The spectrum of beta-thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis.
TL;DR: The β‐thalassaemia mutations in 702 unrelated carriers originating from seven different regions of the Indian subcontinent have been characterized using allele specific priming of the polymerase chain reaction (PCR).
Abstract: The beta-thalassaemia mutations in 702 unrelated carriers originating from seven different regions of the Indian subcontinent have been characterized using allele specific priming of the polymerase chain reaction (PCR). It was possible to identify the mutations in 688 (98%) of the individuals studied. Eleven different mutations were identified, of which five common ones accounted for 93.6%; namely the ones at IVS-1 position 5 (G-C), codons 8/9 (+G), IVS-1 position 1 (G-T), codons 41/42 (-CTTT) and the 619 bp deletion at the 3' end of the gene. The mutations at IVS-2 position 1 (G-A) and codon 30 (G-C), previously undescribed in Asian Indians, were found in two and six individuals respectively. Some regional variation in the distribution of beta-thalassaemia alleles was noted. These findings should prove useful for the development of a first trimester prenatal diagnosis programme based on direct detection of mutations.
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TL;DR: It is argued that malaria selection has operated relatively recently on human populations (within the last 5000 years) and the present distribution of haemoglobinopathies is seen as the result of selection elevating sporadic mutations in local populations.
Abstract: The haemoglobinopathies are the commonest single-gene disorders known, almost certainly because of the protection they provide against malaria, as attested by a number of observations. The geographical distributions of malaria and haemoglobinopathies largely overlap, and microepidemiological surveys confirm the close relationship between them. For two of the commonest disorders, haemoglobin S and alpha(+)-thalassaemia, there is also good clinical evidence for protection against malaria morbidity. However, not all the evidence appears to support this view. In some parts of the world malaria and haemoglobinopathies are not, and never have been, coexistent. It is also difficult to explain why the majority of haemoglobinopathies appear to be recent mutations and are regionally specific. Here we argue that these apparent inconsistencies in the malaria hypothesis are the result of processes such as genetic drift and migration and of demographic changes that have occurred during the past 10,000 years. When these factors are taken into account, selection by malaria remains the force responsible for the prevalence of the haemoglobinopathies.
483 citations
TL;DR: The results presented here can be used as a basis of prenatal diagnosis of β-thalassemia in different regions of Iran and the relative frequency in the north was much higher than other regions, and it lessened toward the south, where the IVS-I-5(G → C) mutation was more common.
Abstract: β-Thalassemia is the most common hereditary disease in Iran. More than two million carriers of β-thalassemia live in Iran. Since the Iranian population is a mixture of different ethnic groups, it is necessary to determine the frequency and distribution of mutations in the different parts of the country. For this purpose, we divided Iran in to eight different regions according to the geographic and ethnic distribution of the population. Over a 10-year period 1,217 β-thalassemia chromosomes of 164 affected patients and 889 unrelated carriers were studied using the amplification refractory mutation system-polymerase chain reaction technique. We detected 81% β-thalassemia mutations in the studied chromosomes. IVS-II-I (G → A) was the predominant mutation found in our study (34%). Its relative frequency in the north was much higher than other regions, and it lessened toward the south, where the IVS-I-5(G → C) mutation was more common. IVS-I-5 (G → C) (7.55%), codons 8/9 (+G) (4.76%), and IVS-I-110 (G → A) (4.7...
168 citations
Cites background from "The spectrum of beta-thalassaemia m..."
...ARMS primers were selected from Old et al (9) and Varawalla et al (10)....
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...Old et al (9) and Varawalla et al (10)....
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TL;DR: Identification of the genetic variants modifying fetal hemoglobin (HbF) production in combination with α globin genotype provide some prediction of disease severity for β thalassemia but generation of a personalized genetic risk score to inform prognosis and guide management requires a larger panel of genetic modifiers yet to be discovered.
Abstract: The remarkable phenotypic diversity of β thalassemia that range from severe anemia and transfusion-dependency, to a clinically asymptomatic state exemplifies how a spectrum of disease severity can be generated in single gene disorders. While the genetic basis for β thalassemia, and how severity of the anemia could be modified at different levels of its pathophysiology have been well documented, therapy remains largely supportive with bone marrow transplant being the only cure. Identification of the genetic variants modifying fetal hemoglobin (HbF) production in combination with α globin genotype provide some prediction of disease severity for β thalassemia but generation of a personalized genetic risk score to inform prognosis and guide management requires a larger panel of genetic modifiers yet to be discovered. Nonetheless, genetic studies have been successful in characterizing the key variants and pathways involved in HbF regulation, providing new therapeutic targets for HbF reactivation. BCL11A has been established as a quantitative repressor, and progress has been made in manipulating its expression using genomic and gene-editing approaches for therapeutic benefits. Recent discoveries and understanding in the mechanisms associated with ineffective and abnormal erythropoiesis have also provided additional therapeutic targets, a couple of which are currently being tested in clinical trials.
128 citations
TL;DR: Although a full spectrum of >1000 mutations causing haemoglobinopathies has been documented, in practice only a limited number are associated with disease states and clinical significance and prior identification of the ethnic origin is thus an important part of the diagnostic strategy.
Abstract: The haemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassaemias) or the synthesis of a structurally abnormal haemoglobin (Hb). In prevalent regions, the thalassaemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and haematological phenotypes. An appreciation of these phenotypes is needed to facilitate the definitive diagnosis of the causative mutations to inform management and counselling. Haematological and biochemical investigations, and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. With the exception of a few rare deletions and rearrangements, the molecular lesions causing haemoglobinopathies are all identifiable by PCR-based techniques. Although a full spectrum of >1000 mutations causing haemoglobinopathies has been documented, in practice only a limited number are associated with disease states and clinical significance. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassaemia mutations. Prior identification of the ethnic origin is thus an important part of the diagnostic strategy which becomes less reliable in the UK because of the large ethnic mix. Although the current approach using a combination of different PCR-based techniques seems to work in most laboratories, practice pressures with the imminent implementation of universal antenatal screening for clinically significant Hb disorders in the UK will require a higher throughput approach for DNA diagnostics in the near future. The complex mutational spectrum and the compactness of the globin genes places them in an ideal position for the different non-gel based analytical platforms.
120 citations
Cites background from "The spectrum of beta-thalassaemia m..."
...For small deletions such as the 619 bp deletion, a common cause of b-thalassaemia in Asian Indians, differential amplification products are generated in the mutant and wild type....
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...With the exception of the 619 bp deletion, which accounts for 20% of the b-thalassaemias in Asian Indians (Varawalla et al., 1991), the common mutations are all point mutations because of single base substitutions, insertion or deletion of a few bases (Thein, 1998; Weatherall & Clegg, 2001)....
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...Hb E/b-thalassaemia is an important cause of b-thalassaemia major and thalassaemia intermedia in South-east Asians and Asian Indians....
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TL;DR: The data are consistent with relatively recent and local origins for most β-thalassaemia mutations and of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution.
Abstract: beta-globin gene haplotypes were determined for 196 normal (beta-A) and 419 thalassaemia (beta-Th) chromosomes of individuals from four different regions of the Indian subcontinent; North-west Pakistan, Gujarat, Punjab and Sindh Analysis of beta-A and beta-Th haplotypes and haplotype-mutation associations in each regional group along with a consideration of Indian history provided information about the origin and spread of beta-thalassaemia mutations on the Indian subcontinent The data are consistent with relatively recent and local origins for most beta-thalassaemia mutations The frequencies of particular alleles differ markedly in various regions and these may be useful population markers Of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution The microepidemiology of beta-thalassaemia in this region reflects considerable ethnic diversity, gene flow from population migration and natural selection by malaria infection
113 citations
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TL;DR: A new method for determining nucleotide sequences in DNA is described, which makes use of the 2',3'-dideoxy and arabinon nucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase.
Abstract: A new method for determining nucleotide sequences in DNA is described. It is similar to the “plus and minus” method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2′,3′-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage ϕX174 and is more rapid and more accurate than either the plus or the minus method.
62,728 citations
TL;DR: The ARMS (Amplification Refractory Mutation System) as discussed by the authors is a system that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis.
Abstract: We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.
2,571 citations
TL;DR: The polymerase chain reaction (PCR) procedure is used to enzymatically amplify a specific segment of the β-globin or HLA-DQα gene in human genomic DNA before hybridization with ASOs, enabling the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple ‘dot blot’ for probe hybridization.
Abstract: Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.
2,040 citations
TL;DR: This work studied the genomic DNA of five patients with β-thalassaemia, and found two previously undescribed mutations, along with three known alleles, including the first natural mutation observed at the cap site of the β-globin gene.
Abstract: Direct sequencing of specific regions of genomic DNA1 became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA2–5. Recently, human mitochondria! DNA was amplified and directly sequenced6. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R. K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with β-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106–107 and the other is an A–C transversion at the cap site (+1) of the β-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.
509 citations
TL;DR: The method, which allows the determination of the mutations in both parental and fetal DNA on the same day, should have wide application to the carrier detection and prenatal diagnosis of monogenic diseases with heterogeneous molecular defects.
314 citations