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.

The population genetics of the haemoglobinopathies.

Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait

Mutation detection is important in all areas of biology. Detection of unknown mutations can involve sequencing of kilobases of DNA, often in many patients. This has lead to the development of methods to screen DNA for mutations as well as methods to detect previously described mutations. This review discusses current methods used for such purposes with special emphasis on genetic diseases of humans. However, savings can be made by similar means in other areas of biology where repetitive or extensive sequencing for comparative purposes needs to be done. This review covers the methods used for detection of unknown mutations, namely the ribonuclease, denaturing gradient-gel electrophoresis, carbodiimide, chemical cleavage, single-strand conformation polymorphism, heteroduplex and sequencing methods. Once mutations have been defined they can be searched for repeatedly by methods referred to as diagnostic methods. Such methods include allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and the artificial introduction of restriction sites. We can now choose from a range of excellent methods, but the choice will usually depend on the background of the laboratory and/or the application in hand. Screening methods are evolving to more satisfactory forms, and the diagnostic methods can be automated to screen whole populations inexpensively.

Current methods of mutation detection

The amplification refractory mutation system (ARMS) is a simple, rapid and reliable method for the detection of any mutation involving single base changes or small deletions. We have applied ARMS methodology to the detection of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Single ARMS tests have been developed for 11 CFTR mutations found in the northwest of England. ARMS reactions for the most common mutations have been multiplexed to give a test which will detect the presence of the delta F508, G551D, G542X, and 621 + 1G----T mutations in a DNA sample. The multiplex test has been validated by the analysis of over 500 previously genotyped samples and has been found to be completely accurate. The rapid detection of the most common mutations has enabled early molecular confirmation of suspected cystic fibrosis in neonates, rapid typing of cystic fibrosis patients and their relatives, and testing of sperm and egg donors.

Development, multiplexing, and application of ARMS tests for common mutations in the CFTR gene.

The β-thalassemias are characterized by a quantitative deficiency of β-globin chains underlaid by a striking heterogeneity of molecular defects. Although most of the molecular lesions involve the structural β gene directly, some down-regulate the gene through distal cis effects, and rare trans-acting mutations have also been identified. Most β-thalassemias are inherited in a Mendelian recessive fashion but there is a subgroup of β-thalassemia alleles that behave as dominant negatives. Unraveling the molecular basis of β-thalassemia has provided a paradigm for understanding of much of human genetics.

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The Molecular Basis of β-Thalassemia

Rapid detection and prenatal diagnosis of β-thalassaemia: studies in Indian and Cypriot populations in the UK

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.

The spectrum of beta-thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis.

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

Analysis of beta-globin gene haplotypes in Asian Indians: origin and spread of beta-thalassaemia on the Indian subcontinent.

Five beta-thalassaemia mutations hitherto undescribed in Asian Indians were identified in beta-thalassaemia carriers originating from the Indian subcontinent by direct sequencing of their beta-globin genes which were amplified by the polymerase chain reaction (PCR). A T-G substitution at IVS 2 position 837, which probably creates an alternative acceptor splice site and a T insertion in codon 88, resulting in a shift in the reading frame with a premature stop codon, are new beta-thalassaemia mutations. The others were framshift codon 5 (-CT), IVS 1 position 110 (G-A) and IVS-1 minus 1 (G-A) which have been described previously in other populations. These results complete the characterization of the beta-thalassaemia mutations in 708 carriers of Asian Indian origin and will enable a comprehensive programme of carrier screening and prenatal diagnosis of beta-thalassaemia in this population.

N. Y. Varawalla

Papers

Rapid detection and prenatal diagnosis of β-thalassaemia: studies in Indian and Cypriot populations in the UK

The spectrum of beta-thalassaemia mutations on the Indian subcontinent: the basis for prenatal diagnosis.

Analysis of beta-globin gene haplotypes in Asian Indians: origin and spread of beta-thalassaemia on the Indian subcontinent.

Rare beta-thalassaemia mutations in Asian indians.

Screening for β‐Thalassemia in Asian Indians by the Amplification‐Refractory Mutation System