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Fetal hemoglobin (Hb F) is characteristic of the fetal development period. However, in some genetic conditions, such as hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia), Hb F continues to be produced in adulthood. We evaluated the frequency of two mutations of HPFH, HPFH-1 and HPFH-2 African, and two mutations in δβ-thalassemia, Sicilian and Spanish, in a Brazilian population. Peripheral blood samples were collected from adults from hospitals and blood centers in southeast and northeast Brazil. These individuals were healthy and without complaints of anemia, but had increased Hb F. Samples were submitted to electrophoretic and chromatographic analyses to quantify Hb F values and, subsequently, to molecular analyses to verify the mutations. In the molecular analysis, 16 of the 60 samples showed a heterozygous profile for the HPFH mutations, two for HPFH-1 and 14 for HPFH-2. In the same sample set, three were heterozygous for Spanish δβ-thalassemia and none were heterozygous for Sicilian δβ- thalassemia. The Hb F values in the HPFH-2 heterozygotes differed from those previously reported for this mutation. In this group, the HPFH mutations were more frequent than the δβ-thalassemia mutations. The finding of these mutations in this Brazilian population reflects the mixing process that occurred during its formation.

Objectives: To assess the clinical significance of the interactions of CAP+1 (A-->C), a silent beta-globin gene mutation, with other beta-thalassemia mutations and globin gene modifiers in north Indians., Methods: The clinical phenotypes associated with compound heterozygosity for the CAP+1 (A-->C) mutation with other beta-thalassemia mutations, together with the potential effect of the genetic modifiers alpha-thalassemia and the Xmn-1(G)gamma C-->T polymorphism were studied in 30 patients. The frequency of the CAP+1 (A-->C) polymorphism was determined and an analysis of the red cell indices, HbA(2) levels, iron status, and alpha-globin genes was carried out in 35 heterozygotes., Results: Based on an analysis of 1075 beta-thalassemia alleles the CAP+1 (A-->C) mutation constituted 3.2% of north Indians. There was a wide spectrum of phenotypic severity in compound heterozygotes; 18 of 30 were transfusion dependent. There was a very high frequency of the -/- genotype of the Xmn-1(G)gamma polymorphism in compound heterozygotes. Analysis of 35 heterozygotes indicated that approximately half were hematologically normal and therefore genuine 'silent' carriers., Conclusions: Compound heterozygotes for CAP+1 (A-->C) and other severe beta-thalassemia alleles are phenotypically severe enough to necessitate appropriate therapy and counseling. The unexpected severity of these interactions may be due, in part, to the high frequency of beta-thalassemia alleles associated with the Xmn-1(G)gamma- allele in Indian populations. It is concluded that the CAP+1 (A-->C) mutation can pose serious difficulties in screening and counseling programs in populations in which it occurs at a significant frequency.

The phenotype of increased Hb A2 typical of beta-thalassaemia (beta-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a delta-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of delta-globin mutations in the Portuguese population we performed a mutational analysis of the delta-globin gene in a group of 51 Portuguese beta-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have delta-globin structural abnormalities. The heterozygosity for the beta(+)IVS-I-6T-->C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical beta-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common beta-thal mutation and the delta(+)Cd27G-->T mutation (Hb A2-Yialousa; HBD:c.82G>T). One of them also presented a novel delta-globin gene promoter mutation,-80G-->A (HBD:c.-130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have delta-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified - Hb A2' (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [delta64(E8)(Gly-->Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different delta-globin mutations were found, being two of them new molecular defects. All delta-alleles identified were found linked to the expected beta-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a beta-thal allele.

In this report we examine the (AC)n(AT)xTy motif residing -530 bp 5' upstream of the beta-globin gene in Chinese thalassaemic patients. This motif is a putative binding site for a repressor protein, termed beta protein 1 (BP1) (Berg et al., Nucleic Acids Res 1989;17:8833-8852). Variations in the (AC)n(AT)xTy repeats affect the binding affinity of BP1, thereby altering the expression of the beta-globin gene. Eight different configurations of this repeat motif are identified in our population of Chinese beta-thalassaemia patients. A (AC)3(AT)7T5 motif was identified among these thalassaemia patients and its influence in beta-globin gene expression was studied using stable transfection assay in murine erythroleukemia (MEL) cells. Our data demonstrated that the (AC)3(AT)7T5 motif has a moderately strong repressor effect on the expression of the cis-linked beta-globin gene. The high affinity of BP1 for this motif may result in the suppression of the transcription of the beta-globin gene (Berg et al., Am J Hematol 1991;36:42-47). We postulate that silencer elements in the beta-globin promoter play an important role in modifying the clinical presentation of the disease., ((c) 2006 Wiley-Liss, Inc.)

Various factors have been described as phenotypic modulators of sickle cell disease, such as levels of fetal hemoglobin (Hb F), presence of alpha-thalassemia (thal), and haplotypes of the beta-globin genes. In order to characterize and determine the frequency of the betaS and betaC mutations and the prevalence of -alpha3.7-thal, 74 patients with sickle cell disease detected during neonatal screening in the State of Pernambuco, Brazil, were studied. The haplotypes of the beta gene and -alpha3.7-thal were determined using polymerase chain reaction (PCR), and specific restriction endonucleases were used to establish the polymorphic sites of the haplotypes. The results showed the high frequency of the Central African Republic (CAR) or Bantu haplotype in the State of Pernambuco, Brazil. The low frequency of the Benin haplotype recorded in this study, in comparison with other states in northeast Brazil, suggests the diversity of origins of Afro-Brazilians in this region.

There are few studies investigating alpha globin gene triplications in beta-thalassemia in Asian Indians and its effect on phenotype, which was the primary aim of this study. Gap-PCR was performed in order to detect common alpha thalassemia determinants (-alpha(3.7), -alpha(4.2) and alpha alpha alpha(anti 3.7) triplication). Alpha-triplication was detected in 15.4% (10/65) of patients with thalassemia intermedia, 8.8% (4/45) of those with thalassemia minor and in 2.7% (2/74) of healthy controls. The severity of jaundice was higher in thalassemia intermedia cases with alpha-triplication and two of the alpha-triplication cases had a marked increase in serum bilirubin following intercurrent illness. Thus, alpha globin gene triplication is important genetic determinant underlying thalassemia intermedia in North Indians. Patients with alpha-triplication may develop prominent jaundice with marked increase in serum bilirubin following antecedent aggravating factors.

We have found an example of the nonsense beta-thalassemia (thal) mutation at codon 37 (TGG-->TAG; Trp-->Stop) in a Chinese family. The fetus, who inherited both parents' beta-thalassemic alleles, was a compound heterozygote for the codons 41/42 (-TCTT) and codon 37 (TGG-->TAG) mutations, and presented with the phenotype of severe beta-thal.

The determination of the prevalence of inherited hemoglobin (Hb) disorders in endemic areas is important in order to develop programs for their control and management. The aim of this study is to determine the prevalence of inherited Hb diseases in Guinea-Bissau which is situated on the west coast of Africa, between Senegal and Guinea. One thousand and fifty-seven blood samples were collected and analyzed with high performance liquid chromatography (HPLC) for detection of beta-thalassemia (thal) and Hb variants, and by gap polymerase chain reaction (gap-PCR) for the detection of deletions in the alpha-globin genes. We found 4.7% children were heterozygous for Hb S [beta6(A3)Glu-->Val, GAG -->GTG], 0.2% were homozygous for Hb S, and 0.3% were heterozygous for Hb C [beta6(A3)Glu-->Lys, GAG -->AAG]. One child had heterozygous beta+-thal, 13.8% were heterozygous for the -alpha3.7 deletion, 1.5% were homozygous for the -alpha3.7 deletion, and 1.5% were heterozygous for the -alpha4.2 deletion. We recommend national screening programs to focus primarily on sickle cell disease, since beta-thal is rare, and the observed alpha-thal deletions are of minor genetic importance.

beta-thalassaemia major, an autosomal recessive hemoglobinopathy, is one of the most common single gene disorders in multi-racial Malaysia. The control of beta-thalassaemia major requires a multi-disciplinary approach that includes population screening, genetic counselling, prenatal diagnosis and the option of termination of affected pregnancies. To achieve this objective, the molecular characterisation of the spectrum of beta-globin gene mutations in each of the affected ethnic groups is required. We studied 88 consecutive unrelated individuals and their respective families with beta-thalassaemia (74 beta-thalassaemia major, 12 HbE-beta-thalassaemia, 2 with HbE homozygotes) and four individuals with beta-thalassaemia trait that contributed a total 180 alleles for study. Using a 2-step molecular diagnostic strategy consisting of amplification refractory mutation system (ARMS) to identify the 8 most common mutations followed by other DNA-based diagnostic techniques, a total of 177 (98.3 per cent) of the 180 beta-thalassaemia alleles were characterised. One out of 91 (1 per cent) of the Chinese alleles, one out of 46 (2.2 per cent) Malay alleles and one out of two Indian alleles remained unknown. A 100 per cent success rate was achieved in studying the Kadazandusun community in this study. A strategy to identify beta-globin gene mutations in Malaysians with beta-thalassaemia is proposed based on this outcome.

beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.

To test the feasibility of developing a diagnostic microarray for a specific disease, we selected all pathogenic changes of the beta-globin gene occurring at a frequency >/=1% in the multi-ethnic Dutch population for analysis. A tagged single-base extension (SBE) approach was used to detect 19 different mutations causing beta-thalassemia or abnormal hemoglobins. In the SBE reaction, the primers were elongated at the 3'site with a fluorescently labeled dideoxyribonucleotide triphosphate (ddNTP) complementary to the mutation, following tag hybridization to a glass or flow-through microarray. We compared the performance of a generic glass array and a porous system, by testing each mutation separately using heterozygous carriers and by screening a cohort of 40 unknown beta-thalassemia carriers and patients. The results were verified by direct sequencing. The microarray system was able to detect 17 beta-globin mutations simultaneously with >95% accuracy in a single SBE reaction. The flow-through array performed slightly better (96%), but the main advantages of the system included real-time data recording and a considerable time saving achieved through a reduced hybridization time.

Two hundred and nine beta-thalassaemia (beta-Thal) alleles of 158 unrelated Myanmar patients (107 HbE-beta-Thal; 51 beta-Thal major) were analysed for beta-globin gene mutations. Amplification refractory mutation system (ARMS) characterized six beta-thal mutations known to Myanmar [betaIVSI-1(G-->T), codon 41/42(-TCTT), betaIVSI-5(G-->C), codon 17(A-->T), betaIVS II-654(C-->T), and -28 Cap (A-->G)] in 166/209 (79.4%) alleles. DNA sequencing of 24 alleles from 43 ARMS-negative samples (20.6%) identified an additional 12 new mutations, to produce a total of 18 different mutations. Nineteen alleles (9.1%) remained for further characterization. The molecular spectrum of Myanmar beta-Thal is wider and more heterogeneous than previously reported.

Objective: To establish an improved, simple and convenient megaprimer PCR method for site-directed mutagenesis(SDM)., Methods: This protocol is based on the design of two different plasmid DNA templates. Template 1 lacks the binding site for reverse flanking primer, and template 2 lacks the binding site for forward flanking primer. This modification avoids amplification of full-length wild-type sequences while two templates, the forward and reverse flanking primers exist simultaneously in one reaction tube. A megaprimer is synthesized in the first PCR reaction using template 1, forward primer and mutagenic primer. The megaprimer that needn't the cumbersome gel purification step is directly added to the second PCR reaction system. The second PCR reaction(PCR 2) containing two stages is performed using template 2, megaprimer, forward and reverse flanking primer. During the first stage of PCR 2, the megaprimer is extended to form the full-length mutation product. In the second stage of PCR 2, the extended megaprimer containing SDM residues is subsequently amplified with the two flanking primers. All of the final PCR products contained the desired mutation., Results: Fifteen types of rare beta-thalassemia mutations in Chinese were obtained using this method. Each of these modified fragments was separately cloned into the pGEM-T vector and sequenced. The desired mutations involved in mutagenesis amplicons were identified in all clones., Conclusion: This improved PCR-based megaprimer method for site-directed mutagenesis is rapid, simple and highly efficient, and the success rate of mutagenesis could reach 100%. Furthermore, this method is suitable for routine application in molecular cloning.

A study of the spectrum of beta-thalassemia mutations in the southern part of the West Bank of the Palestinian Authority revealed the presence of 10 different beta-globin mutations. The study included 41 patients and 54 carriers of beta-thalassemia and sickle cell anemia. The spectrum of mutations observed was typically Mediterranean. However, their relative frequencies was unique. The predominant allele was IVS-I-6 (T-->C), with an exceptionally high frequency of 48.5% for this mutation. The homozygous IVS-I-6 patients had widely variable clinical presentations, from typical transfusion-dependent thalassemia major to non-transfusion-dependent thalassemia intermedia phenotype. Since it is so widespread in these West Bank populations, the IVS-I-6 mutation may date back to ancient times. The nonsense mutation at codon 37 (G-->A) was found at a relatively high frequency of 11.3%, supporting the hypothesis that it originated in this region. The other mutations, at decreasing frequencies ranging from 9.5-1.5%, were: IVS-I-110 (G-->A), frameshift codon 5 (- CT), IVS-I-1 (G-->A), IVS-II-1 (G-->A), Hb S [beta6(A3)Glu-->Val], frameshift codons 8/9 (+G), codon 39 (C-->T), and -30 (T-->A). Our findings will improve health care for the Palestinian population, and also has implications for the study of the origin and spread of thalassemia in the Middle East.

The Great Andamanese are a primitive Negrito tribe of the Andaman and Nicobar Islands, India, with a total population of 37. We studied 29 individuals from eight families from this population for abnormal hemoglobins, G6PD deficiency, DNA haplotypes, and apolipoprotein B (APOB, gene) polymorphism. Hb E was detected in five individuals, the prevalence of Hb E heterozygotes being 14.3%. One individual had beta-thalassemia trait. One female was G6PD deficient and showed the G6PD Orissa mutation. Haplotype analysis of the beta-globin gene cluster showed that the betaE chromosomes were linked to two haplotypes (- - - - - + + and + + - + + + +) representing the framework 1 gene, whereas the betaA chromosomes showed eight different haplotypic patterns corresponding to framework 1 and 3 genes. APOB polymorphism analysis showed that the 631-base-pair (bp) allele was the predominant one with a high homozygosity rate, which could be due to the higher rate of inbreeding in this isolated group. The presence of Hb E and our findings on haplotype analysis supports the hypothesis that the Great Andamanese are reasonably believed to be the surviving representatives of the Negrito race that once flourished in the entire Southeast Asian region in ancient times.

Prevention of beta-thalassemia implies knowledge of the molecular spectrum occurring in the population at risk. This knowledge is necessary, especially when a prevention protocol is applied to a multiethnic population. For this purpose, we have recently analyzed a large population of Iranian patients living in the Province of Hormozgan in Iran, and a small group of Iranian patients living in The Netherlands. We have found a different mutation spectrum in both populations as compared to the data obtained by other authors for the Iranian regions of Tehran, Fars, Sistan Balouchestan, Bushehr, and Khouzestan. The IVS-I-5 (G-->C) is the most frequent mutant in the province of Hormozgan (69%), followed by the IVS-II-1 (G-->A) (9.6%), while the IVS-I-1 (G-->A) was the most frequent defect found in the Iranian population sample in The Netherlands. The IVS-II-745 (C-->G) mutation in cis with the 5'UTR (untranslated region) +20 (C-->T) transition was observed in two unrelated, transfusion-dependent homozygotes, living in the Hormozgan Province where, in contrast with populations living in other provinces of Iran, no IVS-I-110 (G-->A) or IVS-I-1 (G-->A) mutations were found. We report the molecular spectra of our population samples and compare them with the mutation spectra observed in the Iranian populations by other authors. We discuss the severe phenotype of the patients homozygous for the IVS-II-745 (C-->G) mutation, linked in cis to the 5'UTR +20 (C-->T) transition. Molecular analysis using commercial kits is briefly compared with denaturing gradient gel electrophoresis, emphasizing the value of a rapid method of detection for molecular defects in areas where many mutations occur.

This epidemiological study was based on a hematological and a molecular analysis of 310 heterozygous beta thalassemic and 75 carriers of Hb Lepore out of 3,000 microcythemic subjects from the Campania region of Italy. The molecular analysis of beta chains and the deltabeta hybrid gene has shown different beta chain defects, but only the Hb Lepore-Boston-Washington type in association with haplotypes I and V. The prevalence and distribution of these molecular defects in Campania show that they are linked to historical events and to the geographical characteristics of this region.

Introduction: The Comoro archipelago is characterised by a high prevalence of red cell genetic disorders such as G6PD deficiency and haemoglobinopathies, being a region endemic for malaria. Over the last 15 years, the city of Marseilles in France has become the main destination for Comorian immigrants. This Comorian community includes patients with sickle cell disease, sickle cell/beta-thalassaemia trait, thalassaemias and G6PD deficiency., Materials and Methods: Allele frequencies for haemoglobin S, beta-thalassaemia and G6PD deficiency were determined from neonatal and prenatal screenings of the Comorian community. Haemoglobin fractions were detected by isoelectrofocalisation, and the quantitation of HbS, HbA, HbA(2) and HbF was performed by cation exchange high performance liquid chromatography. The molecular study involved 31 alleles carrying the betaS mutation (Cd 6 [A-->T]), six beta-thalassaemic alleles and 17 G6PD-deficient alleles, selected from a group of carriers or affected subjects., Results: Allele frequencies were 3% for haemoglobin S, 1% for beta-thalassaemia trait and 9.5% for G6PD deficiency. Molecular analysis had revealed that the African alleles are predominant, being present in almost all the subjects studied. Mediterranean alleles were found for all the beta-thalassaemia mutations and for three G6PD chromosomes out of 17., Conclusion: These data are consistent with the mixed Arab and African origin of the population of the Comoro Islands, and are of clinical interest in prenatal and newborn screening plans.

Three Indonesian patients with identical genotypes, each compound heterozygotes for Filipino beta(o)-thalassemia/HbE, expressed different clinical severities. One patient has mild disease and is transfusion independent, while the other two are severely affected and transfusion dependent. The size of the Filipino beta(o)-globin gene deletion was confirmed to be 45 kb, resolving conflicting values given in the literature. Neither ameliorating genetic factors such as alpha-globin gene deletions or the XmnI restriction site polymorphism at position -158 upstream of the (G)gamma-globin gene, nor differences in beta-globin gene haplotype, explain the phenotypic variation. These observations have implications for the development of antenatal diagnosis in Indonesia, as at present it is not possible to give an accurate prediction of severity of phenotype for this common genotype., (Copyright 1999 Wiley-Liss, Inc.)

A common beta-thalassemia mutation in Asian populations is the C --> T substitution at position 654 of intron 2, which leads to the activation of two cryptic splicing sites and the incorporation of 73 extra nucleotides into the mutant mRNA. Like most beta-thalassemia mutations, it normally exhibits recessive inheritance. We investigated the unusually severe phenotype in two heterozygotes for this mutation, father and son, who had thalassemia intermedia and an apparent dominant mode of inheritance. An increased level of aberrantly spliced transcript in the reticulocytes of the probands compared with asymptomatic beta654 heterozygotes led us to investigate the production and processing of beta654 RNA. We showed that large amounts of the aberrant beta654 transcript were detectable in erythroblasts from one of the asymptomatic cases. The translation product of this mRNA was not detectable in vivo, and we were unable to demonstrate the translation of the mutant mRNA in a cell-free translation system. Although the reticulocyte alpha:beta mRNA ratios in the two probands were within the range observed in the asymptomatic heterozygotes, globin chain biosynthesis studies showed that the probands had considerably greater alpha:beta chain imbalance. These results imply that the more severe phenotype may be due to a second defect, possibly unlinked to the beta-globin cluster, that acts at the translational or posttranslational level., (Copyright 1998 by The American Society of Hematology)

We report the allelic sequence polymorphism associated with seven beta-thalassaemia mutations. Thirty-two DNAs originating from Algeria and 12 DNAs from Sardinia and Sicily were investigated. Their analysis revealed an association with a unique haplotype for three beta-thalassaemia mutations (-29, IVS-I-2 and IVS-I-1). It seems clear that these mutations have a unicentric origin. The presence of the -29 mutation could be explained by migration and founding effect. However, the local origin of IVS-I-2 seems clear. The four other mutations, FS6, IVS-I-6, IVS-I-110 and stop39 were found to be associated with at least two different sequence haplotypes. The likelihood of so many recurrent nucleotide dimorphisms in different lineages as a consequence of random mutation is very low; it is supported neither by the analysis of equivalent regions in other primates, nor by the presence of highly mutable sites such as CpG dinucleotides. The fact that these mutations are found exclusively in the Mediterranean area is not in favour of a recurrent origin of the mutation. The diversity is far more important for the preponderant thalassaemia mutations of the Mediterranean area and is higher in the 5' part of the beta-globin gene. Hence, the IVS-I-110, the preponderant beta-thalassaemia in the Eastern Mediterranean, probably emerged in the extension of the fertile crescent. For the stop39, all the data support the hypothesis of a West-Mediterranean origin. The diversity of haplotypes would then be generated by recombination events (crossing-over or gene conversions) between the original beta-thalassaemia chromosome and the other chomosomal structures present in the normal population., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

We have systematically analyzed beta-thalassemia genes using polymerase chain reaction-related techniques, dot-blot hybridization with oligonucleotide probes, allele specific-polymerase chain reaction, and sequencing of amplified DNA fragments from 41 unrelated patients, including 37 beta-thalassemia homozygotes, three with beta-thalassemia/Hb E, and one with beta-thalassemia/Hb S. Four different beta-thalassemia mutations were detected in 78 alleles. These are the IVS-I-5 (G-->C), codon 30 (AGG-->ACG) [also indicated as IVS-I (-1)], IVS-I-1 (G-->A), and codons 41/42 (-TTCT) mutations. The distribution of the beta-thalassemia mutations in the Maldives is 58 alleles (74.3%) with the IVS-I-5 (G-->C) mutation, 12 (15.4%) with the codon 30 (AGG-->ACG) mutation, seven (9%) with the IVS-I-1 (G-->A) mutation, and one with the codons 41/42 (-TTCT) mutation. The first three mutations account for 98.7% of the total number of beta-thalassemia chromosomes studied. These mutations are clustered in the region spanning 6 bp around the junction of exon 1 and the first intervening sequence of the beta-globin gene. These observations have significant implications for setting up a thalassemia prevention and control program in the Maldives. Analysis of haplotypes and frameworks of chromosomes bearing each beta-thalassemia mutation suggested that the origin and spread of these mutations were reflected by the historical record.

Two Mexican mestizo families with Hb Lepore Washington-Boston are described. One family is from Cordova, in the State of Veracruz, in the East coast of Mexico: the proband is a 44-year old asymptomatic male with italian ancestors; the other family is from the city of Durango, State of Durango, in the northwestern part of the country: the propositus is a 32-year old pregnant female with French ancestors. In both cases the Hb Lepore was identified by alkaline electrophoresis and characterized by high performance liquid chromatography and PCR with specific probes flanking the deletion frame. The beta-haplotype in both families was +(-)-(-)-(++), the commonest beta-haplotype reported with this mutation. This paper describes the first cases of this entity in Mexico.

Purpose: Considering the importance of type beta thalassaemias as hereditary syndromes of high significance in different populations of Mediterranean origin and, by extension, in the Brazilian population, the objective of the present study was to determine by PCR/DGGE the gene structures responsible for neutral polymorphisms (frameworks) observed in the human beta globin gene associated with the mutations responsible for type beta thalassaemias in a sample of the Brazilian population and, more specifically, of the population of the State of São Paulo., Patients and Methods: Thirty individuals with beta thalassaemic mutations were analyzed: 22 mutations were in codon 39 (C-->T), 5 in IVS1-110 (G-->A), 2 in IVS1-6 (T-->C) and 1 in IVS1-1 (G-->A). DNA was extracted and selective amplification was performed by PCR extending from position IVS1 nt 46 to IVS2 nt 126 (474 pb). The product was then analyzed by polyacrylamide gel electrophoresis on a denaturing 10-60% urea/formamide gradient., Results: The results demonstrated that, as expected, the mutations responsible for type beta thalassaemia observed in this population are of Mediterranean origin, with 73% distribution represented by codon 39, 17% by IVS1-110, 7% by IVS1-6 and 3% by IVS1-1. In turn, framework distribution seems to indicate a higher frequency of Fr 1-1 in codon 39 and IVS1-110, of Fr 1-3 in IVS1-6 and of Fr 1-2 in IVS1-1., Conclusions: These results permit us to conclude that gene amplification by PCR followed by DGGE is an appropriate method for the separation of DNA molecules that differ even by a single base change and therefore can be utilized to detect the alterations observed in the human beta globin gene. This methodology shows that, using only a pair of primers, it is possible to define the frameworks that are observed in the beta globin gene.

In order to perform genetic counselling and prenatal diagnosis of Hb-S-beta-thalassemia disease and beta-thalassemia, we have delineated the spectrum of beta-thalassemia alleles in the Guadeloupean population. A sample of 63 unrelated families was analyzed including 70 beta-thalassemia carriers, 52 Hb-S-beta-thalassemia, and 8 patients with different beta-thalassemic hemoglobinopathies. Among the eleven mutations identified, four of them [-29 (A --> G), IVS-I-5 (G --> A), IVS-II-1 (G --> A), and IVS-I-5 (G --> C)] account for 77.6% of the beta-thalassemia chromosomes present in the studied families. The seven other variants, CD 24 (T --> A), IVS-I-2 (T --> C), Poly A (T --> C), -88 (C --> T), IVS- 11-849 (A --> G), Hb E, and Hb Lepore are less frequent. As a result, Hb S-beta+-thalassemia type 1 (low Hb A values: 5-15%) together with Hb S-beta(omicron)-thalassemia phenotypes are as frequent as Hb S-beta+-thalassemia type 2 (high Hb A values: 20-30%) in the Guadeloupean population. Patients with Hb S-beta+-thalassemia type 2 have milder hematological manifestations of the disease compared to patients with Hb S-beta(omicron)-thalassemia and Hb S-beta+-thalassemia type 1. This first report on the type and nature of beta-thalassemia mutations in Guadeloupe shows that prenatal diagnosis of Hb S-beta-thalassemia and beta-thalassemia should be feasible by direct detection of point mutation in most cases.

We have identified two individuals of Greek Cypriot origin with thalassemia intermedia. Molecular analysis has shown that each individual is a compound heterozygote for a previously described beta zero thalassemia allele and a novel mutation, C-->G in position +33, in the 5' untranslated region of the beta globin gene. In both families the beta +33 allele is associated with the same beta haplotype (-++- ) suggesting that it is likely to be of a single origin, beta-cDNAs from normal and mutant beta alleles were isolated from peripheral blood reticulocytes using the technique of reverse transcription-polymerase chain reaction. Because the beta +33 (C-->G) mutation creates a cutting site for the restriction enzyme NlalV, we could demonstrate by differential restriction analysis that the beta gene with +33 mutation showed 25% to 35% residual activity compared with normal. The additive effect of this moderate deficit in beta globin production with the beta zero thalassemia mutation would explain the clinical phenotypes observed in the two probands. In contrast, two siblings of one proband who were also compound heterozygotes for the same beta thalassemia mutations, as well as heterozygotes for a nondeletional alpha thalassemia variant, and two other compound heterozygotes for the beta +33 and a beta+ thalassemia allele were completely asymptomatic. Individuals heterozygous for the beta +33 C-G mutation alone are clinically and hematologically silent, with normal red blood cell indices and normal levels of hemoglobin (Hb) A2. A direct relationship between genotypic and phenotypic severity is clearly demonstrated in these cases with obvious implications for prenatal diagnosis.

We present an analysis of the thalassemic alleles observed in Mexican mestizos. In 18 unrelated patients with mild to severe hemolytic anemia we saw 16 with beta-thal and two with alpha-thal and identified 25 chromosomes with 14 different alleles (10 for beta-thal), predominating the Mediterranean type (seven beta-thal and two alpha-thal). The most common mutation was the nonsense Cd 39 observed in seven chromosomes (28%); the other mutants were three Asian alleles, one beta-thal Indian (IVS-1 nt 5 G-->C), two Southeast Asian alpha-thal (-SEA and the -FIL), one Kurdish Jew (-28 A-->C) and one Mexican (Cd 11 -T). These findings suggest a marked molecular heterogeneity in the thalassemia genes in Mexico.

The beta-thalassemias are a heterogeneous group with respect to molecular pathogenesis, and different populations and ethnic groups differ with respect to the predominating mutations. This variable spectrum of beta-thalassemia mutations has resulted in extensive studies in each population and ethnic group to identify the major mutations. In this study we investigated the prevalence of 14 mutations in 253 beta-thalassemia patients drawn from eight Arab countries (i.e. Jordan, Egypt, Syria, Lebanon, Yemen, and Saudi Arabia), living in Saudi Arabia and attending Ministry of Health hospitals. The mutations investigated included IVS-I-110 (G-->A), IVS-II-1 (G-->A), IVS-I-5 (G-->C), codon 39 (C-->T), IVS-I-1 (G-->A), frameshift at codons 8/9 (+G), frameshift at codons 41/42 (-TTCT), codon 15 (TGG-->TAG), IVS-I-6 (T-->C), frameshift at codon 16 (-C), IVS-II-745 (C-->G), codon 6(-A), IVS-I, 3' end (-25 bp), and Cap +1 (A-->C). The most frequently encountered mutations were IVS-I-110 and IVS-II-1 which were identified in the population of each Arab country. The IVS-I-1 and IVS-II-745 mutations were encountered in Jordanians, Egyptians, and Syrians. The IVS-I-5, codon 39, codon 6, IVS-I, 3' end (-25 bp), and Cap +1 mutations were encountered only in Saudis and not in other Arabs, except codon 39 which was present in the Syrians and Lebanese. Other mutations were generally rare and not specific to any Arab ethnic group. This paper presents preliminary data on the prevalence of 14 mutations in the Arab populations and shows wide variation in the molecular basis of beta-thalassemia in different Arab ethnic groups. Further detailed studies to identify the entire spectrum of beta-thalassemia mutations are stressed.

The gene frequency of beta-thalassemia among Filipinos is estimated to be 0.02, although little is known about the mutations involved. Recently, an extensive beta-thalassemia deletion was reported in several unrelated individuals of Filipino descent. The deletion begins approximately 4 kb upstream of the beta-globin gene, and extends 3' beyond the beta-globin gene. In this report, we identify the 5' and 3' deletion endpoints and present a polymerase chain reaction (PCR) strategy for rapid DNA diagnosis of the Filipino beta-thalassemia deletion.

A considerable number of deletions of variable size and position that involve the beta-globin gene complex on chromosome 11 are associated with the clinical entities of hereditary persistence of fetal hemoglobin (HPFH) and delta beta thalassemia. Specific deletions appear to be associated with consistent phenotypes and some are known to be recurrent. To facilitate the molecular diagnosis of uncharacterized patients with HPFH and delta beta thalassemia, oligonucleotide primers have been designed to enzymatically amplify deletion-specific products for nine known deletions, which include those responsible for HPFH-1, HPFH-2, HPFH-3, Spanish (delta beta)zero thalassemia, hemoglobin (Hb) Lepore, Sicilian (delta beta)zero thalassemia, Chinese G gamma(A gamma delta beta)zero thalassemia, Asian-Indian inversion-deletion G gamma(A gamma delta beta)zero thalassemia, and Turkish inversion-deletion (delta beta)zero thalassemia. Using this approach, we have successfully characterized the molecular basis for delta beta thalassemia in 23 individuals from 16 families of diverse ethnic origins. Thirteen individuals from this group were shown to be heterozygous for the 13.4-kb Sicilian deletion, two were heterozygous for the 100-kb Chinese G gamma(A gamma delta beta)zero deletion, four were heterozygous for the Turkish form of inversion-deletion delta beta thalassemia, and three were heterozygous for the Asian-Indian form of inversion-deletion G gamma(A gamma delta beta)zero thalassemia. One Vietnamese subject was heterozygous for a 12.6-kb deletion, which we have fully characterized at the molecular level. Sequence analysis of the breakpoint regions of the Chinese deletion and the Turkish rearrangement indicates that, in each case, the mutation is likely to have arisen from a single origin. This hypothesis is supported by the evident geographical clustering of the various deletions described here.

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.

The distribution of restriction fragment length polymorphism (RFLP) at the BamH1 site of the beta-globin gene was investigated in the Chinese, Indian, and Malay race in Singapore. The sample comprised of 183 normal individuals and 35 beta-thalassemia carriers in which 13 were couples with at least one beta-major child. The results from this study indicate that BamH1 polymorphism will be informative in 22% of pregnancies at risk for beta-thalassemia major in Chinese, 19% in Malays and 7% in Indians. In prenatal diagnosis using BamH1 polymorphism for one beta-major affected family, the fetus was diagnosed to be normal or beta-carrier. The validity of BamH1 polymorphism in the exclusion of beta-thalassemia major was subsequently confirmed at birth by globin chain biosynthesis.

This study is to identify the various beta-thalassemic alleles in the United Arab Emirates (UAE), and compare them with the UAE residents from neighboring countries suffering from the same problem. Gene amplification, dot-blot hybridization with synthetic probes, restriction enzyme analyses, and sequencing were the tools used. Thirteen different mutations were observed in the UAE patients and seventeen mutations in the non-locals. The IVS-I-5 (G-->C) Asian Indian mutation was the most frequent mutation in both groups. Homozygous mutations in both groups were relatively higher than double heterozygous mutations.

We report the characterization of seven different beta-thalassemia mutations in 131 newly diagnosed Portuguese beta-thalassemia heterozygotes. Methodology included the detection of abnormal fragments by agar gel electrophoresis of PCR-amplified DNA fragments after digestion with specific restriction endonucleases, as well as hybridization with synthetic nucleotide probes and sequencing of amplified DNA. Four mutations, including the newly discovered TGG-->TGA change at codon 15, occurred in excess of 10% and accounted for some 90% of the beta-thalassemia alleles in this population. The geographical distribution is uneven; the TGG-->TGA mutation at codon 15 was primarily observed in the coastal region north of Lisbon, while the IVS-I-6 (T-->C) mutation was confined to the central part of the country.

The Chinese G gamma+(A gamma delta beta)zero-thalassemia is caused by a deletion of more than 80 kilobases. It has a beta(+)-thalassemia phenotype and should be differentiated from other mutations causing beta-thalassemia. Using polymerase chain reaction with three oligonucleotide primers bridging the breakpoints, the deletion can be detected easily. The method is useful in the genetic counseling and prenatal diagnosis of the at-risk families.

We have analysed the molecular basis of beta-thalassaemia in 22 Anglo-Saxon individuals, all of whom were heterozygous for beta-thalassaemia except for one, who was a compound heterozygote. Using a combination of allele-specific priming of the polymerase chain reaction (PCR) and direct sequencing of genomic DNA amplified by the PCR, 20/23 beta-thalassaemic genes were characterized. Nine different mutations were identified; four are commonly found in the Mediterranean, one in Asia, one has been described previously in both Europe and Asia, and three are rare mutations associated with a dominant beta-thalassaemia phenotype. In three individuals the mutation remains uncharacterized despite sequence analysis of the beta-globin gene and its immediate flanking regions. We report our findings and discuss the diversity of these mutations.

A rapid, simple, cost-effective, non-radioactive method for detection of the most common mutations causing beta-thalassemia in Mediterranean people has been developed by combining multiplexing with the amplification refractory system. This approach, the multiplex amplification refractory mutation system (MARMS), provides an easy assay for direct detection of normal and mutant beta-globin genes in homozygotes and heterozygotes. The strategy involves multiplex PCR of four of the five regions of interest within the beta-globin gene in a single reaction containing a common oligoprimer and either the normal or mutant oligonucleotides corresponding to IVS-1 nucleotide 1 or IVS-1 nucleotide 6, IVS-1 nucleotide 110, codon 39, and IVS-2 nucleotide 1 regions. Primers are chosen so that the sizes of the four PCR products differ, thereby facilitating detection on agarose gels following amplification. Patient samples are primed with either four normal or four mutant oligonucleotide mixtures and the common oligoprimer, and PCR products run in parallel on gels to detect band presence or absence. This approach simplifies mutation detection and shows promise for automation employing fluorescent-tagged primers.

Thirty tajiks, whose relatives had beta-thalassemia traits (revealed in previous investigations by determination of the HbA-2 and HbF levels) were selected to screen beta-thalassemia mutations. DNA samples from each individual were subjected to the PCR (polymerase chain reaction) to amplify the 635 bp beta-globin gene fragment. One additional band was detected in three samples after the amplified fragment underwent electrophoresis in 2% agarose gel and the EtBr was stained, and two additional ones were revealed by 6% PAAGE and staining of the EtBr. All additional bands migrated more slowly than appropriate 635 bp fragment. It is supposed that additional bands are heteroduplexes formed from the wild type chains and mutated chains carrying a deletion or insertion. The 4 bp deletion of the 41-42 (-tctt) was detected after the direct sequencing of the amplified fragments. This mutation is common among Chinese but it was not revealed in the Middle Asia populations. The mutation can be easily screened using the PCR and electrophoresis in 2% agarose gel or PAAG of the amplified beta-globin gene fragments.

1. Clinical, hematological, genetic and peripheral blood globin synthesis studies were carried out on 17 symptomatic Brazilian thalassemics and their parents who live in the northeast of São Paulo State. The group included 8 beta zero-thalassemia homozygotes, 7 carriers of at least one beta + gene, one delta beta zero/beta zero-thalassemia double heterozygote and one beta zero homozygote also carrying the alpha-chain variant Hb Hasharon (alpha 2(47) His beta 2). 2. The mean non-alpha/alpha ratio for globin biosynthesis of the patients lacking HbA (beta zero homozygotes and delta beta zero/beta zero double heterozygotes) was 0.26 +/- 0.11 (mean +/- SD), which is not statistically different from the value of 0.32 +/- 0.06 obtained for the carriers of at least one beta + gene. In contrast, the mean non-alpha/alpha ratio for the thalassemia major patients (0.22 +/- 0.07) was significantly lower than that obtained for the milder cases (0.34 +/- 0.06) although the beta/alpha ratios for the parents of the two groups were similar. 3. The heterogeneity within this group of Brazilian patients having two thalassemic genes, i.e. 60% who are beta zero homozygotes and 40% who are carriers of at least one beta + gene, is consistent with the Italian origin of most of these patients.