Normal Beta-globin Gene Research Articles

Lentiviral vector containing beta-globin gene for beta thalassemia gene therapy

Background and aimBeta thalassemia is a common monogenic disorder caused by partial or complete reduction of beta globin chains synthesis. In recent years allogeneic bone marrow transplantation (BMT) has been considered to be the successful cure for patients with thalassemia major, however this is restricted due to limited number of HLA-matched donors. Therefore, molecular approaches including gene therapy for direct normal beta globin gene transmission seem quite promising to cure thalassemia. The goal of this study was to evaluate the beta globin gene expression in K562 cell line, as a model of hematopoietic cell, transduced with lentiviral vector carrying normal beta globin gene. MethodsFor our purpose, we designed the DEST Lentiviral vector (3rd generation) carrying normal beta globin gene and its promoter and packaged in LentiX-293T cell line. The K562 cell line was transduced by packaged lentivirus containing β-globin cassette (pLenti-HBB). Bulk cells suspension form K562 cells were diluted and seeded into 96-well micro plate by manual pipetting. After single cell expansion, β-globin protein expression level in single cell clones was determined by flowcytometry and Western blotting. ResultsOur results showed that we have successfully packaged and generated lentivirus in LentiX-293T cell line. Flowcytometry analysis showed that beta globin protein expression achieved 20.3% in bulk population of K562 cells in MOI = 20 and 74.4% in F2 clone. ConclusionThese data indicated that vector used in this study is efficient in vitro and could be applied in gene therapy in patient's hematopoietic stem cell. The final goal of this study is to examine designed vector in hematopoietic stem cells promising therapeutic strategy for beta thalassemia and sickle cell disease.

Gene Therapy for Thalassemia and Sickle Cell Anemia Poses Acceptable Toxicity and Efficacy: Clinical Outcomes in Forty-Seven Cases

Background:Sickle cell disorder (SCD) and transfusion dependent beta-thalassemia (TDT) are inherited disorders of β-globin genes leading to ineffective erythropoiesis, hemolysis and multi-organ damage. Though allogenic hematopoietic cell transplantation (allo-HCT) in the only potentially curative therapy, lack of full HLA-matched donor, increased risk of myeloablative conditioning (MAC) regimen related toxicity, poor prognosis in older age groups (>14 years), and a high risk of infections and graft versus host disease (GVHD) culminating into a high transplant related mortality (TRM) preclude HCT as a treatment option for many patients.Gene therapy has been proposed as a relatively new treatment option for these monogenic disorders. It involves transfection of harvested CD34+ hematopoietic stem cells (HSC) with viral vector to transfer a normal beta globin gene, which are then transplanted back to the autologous donor. However, the available data on gene therapy is extremely variable. The purpose of this review is to evaluate the risks and outcomes of SCD and TDT patients undergoing gene therapy.Methods:A systematic search of databases using PubMed, Embase, Web of science, Cochrane database and Clinicaltrials.gov was performed on with no restrictions of language or time period. A total of 4312 articles were identified and after duplicates removal, 3508 articles were screened for relevance. Clinical studies reporting the most recent follow up results about the efficacy and safety of gene therapy in patients with hemoglobinopathies were considered for inclusion. Their quality assessment was done using the Cochrane tool, and bias was evaluated regarding the study selection, performance, detection, attrition and the reporting of the clinical trials.RESULTS (table-1):Only 7 articles qualified for the strict selection criteria with an N of 47: six studies were phase I trials (Cavazzana, M. et al 2010, Boulad, F. et al 2013, Marktel, S. et al 2017, Kwiatkowski, J. et al 2017, Cavazzana, M. et al 2017, Kanter, J. et al 2017) and one was a phase III trial (Walters, M. et al 2017). Thirty five patients (74.4%) had TDT while 12 patients (25.5%) had SCD. All these patients did not have a suitable HLA-matched HCT donor. The length of follow up was heterogeneous owing to the step wise enrollment of participants and ranged from 2-36 months. Except for 4 cases, the age of included population was >13 years. Lentivirus BB305 encoding the HbAT87Q globin gene was used for gene delivery in 81% patients (n=38). The normal beta globin gene was transduced in remaining 9 TDT cases and it involved transduction using TNS 9.3.55 vector in two cases and with GLOBE lentivirus in 7 cases. MAC with busulfan was used in 81% cases (n=38), while treosulfan+thiotepa was used in 7 cases. The non-myeloablative (NMA) conditioning with busulfan was used in only 2 cases (both TDT). Bone marrow harvest was the source of HSCs in all SCD cases. Except for 3 SCD cases in HGB-204 trial and 3 TDT cases in HGB-207 which measured the potential improvement in outcome by an increase in DP-VCN, the range of drug vector copy number (DP-VCN) was 0.3-1.5 copies per diploid genome. The total hemoglobin (HB) level at last follow up was reported for 62% cases (n=29) and it was >8.8 g/dl in all cases. Twenty-one (60%) thalassemia cases were in a transfusion (Tx) free state at last follow up, including the 7 cases (20%) who developed a complete independence from transfusions. No such Tx-free state was noted in SCD cases although they developed a reduction in the Tx-requirement and 75% SCD cases (n=9) had a 30-100% reduction in the frequency of VOCs. All the cases were alive at their recent follow up with manageable toxicity form conditioning. Acceptable safety until the median follow-up from the 7 trials was established in all cases, with no reported development of post-treatment leukemia or new malignancy in these trials.DISCUSSION:In this largest series of gene therapy for the treatment of hemoglobinopathies (to our knowledge), we find it to be clinically effective in terms of reduction in transfusion needs; data to suggest an improved OS and disease free survival is promising, however long term data is awaited. Gene therapy appears to bypass the limitations faced by allo-HCT especially a high early TRM. Randomized trials reporting the efficacy and safety outcomes with longer follow ups are urgently required. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bench to Bedside-Gene Therapy for Thalassemia and Sickle Cell Disease

The beta-hemoglobinopathies (beta-thalassemia and sickle cell disease) are the most prevalent inherited disorders worldwide and affect millions. Patients with beta-thalassemia major cannot survive without monthly, lifelong transfusions together with iron chelation therapy, and severe cases of sickle cell disease suffer from multiple life-threatening complications. Both categories of patients often have a shortened life expectancy in spite of supportive therapies, which impose an enormous financial burden on affected countries. The only available curative therapy is allogeneic hematopoietic stem cell transplantation, although most patients do not have an HLA-matched sibling donor, and those who do still risk graft rejection or graft-versus-host disease with associated morbidity and early mortality. This is why autologous gene therapy, by ex vivo transfer into the patient's own hematopoietic stem cells of a derivative of the normal beta-globin gene whose expression is appropriately regulated, is an attractive novel therapeutic modality. In addition, the very large number of known mutations causing beta-thalassemia makes gene therapy by gene addition ideally suited for regulatory product approval rather than the many mutation-specific products that would be required for site-specific gene correction. However, gene therapy of these disorders is especially challenging given the requirement for massive hemoglobin production in a lineage specific manner and the lack of selective advantage for corrected hematopoietic stem cells. During the past two decades, we and others have devised lentiviral vectors and applicable protocols to achieve the permanent correction of mouse models of the beta-hemoglobinopathies. The first approved human clinical trial worldwide resulted in the conversion to transfusion-independence of a patient with severe betaE/beta0-thalassemia, who required monthly transfusions since early childhood. This patient demonstrated prolonged transfusion-independent for over 8 years after gene therapy, and the initially identified partially dominant integration site (HMGA2) is no longer preeminent. A further optimized vector with high-grade purification is now being used in subsequent multi-center clinical trials in the USA, France, Australia, and Thailand both for beta-thalassemia major and severe sickle cell disease. As of the last public disclosure in June 2015, 34 subjects have been enrolled, of whom 12 and 2 patients with beta-thalassemia major and severe sickle cell disease, respectively, have undergone the gene therapy procedure. Currently analysable patients with beta-thalassemia major have rapidly decreased their transfusional needs or become completely transfusion-independent, often with near normal hemoglobin values and polyclonal distribution of vector bearing progenitors. Interestingly, conversion to transfusion-independence also occurred in a Cooley's anemia patient of the beta0/beta0 genotype. With regard to the first analysable patient with sickle cell disease, the anti-sickling globin variant utilized in the vector (βT87Q) is expressed at 40% level of all hemoglobin chains - a level well above the expected sickling inhibitory threshold - resulting in improvements in disease-specific biological markers and no hospitalization for sickle cell complications or acute episode despite weaning off transfusions, which this patient was receiving regularly for the prevention of stroke relapse. Prospects for bringing this novel therapeutic approach to medical practice and for complementary approaches to increase safety and efficacy will be discussed. DisclosuresLeboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Accumulation of gamma-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine.

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.

Expression of beta-globin in primary erythroid progenitors of beta-thalassemia patients using an SV40-based gene delivery system.

SV40-based vectors are very efficient in gene delivery into human hematopoietic cells. In the present work, we investigated the expression of constructs carrying the human beta-globin gene that were delivered as beta-globin pseudovirions. Expression studies were performed by RNA analysis of primary human erythroid progenitors cultivated from peripheral blood of beta(0)-thalassemia patients who are unable to produce normal beta-globin RNA. This erythroid culture system recapitulates in vitro the process of growth, differentiation, and maturation of authentic erythroid precursors. The progenitors were induced to differentiate by the addition of erythropoietin (EPO). Five days later, the cells were infected with pseudovirions containing the normal beta-globin gene, and RNA was harvested on day 8. The results showed significant levels of normal beta-globin gene mRNA. A small DNA fragment derived from the 5'-region of the HSII element of the human beta-globin locus control region (LCR) enhanced expression of the linked beta-globin gene 20-30-fold. Normal beta-globin mRNA expression was in direct correlation to the multiplicity of infection. These studies suggest the potential feasibility of using the beta-globin delivery system for gene therapy of beta-thalassemia.

Hematological effects of atypical and Cameroon beta-globin gene haplotypes in adult sickle cell anemia.

To examine the effects of unusual or atypical beta-globin gene cluster haplotypes on the hematological features and Hb F levels of sickle cell anemia, we studied African Americans who had an atypical or Cameroon haplotype chromosome in association with a typical haplotype. We identified over 20 atypical haplotypes. The distribution of 5' sub-haplotypes of the atypical chromosomes mirrored the distribution of common haplotypes in African Americans with sickle cell anemia. Neither 5' nor 3' subhaplotypes of the atypical chromosomes affected Hb F levels, packed cell volume, or mean corpuscular volume in individuals with a Benin chromosome. That the 5' subhaplotype is unaffected might be a consequence of the small numbers of Senegal 5' subhaplotypes in our sample, the need for linkage of both 5' and 3' subhaplotypes of any haplotype for an effect on Hb F to be present, or the likelihood that a normal beta-globin gene contributed the 5' subhaplotypes of some atypical haplotypes.

Hematological effects of atypical and cameroon β-globin gene haplotypes in adult sickle cell anemia

To examine the effects of unusual or atypical beta-globin gene cluster haplotypes on the hematological features and Hb F levels of sickle cell anemia, we studied African Americans who had an atypical or Cameroon haplotype chromosome in association with a typical haplotype. We identified over 20 atypical haplotypes. The distribution of 5' sub-haplotypes of the atypical chromosomes mirrored the distribution of common haplotypes in African Americans with sickle cell anemia. Neither 5' nor 3' subhaplotypes of the atypical chromosomes affected Hb F levels, packed cell volume, or mean corpuscular volume in individuals with a Benin chromosome. That the 5' subhaplotype is unaffected might be a consequence of the small numbers of Senegal 5' subhaplotypes in our sample, the need for linkage of both 5' and 3' subhaplotypes of any haplotype for an effect on Hb F to be present, or the likelihood that a normal beta-globin gene contributed the 5' subhaplotypes of some atypical haplotypes.

Heterozygous beta-thalassemia with thalassemia intermedia phenotype.

In this study we investigated the molecular bases of the beta-thalassemia intermedia phenotype in six patients belonging to two unrelated families of Sardinian descent. Sequence analysis of the beta globin gene from these patients detected, as the sole abnormality, the heterozygosity for the codon 39 nonsense mutation. The A gamma and Ggamma promoters as well as the HS2 and HS3 core sequences of the beta globin LCR from these patients, did not show any non-polymorphic nucleotide variation from the consensus sequence. One of the parents was heterozygous for codon 39 nonsense mutation but showed the beta-thalassemia carrier phenotype; the other was hematologically normal and had an entirely normal beta globin gene sequence. In both families, other members showed the typical hematological phenotype, clinically silent, of heterozygous beta thalassemia. To explain the thalassemia intermedia phenotype, we postulated the presence of an unknown molecular defect interacting with the beta globin gene mutation. Haplotype analysis excluded that this postulated defect lies in the beta globin gene cluster.

Isolation, genomic structure, and expression of human erythroid Krüppel-like factor (EKLF).

Erythroid Krüppel-like factor (EKLF) is an essential transcriptional activator that directs high-level expression of the adult beta-globin promoter by binding to its CACCC element, one of a trio of highly conserved sequences present in erythroid cell-specific promoters and enhancers. This report describes the isolation and characterization of the human homolog of murine EKLF. The human EKLF transcription unit shares a number of structural properties with its marine counterpart. Human EKLF is contained within 3 kb of genomic DNA, and its coding region is interrupted by two introns whose locations are conserved with the murine gene. The three zinc fingers share >90% sequence similarity with and are predicted to bind the same target sequence as the mouse EKLF. The rest of the protein is proline-rich and retains approximately 70% sequence similarity to the mouse gene. Human EKLF is expressed in bone marrow and HEL, JK1, and OCIM1 erythroleukemic cell lines but not in K562 nor in myeloid or lymphoid cell lines. These results indicate that the genomic structure and erythroid-restricted expression of EKLF are highly conserved between the murine and human homologues. Availability of human EKLF will enable initiation of studies to molecularly assess whether it is functionally compromised in those cases of beta-thalassemia that contain a normal beta-globin gene locus.

Nondeletional type of hereditary persistence of fetal haemoglobin: molecular characterization of three unrelated Thai HPFH.

The beta-globin gene clusters of three unrelated Thai families with a nondeletional type of hereditary persistence of fetal haemoglobin (HPFH) were studied using polymerase chain reaction-related techniques. All appeared to have normal nucleotide sequences from the Cap site to position -400 of both the G gamma- and A gamma-globin genes. Two individuals suspected of having a beta-thalassaemia gene linked to the high HbF condition also had a normal beta-globin gene sequence, spanning from position -108 from the Cap site to the polyadenylation site. Deletion of four nucleotides, AGCA, at positions -225 to -222 of one A gamma-globin allele was detected in one subject and was confirmed by dot-blot hybridization. Restriction fragment length polymorphisms in the beta-globin gene cluster showed that the 5' haplotype (-+-++) and the presence (+) of an Xmm 1 polymorphic site at -158 of the G gamma-globin gene are associated with the high F phenotype in these families. Direct sequencing of the 5' hypersensitive-2 (5' HS-2) site of the locus control region (LCR) showed that this Xmn 1 (+) site is also linked to a specific rearrangement of TA repeats (TA)9CACATATACG(TA)10, in HS-2 segment.

A beta-thalassaemia phenotype not linked to the beta-globin cluster in an Italian family.

This paper describes a family of Central Italian origin in which three patients in two generations had either thalassaemia intermedia or a late presenting form of thalassaemia major. Sequence analysis of the patients' DNA revealed that only one of the beta-globin genes was affected by a beta-thalassaemia mutation (the codon 39 nonsense mutation), the other being completely normal, apart from the complex rearrangement (-T +ATA) at position -530 5' to the CAP site of the beta-globin gene, which has uncertain clinical significance. Haematologically, all these patients were characterized by unusually low HbF levels (1.8-7.3%) for a beta-thalassaemia major or intermedia phenotype. The mother of the two patients with thalassaemia intermedia was heterozygous for beta-thalassaemia (codon 39 nonsense mutation), while the father had thalassaemia-like red cell indices, an increased alpha/non alpha chain synthesis ratio, a slight increase of HbF and a low HbA2 level, but showed entirely normal beta-globin gene sequences, apart from the complex rearrangement (-T +ATA) at position -530 5' to the CAP site. One of the thalassaemia intermedia patients married a normal woman and they had a child with thalassaemia major who inherited only the codon 39 nonsense mutation but not the complex rearrangement at position -530. The clinical phenotype of thalassaemia-intermedia or major in the patients from this family may be explained by postulating the inheritance of the double heterozygous state for beta-thalassaemia and for a mutation in a gene coding for an erythroid-specific DNA binding protein which may impair the function of the normal beta-globin gene. Heterozygosity for this postulated mutation (father of the patients with thalassaemia intermedia) may result in the production of a beta-thalassaemia carrier state with normal HbA2 level.

The proximal element of the beta globin locus control region is not functionally required in vivo.

In addition to local sequence elements the regulation of the high-level, development- and tissue-specific expression of the human beta globin gene cluster appears to require distant regulatory sequences which have been termed locus control region. In the chromatin of erythroid cells the locus control region is characterized by four DNaseI hypersensitive sites that are located 6-18 kb 5' of the epsilon globin gene. The definition of the sequences minimally required for locus control region activity is likely to further the understanding of its physiology and will be of interest for the development of somatic gene therapy strategies of the hemoglobinopathies. We present here the analysis of a family with a 3,030-bp deletion of sequences upstream of the epsilon globin gene including the most 3' locus control region element and cosegregating beta(0) thalassemia. The deletion is linked in cis to a structurally and functionally normal beta globin gene. The proximal element of the locus control region does not therefore appear to be necessary for beta globin gene activity in vivo.

β-Globin Enhancers Target Expression of a Heterologous Gene to Erythroid Tissues of Transgenic Mice

To examine the role of human beta-globin enhancers in tissue-specific and developmental regulation, a hybrid beta-globin-simian virus 40 gene was analyzed in transgenic mice. A beta-globin DNA fragment containing two previously defined enhancers stimulated transcription from the simian virus 40 promoter in a tissue- and stage-specific pattern similar to that of the normal beta-globin gene. These results help to define the functions of beta-globin regulatory elements and suggest an approach for targeted expression of heterologous genes in erythroid cells in vivo.

Beta-globin enhancers target expression of a heterologous gene to erythroid tissues of transgenic mice.

To examine the role of human beta-globin enhancers in tissue-specific and developmental regulation, a hybrid beta-globin-simian virus 40 gene was analyzed in transgenic mice. A beta-globin DNA fragment containing two previously defined enhancers stimulated transcription from the simian virus 40 promoter in a tissue- and stage-specific pattern similar to that of the normal beta-globin gene. These results help to define the functions of beta-globin regulatory elements and suggest an approach for targeted expression of heterologous genes in erythroid cells in vivo.

A β-Thalassemia Gene Caused by a 290-Base Pair Deletion: Analysis by Direct Sequencing of Enzymatically Amplified DNA

The base composition around a recently detected deletion in the human beta-globin gene was determined by direct DNA sequencing of an enzymatically amplified DNA segment. The deletion removes 290 base pairs (bp), including the entire exon 1 and the mRNA cap site. In the vicinity of the deletion endpoints, the normal beta-globin gene contains direct and inverted repeats which may have taken part in generation of this deletion.

A beta-thalassemia gene caused by a 290-base pair deletion: analysis by direct sequencing of enzymatically amplified DNA

The base composition around a recently detected deletion in the human beta-globin gene was determined by direct DNA sequencing of an enzymatically amplified DNA segment. The deletion removes 290 base pairs (bp), including the entire exon 1 and the mRNA cap site. In the vicinity of the deletion endpoints, the normal beta-globin gene contains direct and inverted repeats which may have taken part in generation of this deletion.

Beta-globin gene linked DNA haplotypes and frameworks in three South-East Asian populations.

DNA haplotypes and frameworks (numbers in parenthesis) linked to the beta-globin gene were determined by restriction fragment analysis using eight restriction endonucleases on 86 (97) chromosomes bearing the normal beta-globin gene (HBB*A) and 108 (118) chromosomes bearing HBB*E in subjects homozygous for HBB*A or HBB*E from three South-East Asian populations with high HBB*E frequencies (northern Thailand, north-eastern Thailand and Cambodia). A systematic nomenclature for beta-globin gene-linked haplotype characterized by six polymorphic sites is introduced. In all populations, HBB*A occurred preferentially (greater than 80%) in linkage with the haplotype 41 (+----+) and all three frameworks described by Antonarakis et al. (1982). In contrast, almost 80% of the HBB*E genes occurred with the haplotype 27 (-+- ). In northern and north-eastern Thailand, HBB*E was present almost exclusively in frame-work 2; HBB*E in framework 3 (Asian) was limited to the Khmer population of Cambodia, and the frequency of HBB*E-linked framework 3 increased from the west to the east in this country.

Molecular characterization of a beta zero-thalassemia resulting from a 1.4 kilobase deletion

We report the characterization of a beta zero-thalassemia in an American Black with unusually high HbA2 and HbF levels. Genomic southern analysis indicated that the individual was heterozygous for a deletion that began within the second intervening sequence of the beta- globin gene and extended approximately 1.4 kb in the 5′ direction. A clone spanning the breakpoint on the abnormal chromosome was isolated and further mapped, and the deletion joint was sequenced. Comparison of the normal beta-globin gene and its 5′ flanking region with the deletion joint sequence indicated that the 5′ breakpoint for this deletion was 484 base pairs (bp) 5′ to the transcriptional start site for the beta-globin gene and the 3′ breakpoint was 908 bp into the beta- globin gene; the deletion removed a total of 1,393 bp. Comparison of the normal 5′ and 3′ breakpoint sequences indicated that this deletion was the result of a “clean” nonhomologous breakage and reunion event; ie, no spurious bases were added during the recombinational event. Analysis of the breakpoints of this deletion together with the breakpoints of two other small deletions involving the beta-globin gene suggests that the breakpoints may occur at DNA polymerase alpha pause sites.

Molecular Characterization of a β0 -Thalassemia Resulting From a 1.4 Kilobase Deletion

We report the characterization of a beta zero-thalassemia in an American Black with unusually high HbA2 and HbF levels. Genomic southern analysis indicated that the individual was heterozygous for a deletion that began within the second intervening sequence of the beta-globin gene and extended approximately 1.4 kb in the 5' direction. A clone spanning the breakpoint on the abnormal chromosome was isolated and further mapped, and the deletion joint was sequenced. Comparison of the normal beta-globin gene and its 5' flanking region with the deletion joint sequence indicated that the 5' breakpoint for this deletion was 484 base pairs (bp) 5' to the transcriptional start site for the beta-globin gene and the 3' breakpoint was 908 bp into the beta-globin gene; the deletion removed a total of 1,393 bp. Comparison of the normal 5' and 3' breakpoint sequences indicated that this deletion was the result of a "clean" nonhomologous breakage and reunion event; ie, no spurious bases were added during the recombinational event. Analysis of the breakpoints of this deletion together with the breakpoints of two other small deletions involving the beta-globin gene suggests that the breakpoints may occur at DNA polymerase alpha pause sites.

Haemoglobin Freiburg: direct detection by synthetic oligonucleotide probes.

The molecular defect leading to Haemoglobin (Hb) Freiburg has been analysed using synthetic oligonucleotides. Oligonucleotide probes 19 residues and 16 residues long, corresponding to the normal and mutant beta-globin gene sequences, respectively, were used to develop a direct assay for the beta F-globin gene, which codes for an unstable haemoglobin. Under the conditions described here the use of the respective synthetic oligonucleotides should aid in the determination of all Hb Freiburg genotypes in families at risk with a high level of confidence.