in nucleotide 1234 to an adenine that results in the replacement of the normal alanine (GCT) with a threonine (ACT). The proposita was heterozygous, and this mutation was not present in her parents and her sister, compatible with a neomutation. This is the first report of TRbeta gene mutation causing RTH in an individual of Thai origin.     

Studies of left-ventricular function in anemia due to beta-thalassemia

Left-ventricular (LV) function was studied in 23 patients with anemia due to beta-thalassemia, of whom seven had thalassemia intermedia and the remainder thalassemia major. Two-thirds of the patients wih thalassemia intermedia and almost all the patients with thalassemia major were in clinical congestive heart failure. Despite this, resting measurements of ventricular size and systolic ventricular function were normal, indicating high-output cardiac failure. However, effort testing showed a flat response or decrease in the LV shortening fraction in patients with thalassemia major, and serial studies showed a decrease in the shortening fraction over a 4-yr period in some patients. LV diastolic function was studied by calculating peak LV filling rate and the pattern of LV filling in early diastole. Three patient with thalassemia major showed a pattern indicating abnormal LV distension. Since LV end-diastolic dimension was increased, volume overload was present in all patients. The results indicate that the following factors contribute to the genesis of cardiac failure in beta-thalassemia: 1) diminished response of systolic ventricular performance to exercise and later at rest; 2) ventricular volume overload; and 3) abnormal ventricular distension in diastole. Although the ventricular filling suggests abnormal LV compliance, the effect of right-ventricular volume overload or a pericardial factor cannot be excluded.     

Pre-rupture diagnosis and management of rudimentary horn pregnancy in the first trimester

We analyzed the hemoglobins of a Japanese girl with beta-thalassemia and those of her immediate family. DNA sequencing of the cloned beta-globin gene from this patient revealed a point mutation at the IVS-I position 1 (G-->T). This rare point mutation has been found in Asian Indians, but this is the first reported Japanese case.     

Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes

About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched anti-sense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells.