REVIEW ARTICLE Thalassemic Hemoglobinopathies MARTIN H. STEINBERG, MD, From the Veterans Administration Medical Center and Department and JUNIUS G. ADAMS, PhD of Medicine, University of Mississippi School of Medicine, Jackson, Mississippi Hemoglobinopathies are due to changes in the normal produce a hemoglobinopathy with features of thalasse- amino acid sequence of globin. Thalassemias result mia. In this review the authors discuss such disorders from imbalance in the normal coordinated synthesis of and include the Hb Lepore and Constant Spring vari- the globin subunits that make up the hemoglobin tet- ants, hyper-unstable globins, mutations which create ramex It is now apparent that a single globin gene can alternative sites for mRNA splicing, and amino acid have coding region mutations which simultaneously substitutions likely to be associated with an additional produce a structural defect (hemoglobinopathy) and a thalassemia lesion within the same gene. (Am J Pathol biosynthetic defect (thalassemia). It is likely that two 1983, 113:396-409) distinct mutations within the same gene can occur and THE GENES that specify the structure and direct the junction for splicing to cleanly and faithfully take synthesis of globin have been assigned to specific place. I chromosomal regions,"2 mapped by restriction en- Hemoglobinopathies are disorders of the primary donuclease analysis,3-6 and completely sequenced.'-" structure of globin and are most often due to the The a and a-like embryonic C genes are located on the substitution of a single amino acid.18 The biosyn- short arm of Chromosome 161 as shown in Figure la. thesis of structural variants is generally normal except The a-globin genes are duplicated, as are most globin for hemoglobinopathies caused by 6(3-fusion genes genes.3 The short arm of Chromosome 11 contains (Lepore hemoglobins)18"9 or by mutations in the the P-like globin gene cluster2 that includes the em- a-globin chain termination codon (Constant Spring bryonic E-gene, the paired y-globin genes and adult 6- hemoglobins).'18"9 In distinction, the thalassemias and P-globin genes (Figure lb). Several varieties of result from disruption of the usual coordinated syn- thalassemia involve deletion of globin genes or of thesis of the globin chains that comprise tetrameric portions of these genes. The extent of deletion in hemoglobin.'9 With the above exceptions, the pri- these varieties of thalassemia is illustrated beneath mary structure of globin in thalassemia was con- each gene cluster. sidered to be normal,20 the clinical manifestations of A more detailed picture of the structure of a globin the disease a result of suboptimal synthesis of the in- gene is shown in Figure 2, along with the process of volved globin chain. gene transcription, mRNA processing, and mRNA The thalassemia phenotype includes combinations translation. Of special note for this review is the and varying degrees of hypochromia and microcy- process of RNA splicing wherein the intervening tosis, anemia, reticulocytosis, splenomegaly, and sequences (introns) are cleanly excised from precursor erythroid bone marrow hyperplasia. The most prev- mRNA and the coding segments (exons) are ligated to alent and clinically important thalassemias are the a- form a continuous stretch of mRNA that codes for globin. This process is complex, not yet fully under- stood, and may take place by more than one mech- Supported by Research Funds of the Veterans Adminis- tration and the Mississippi Affiliate of the American Heart anism. 14-16 It is generally believed that specific nucleo- Association. tide sequences are required at the 5' exon-intron Address reprint requests to Martin H. Steinberg, MD (donor) junction and at the 3' intron-exon (acceptor) (151), VA Medical Center, Jackson, MS 39216. 396 Vol. 113 * No. 3 THALASSEMIC HEMOGLOBINOPATHIES 397 CHROMOSOME 16 KbpO I1 20 30 5' *i - Et I -ML 3' C2 *c' lfa, a2 a, M-ED- _, ,NErASIAN, MEE= MED MED CHROMOSOME II Kb 0 10 20 30 40 50 60 5' 3'5I _ -o - se alu_ *12 Gy Ay */1 8 R CLEPORE1 Gd-- AY (,§0)0-THAO _ y HPFI AlHPF±~~ (61)0 THA~ (Y,13)0 THAL7 (Y613n0 THAL~~ (6) THAL~ Figure la-The a-like gene complex showing the duplicated a-genes, a2 and a, and the deletions described in a-thalassemia. The open boxes indicate the possible limits of deletion, while the hatched boxes indicate the maximum extent. Broken edges indicate that the maximum ex- tent is not known. The first two types are seen in blacks. Med refers to the presence of this lesion in Mediterranean populations and Asians to Southeast Asian peoples. b-The p-like gene complex showing the embryonic E gene the duplicated fetal y genes, Gy and Ay and the adult d and p genes with the known varieties of deletion p-thalassemia. HPFH refers to the hereditary persistence of fetal hemoglobin, a thalas- semia-like condition. Wa, Pp,, wPP2 and yC, refer to pseudogenes which have base sequences homologous to expressed genes but due to a variety of sequence changes are incapable of producing a recognizable globin. (Modified from Weatherall and Clegg.'3) and P-thalassemias. Both groups encompass a large genes results in intrauterine or neonatal death (hy- and still growing number of molecular defects that drops fetalis). result in suboptimal globin synthesis. Thalassemia In addition to deletion of genetic material,21-28 can result in either no globin synthesis (31 or ao) or molecular lesions associated with thalassemia include diminished globin synthesis (f3 or a+) directed by the nucleotide substitutions in conserved sequences 5' to defective gene(s).19 As mentioned previously, deletion the protein coding portion of -the gene, which may of genes or portions of genes is a mechanism of pro- "down-regulate" gene transcription2930; intervening ducing thalassemia. Gene deletion is the major cause sequence nucleotide substitutions or deletions that of a-thalassemia. Deletion of one of the four a-glo- alter existing sites or create new sites for intron ex- bin genes results in a clinically normal phenotype cision and exon splicing, impairing mRNA process- referred to as the "silent carrier" or heterozygous ing31-36; and nonsense mutations which introduce a-thalassemia-2. Deletion of two a-globin genes by stop codons into the coding portion of the gene.37-39 either loss of one gene from each chromosome (ho- There have been a number of recent reviews of the mozygous a-thalassemia-2) or both genes from a molecular biology of the thalassemias. 13.4044 single chromosome (heterozygous a-thalassemia-1) For many years structural and thalassemic genes produces a clinically mild phenotype with microcy- were considered to be so closely linked that the pres- tosis and minimal anemia. Deletion of three a-globin ence of one of these mutations on a particular genes results in HbH disease, characterized by a clini- chromosome precluded the presence of the other on cally significant hemolytic anemia. HbH, a tetramer the same chromosome. This concept began to erode of four P-chains, is present in the red cells of individ- with the findings of hemoglobin variants with two uals with this disorder. Deletion of all four a-globin separate amino acid substitutions such as HbC Har- 398 STEINBERG AND ADAMS AJP - December 1983 DNA structure of globin. These alterations may be the a. 5' - - 1 primary cause of thalassemia or be incidental to an t Transcription associated thalassemia lesion. We include in our Nm RNA discussion the Lepore and Constant Spring-like de- b. fects, hyper-unstable globins, coding region defects Processing creating alternative sites for mRNA splicing with associated amino acid substitutions, silent coding C. CAP AAAA... region mutations creating alternative splice sites, and amino acid substitutions which are likely to be as- mRNA sociated with an additional thalassemia mutation on d. CAP F AAAA... the same chromosome. I Translation T Globin The Lepore and Constant Spring-like Hemoglobins -Untranslated These P-like and a-globin variants are considered -Tronslated together because both have extensive differences be- -- Introns tween the structure of the variant and normal globins. Figure 2a-The nucleotide sequences that specify the structure of globin are encoded in discontinuous blocks of DNA called exons. The These differences are caused in the case of Lepore three globin exons are interrupted by two intervening sequences, or hemoglobins by nonhomologous crossing over be- introns; 5' and 3' to the coding sequences are untranslated portions of DNA. The 5' sequences contain specific signals which in part deter- tween 6 and P genes and in the Constant Spring mu- mine the frequency and fidelity of transcriptional events and have tants by termination codon mutation and elongated been termed "promoters." b-In an enzymatically governed pro- cess the entire gene is transcribed into a large nuclear RNA copy, a-globin chains. These variants have been recognized which contains untranslated sequences and introns. c-This as being associated with the thalassemia phenotype large RNA is processed to yield a smaller mRNA, which can be trans- ported to the cytoplasm. During processing the introns are cleanly for a number of years, have been the subject of con- excised and the exons ligated in a process called splicing. A special siderable study, and will not be covered in detail. cap nucleotide is added, and a poly(A) tail attached, which promotes the translational efficiency and stability of mRNA. The above events The Lepore hemoglobins, the first structural vari- are all intranuclear. d-mRNA is translated into globin on the ants to with the thalassemia polyribosomes of the cell cytoplasm. This involves the interplay of be associated phenotype,49 a number of initiation and elongation factors and transfer RNAs that consist of 3 known different 6(3-fusion chains,50-53 convey the specified amino acid to the growing globin polypeptide.