Genetic diseases of hemoglobin 19 Genetic diseases of hemoglobin: diagnostic methods for elucidating β-thalassemia mutations S.Tuzmen, 1 A. N. Schechter 1 1 Laboratory of Chemical Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA INTRODUCTION n the last two decades, a vast array of molecular tools for the diagnosis of genetic diseases of hemoglobin have been developed that allow definitive identification of the DNA mutations underlying each of these diverse but prevalent diseases. It is the purpose of this article to review these new tools which can be used for individual diagnoses, analyses of fetal tissue or in screening and epidemiological studies. Although these techniques have been developed in high technology settings, recent technical advances are mak- ing their application simpler and cheaper so that in the near future they will likely be available in many more parts of the world, especially those areas in which these diseases are most prevalent.This review updates previous reviews by Cao and Rosatelli, 1 Embury 2 and Cao et al., 3 on the methodolo- gies used to elucidate the various β-thalassemia genetic lesions. α-thalassemia syndromes have been reviewed else- where. 4,5 HEMOGLOBINOPATHIES AND THALASSEMIAS Inherited abnormalities of the hemoglobin tetramer may be divided into two categories: those that are characterized by structural anomalies of the hemoglobin chains, and others that result from an array of molecular defects that either reduce or completely abolish the synthesis of one or more of the polypeptide chains of the hemoglobin molecule. The term ‘hemoglobinopathy’ refers to the former disorders, whereas the latter defines the term ‘thalassemia’. 6 The hemo- globinopathies may be further divided into functionally dis- tinctive groups, and they can be classified according to their abnormalities.To date, nearly 700 mutant alleles have been characterized (http://globin.cse.psu.edu). 7 These structural anomalies are regionally specific and in most instances the geographical and ethnic distributions have been described. Structural alterations may include amino acid substitutions, deletions, and insertions; chain fusions may also form from two different polypeptide chains. Many of these mutations are functionally normal and, therefore considered as clini- cally silent. The thalassemia syndromes are a diverse group of inher- ited disorders that can be characterized according to their insufficient synthesis or absent production of one or more of the globin chains.They are classified into α, β, γ, δβ, δ and εγδβ thalassemias depending on the globin chain or chains which are affected. The β-thalassemias refer to that group of inher- ited hemoglobin disorders which are characterized by a reduced synthesis (β + -thalassemia) or absence (β 0 -- thalassemia) of β-globin chain production. 8 This leads to an imbalance of the ratio of α/non α-globin synthesis, which is the major factor in determining the severity of the disease in the β-thalassemia syndromes. 9 In β-thalassemia, normal syn- thesis of α-globin from the unaffected α-gene continues resulting in the accumulation, within erythroid cells, of a pool of free α-chains.The free α-chains are not able to form viable tetramers, and instead precipitate in the red cell precursors forming inclusion bodies.These inclusions are responsible for the intramedullary destruction of the erythroid precursors and hence the ineffective erythropoiesis that characterizes β- thalassemia.Although the anemia in β-thalassemia is primarily due to ineffective erythropoiesis, there is also a hemolytic component, which is related to the destruction of mature red cells containing β-chain inclusions in the circulation.To date, over 200 mutations have been described to cause β-tha- lassemia and related disorders. 10 The large majority of muta- tions causing β-thalassemia are primarily point mutations, others include deletions or addition of nucleotides (Fig. 1). Lists of mutations causing β-thalassemia are also available at the globin gene server (http:// globin.cse.psu.edu), and the Human Gene Mutation Database (HGMD) (http://www. uwcm.ac.uk/uwcm/mg/ hgmd0.html). Population studies demonstrated that approximately 25 mutations represent the vast majority of the β-thalassemia alleles in all populations at risk. 11 The mutations are population-specific and each population at risk for this dis- ease has a small number of common mutations as well as a variable number of rare cases. β-thalassemia lesions have been shown to strongly correlate with particular configura- tion of restriction fragment length polymorphisms (RFLPs) or haplotypes, along the β-globin gene cluster.Within each population there is a limited number of haplotypes, which demonstrates the independent origin of β-thalassemia in sev- eral populations. 12 Haplotype analysis has been useful for studying the origin and distribution of specific thalassemia mutations and as a guide to predicting which mutations are likely to occur in a given population based on linkage between the mutations and the polymorphisms. 13 Figure 2 illustrates the β-globin gene cluster and its DNA polymor- phisms in β-thalassemia chromosomes of Mediterranean ori- gin used in the construction of the β-globin haplotypes. 13 The thalassemias are, worldwide, the commonest single gene disorders, causing a major public health problem espe- cially in the Mediterranean area, the Middle East, the Indian subcontinent, the Far East, tropical Africa and the Caribbean. Nowadays, owing to the rapid population flow, they are widespread, occurring also in continental Europe, North and South America (http://www.who.int/ncd/hgn/haemogl. htm). Figure 3 illustrates the population distribution of the preva- lent β-thalassemia mutations in Italy, 14 Greece, 15 Turkey, 16 Cyprus, 17 India, 18 China 19 and Indonesia. 20 The best available approximate estimate of affected individuals indicates that 250 million people, 4.5% of the world population, are het- erozygous for a defective globin gene and that at least 300,000 lethally affected homozygotes are born annually, I  2001 Harcourt Publishers Ltd Blood Reviews (2001) 15, 19–29 doi: 10.1054/blre.2001.0147, available online at http://www.idealibrary.com on