REVIEW ARTICLE Thalassemic

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 with features of thalasse- amino acid sequence of . 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 tet- ants, hyper-unstable , 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 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 contains (Lepore )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, , reticulocytosis, , 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

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(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 , 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 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 . 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 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 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. although additional examples are possible (Table 1). The completed a and P-chains, which are made on separate ribo- somes, are released, groups inserted, and tetrameric hemo- The crossing over event deletes the normal P-globin globin formed. gene from the chromosome containing the fusion gene.5'54 This, in addition to the reduced synthesis of lem45 and Hb Arlington Park,46 the suggestion that Lepore globin, leads in heterozygotes, to hematologic a-globin genes may be duplicated,47 and the demon- features of P-thalassemia trait.19 It is not clear what stration of a-chain locus duplication by the presence underlies the reduced synthesis of Lepore hemoglo- of HbG Buda, HbJ Pest, and HbA in a single individ- bins, which, in some respects, resembles the synthesis ual.48 From our current, very complete, understand- of the normal HbA2.5559 Since the amino terminal ing of the location, organization, and nucleotide portion of Lepore globin is derived from the 6-globin sequences of the globin genes and with our expanding gene which is inherently a poorly expressed gene knowledge of the molecular lesions of thalassemia, it when compared with the P-chain gene, from which it can be predicted that mutations causing both a hemo- arose, it is possible that the 6-chain and Lepore hemo- globinopathy and thalassemia might be present with- globin genes both contain the same changes which in the same gene. There is now a great deal of evi- impair the efficiency of transcription. The CCAAT dence that mutations in the protein coding portion of box of the 6-globin gene, a region which affects the the gene may produce the thalassemia phenotype. frequency of transcriptional events, contains a single The mechanisms appear varied. In addition, other nucleotide change (CCAAC) from that of the P-globin variant globins appear to be associated with a sepa- gene.9 Hb Parchman, a 6P6 variant which probably rate thalassemia producing mutation on the same arose by a double nonhomologous crossover, is syn- chromosome. Thus the distinction between hemoglo- thesized like normal 6-chain but probably contains a binopathies and thalassemia becomes unclear and the P first intervening sequence (IVS-1).60 Thus, changes particular clinical syndrome determined by the type in 6 or Lepore IVS-1 are alone unlikely to explain the and place of the coding region mutation. reduced transcription of these globins. There is evi- In this review, we will discuss thalassemia phe- dence suggesting that 6 and 6p mRNA is unstable.57 notypes associated with alterations of the primary It has also been demonstrated that there is defective Vol. 113 * No. 3 THALASSEMIC HEMOGLOBINOPATHIES 399 Table 1 - Features of the Lepore and Constant Spring-like Hemoglobins Site of Variant abnormality % in heterozygote Clinical features Lepore Boston 687-pl116 10-12 ,3-thalassemia trait Lepore Baltimore d50-P86 10-12 p-thalassemia trait Lepore Hollandia 622-p50 10-12 p-thalassemia trait Constant Spring a142 UAA-.CAA(gln) 0.2-1.7 a-thalassemia-2 Koya Dora a142 UAA-UCA(ser) 0.5-10 c-thalassemia-2 Icaria 6142 UAA-.AAA(lys) <1 a-thalassemia-2 Seal Rock a142 UAA- GAA(glu) 2 a-thalassemia-2 accumulation of 6p mRNA in gene transfer systems synthesis of Hb Indianapolis with rapid posttransla- primed with "Lepore" genes constructed in vitro.6" tional degradation is the observation that pIndianapolis Hb Constant Spring was first definitively described made up 35% of non-a-radioactivity when reticulo- by Milner and his co-workers62,63 and was followed by cyte mRNA was translated in a wheat germ cell-free the finding of several other variants with similar char- system and that incubation of reticulocytes at 22 C acteristics (Table 1). In this family of a-globin mu- led to a 30% increase in pIndianapolis radioactivity, tants, a nucleotide change in the termination codon when compared with incubation at 37 C.73 An in- which follows the last translated codon allows con- triguing and still unsolved question is why heterozy- tinued translation of a-mRNA until a subsequent in- gotes with Hb Indianapolis appear to have severe phase termination codon appears.63 This extends the P-thalassemia, while individuals heterozygous for a-chain by 31 amino acid residues. The structure of (O-thalassemia or other unstable P-chain variants, the four known termination codon variants differ at having a similar net accumulation of P-chain, have amino acid residue 142, the site of the expected ter- minimal disease. It may be that the erythrocyte mem- mination codon.64-66 The remaining 30 extra residues brane is damaged by both the accumulation of exces- are identical and predicted by the sequence of the 3' sive a-globin and the extremely rapid catabolism of non-coding mRNA and DNA." 67 An in-phase termi- pIndianaPolis chains.73 nation codon occurs following the 31st additional It is not unreasonable to assume that in other pa- amino acid.67 Hb Constant Spring mRNA may be tients with the phenotype of P-thalassemia the disease unstable, leading to reduced globin synthesis.19 The is produced by a similar mechanism. The detection of presence of an Hb Constant Spring gene causes the these variants could be extremely difficult if the muta- hematologic features of heterozygous a-thalassemia- tion was chromatographically "silent." the dominant 2. When present with the a-thalassemia-1 chromo- transmission of severe thalassemia, which appeared some, which has deletion of both a-globin genes, to result from a new mutation, might provide a clue HbH disease occurs. Messenger RNA studies demon- to the presence of this type of defect. strated decreased amounts of a-mRNA which was Hemoglobin Quong Sze (a125 leu,pro), an a-chain probably unstable, because the amount of a-mRNA variant that produces a thalassemic phenotype by a was greater in bone marrow cells than in reticulo- mechanism conceptually similar to Hb Indianapolis, cytes. 68-70 was found in quite a different way.74 A patient with The clinical and biochemical properties of Lepore HbH disease was noted to have deletion of only two and Constant Spring hemoglobins have been reviewed a-globin genes.75 HbH disease is typically caused by in detail by Weatherall and Clegg.'9 deletion of three of the four a-genes or deletion of two a genes plus the presence of Hb Constant Spring. The Hyper-unstable Hemoglobins It was postulated that the chromosome having two a- globin genes contained one gene that was "dysfunc- Hb Indianapolis ((3112 cys'arg) provided the first evidence that a single amino acid substitution could produce the findings of typical thalassemia.71.72 This Table 2-The Hyper-Unstable Variants variant is synthesized at a near normal rate but is so Method of Clinical rapidly catabolized that the half-life of the plndianapolis Variant Mutation detection features chain is less than 10 minutes.71 Thus, Hb Indianapolis Indianapolis p112cys- arg Radiolabeled peptide Severe p- is undetectable in the hemolysate by all the commonly and amino acid thalassemia analysis used techniques,72 and it is not known whether the Quong Sze a125leu- pro Gene sequencing a-thalassemia-1 Plndianapolis chain binds heme or participates in tet- and radiolabeled ramer formation. Further supporting the near normal protein 400 STEINBERG AND ADAMS AJP * December 1983 tional."75 When both a genes of this chromosome Variant Hemoglobins Creating Alternative Sites were cloned and their nucleotide sequence ascer- for Globin mRNA Splicing tained, a CTG (leucine) codon was changed to CCG (proline) at coding position 125 of the a2 gene.74 HbE, a P-globin variant (f326 glu-lys) common in When the a-globin corresponding to the a gene se- Southeast Asians is associated with some unusual quence was searched for, no protein was detected by properties. Its proportion (25-30%7o) in heterozygotes several electrophoretic techniques; however, a rapidly is less than expected for stable P-chain variants and degrading a-globin band was seen when reticulocytes mild microcytosis is typical in these individuals were labeled with 3H-leucine. a2- and a,-mRNAs were (MCV-75fl).19'89 Homozygotes for HbE have mild present in the expected proportions, indicating that hemolytic anemia whereas individuals who are mixed both genes were transcribed normally and translation heterozygotes for HbE and p-thalassemia may have of both mRNAs in a reticulocyte lysate cell-free sys- the phenotype of severe P-thalassemia.90-95 These tem was equal.76 However, a2 mRNA (aQuong Sze) was paradoxical observations led to the hypothesis that degraded more rapidly than the normal mRNA from the ,E gene behaved as a mild P+-thalassemia allele. the a1 gene.76 The insertion of a proline residue into This hypothesis was confirmed by finding reduced the a-helix disrupts the integrity of the helix and, in (3E-chain synthesis,96'97 a deficit of PE-mRNA,96.97 the case of Hb Quong Sze, is also likely to interfere and instability of pE-mRNA.98 The sole pathologic with the afl, interchain contact. A hyper-unstable change in two cloned pE genes was located in the globin is produced, and the features of a-thalassemia codon for amino acid 26, where the expected transi- emerge. tion from GAG to AAG was found.99 However, when In both the example of Hb Indianapolis and Hb a cloned pE gene was examined in an in vivo expres- Quong Sze, it is not entirely clear why the phenotype sion system, the codon change activated an alterna- of the thalassemia rather than that of the hemolytic tive site for pE-mRNA splicing (Table 3), so that in anemia characteristic of unstable hemoglobins exists. addition to normally spliced mRNA, which cleanly There have been many severely unstable P-globin vari- excises IVS-1, an additional mRNA which utilized ants described,77-84 some of which comprise only this alternative site, resulting in a deletion of a por- 1-2Oo of the hemolysate.85 Yet in none of these cases tion of the first exon, was present.99 There was also an are features of thalassemia evident. In some respects, increased quantity of mRNA which contained the the hematologic findings in simple heterozygotes for first intron. Thus, the 3E mutation retards normal Hb Quong Sze are very analogous to those in hetero- IVS-1 excision and induces abnormal mRNA splic- zygotes for Hb Indianapolis. Only one of four a-glo- ing, both of which prevent the normal accumulation bin genes is abnormal; yet the hematologic picture of 3E-mRNA and synthesis of 3E-globin. The muta- mimics that seen when two of four a-globin genes are tion in the protein coding portion of the 1E gene is missing (a-thalassemia-1 or homozygous a-thalas- therefore responsible for the thalassemic phenotype semia-2). Perhaps other a-thalassemia phenotypes associated with HbE. associated with intact a-loci, as determined by restric- The hematologic findings associated with Hb tion endonuclease gene mapping,86 will turn out to Knossos (p27 aa-ser) appears similar to HbE in many have identical or similar types of structural variants. respects.'00'101 Heterozygotes have the picture of mild It is interesting that single "dysfunctional" a-genes Pf-thalassemia but with a low HbA2 level, while mixed appear to lead to more severe a-thalassemia than do heterozygotes with p-thalassemia have severe thalas- single deleted genes.87'88 semia. As with the HbE mutation, the site and nature

Table 3-Hemoglobin Variants Which Cause or Are Likely to Cause Alternative Splicing of Globin mRNA Alternative Variant Mutation % in heterozygote l/p Ratio sequence Splice site HbE p26 glu-Iys 25-30 1.5 GGTAAGG 5', IVS-1 (GAG- AAG) Hb Knossos P27 ala-ser 33 1.5-2.5 GGTGAGTCC 5', IVS-1 (GCC-TCC) Hb Queens a34 leu-arg 12 "Balanced" TTCCGG 3', IVS-1 (CTG-.CGG) Vol. 113 * No. 3 THALASSEMIC HEMOGLOBINOPATHIES 401 IVS I IVS I1 I a t~~~~i li I 1 2 34 567 8

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THALASSEMIC PHENOTYPE

Figure 3-Alpha- and P-globin showing the site in the protein where the intervening sequences (IVS) were removed and the location of hemoglobin variants which have been associated with the phenotype of thalassernia ( ), an elevated HbA2 (, ), or microcytosis (t). The clustering of these variants about the sites of IVSs is striking, especially in the case of p-IVS-1. The large proportion of unstable variants with altered oxygen affinity is related to the location of certain amino acid residues which play critical roles in maintaining molecular stability and cooperativity about exon-intron junctions. This may be related to the evolution of globin.'56 a-Globin: 1) Hb Fort Worth,122 2) Hb Queens,'28"29 3) Hb Titusville,'33 4) Hb G Georgia,'34135 5) Hb Suan Dok,'4' 6) Hb Petah Tikva,"2 7) Hb Strumica,'55 8) Hb Quong Sze.74 (-Globin: 1) Hb Belfast,'50 2) Hb Riverdale- Bronx,83 3) Hb Moscva,79 4) Hb Henri Mondor,105 5) HbE,89-95 6) Hb Knossos,°°0-1'0 7) Hb Volga,80 8) Hb Genova,78 9) Hb Tacoma,'08 10) Hb Perth- Abraham Lincoln,'09 11) Hb Luftin,'07 12) Hb Shepherds Bush,'5' 13) Hb Vicksburg,'45 14) Hb Newcastle,'52 15) Hb Kansas,"2 16) Hb Southhampton,82 17) Hb Indianapolis,71'72 18) Hb Madrid,'53 19) Hb Wien,'54 20) Hb North Shore.'47-'49 of the Hb Knossos transversion (GCC- -TCC) may tide substitution at coding positionr 24 of the P-gene'03 also produce an alternative splice site (Table 3). all appear to create a sequence that resembles the There are conserved, or consensus, nucleotide usual donor site. In the instances of HbE95 and the sequences located at intron-exon boundaries that P-24 lesion,103 abnormal mRNA splicing has been appear to play a major role in the recognition process demonstrated by SI nuclease mapping experiments, for intron excision and exon ligation (mRNA splic- and affected individuals have the Pf-thalassemia ing).14-15-17-102 Extensive analysis of these regions by phenotype. The critical studies to prove that Hb Mount has shown that the 5' (donor) sequence is Knossos is also associated with aberrant RNA splic- 102 A.CAG/GTAAGT.G A, C, G, and T represent adenine, ing have not yet been reported. cytosine, guanine, and thymine, respectively. The We have completed an analysis of variant hemoglo- slash mark represents the actual site of excision-liga- bins caused by mutations which are close to the splice tion. The 3' (acceptor) sequence is (T)nNCAG/G, junctions of a and P-IVS-1 and IVS-2 to see whether where N can be any base. 102 The lower case n indicates they are associated with the thalassemia phenotype that the pyrimidine (C or T) stretch is variable in and whether the nucleotide change introduces a sig- length. In Mounts' analysis, n was 11 or greater.102 nal that might promote alternative RNA splicing HbE, Hb Knossos and the case of a silent nucleo- (Figure 3). Other than those already discussed, there 402 STEINBERG AND ADAMS AJP * December 1983 Table 4- 3-IVS-1 and P-IVS-2 Acceptor and Donor Site Variants Clinical* Name Residue Substitution Codon features Reference p-IVS-i Riverdale-Bronx 24 GLY - ARG GGT - CGT Unstable, tA2 83 Savannah 24 GLY - VAL GGT - GTT Unstable 84 Moscva 24 GLY - ASP GGT - GAT Unstable, tA2 79 G-Taiwan 25 GLY - ARG GGT - CGT Normal 109 HbE 26 GLU - LYS GAG - AAG Thalassemia 89-95 Henri Mondor 26 GLU - VAL GAG - GUG Unstable 105 microcytosis Knossos 27 ALA - SER GCC - UCC Thalassemia 100-101 Volga 27 ALA - ASP GCC -GAC Unstable, tA2 80 St. Louis 28 LEU - GLN CTG - CAG Unstable 106 MetHb tO2 affinity Genova 28 LEU - PRO CTG - CCG Unstable, tA2 78 Luftin 29 GLY - ASP GGC - GAC Unstable 107 Tacoma 30 ARG - SER AGG - AG; tO2 affinity, tA2 108 Perth-Abraham 32 LEU - PRO CTG - CCG Unstable, tA2 109 Lincoln Castilla 32 LEU - ARG CTG - CGG Unstable 110 P-IVS-2 Richmond 102 ASN - LYS AAC - AAR Normal 111 Kansas 102 ASN - THR AAC - ACC +02 affinity, tA2 112 Beth Israel 102 ASN -'- SER AAC - AGC Cyanosis 113 CTC Heathrow 103 PHE - LEU TTC --TTA tO2 affinity 114 TTG Camperdown 104 ARG - SER AGG - AG? Normal 115 Sherwood Forest 104 ARG - THR AGG - ACG Normal 116 Southampton 106 LEU - PRO CTG - CCG Unstable, tA2 82 Tubingen 106 LEU --GLN CTG - CAG Unstable 117 Burke 107 GLY - ARG GGC - CGC Unstable 81 $02 affinity Yoshizuka 108 ASN - ASP AAC - GAC 402 affinity 118 San Diego 109 VAL - MET GTG - ATG tO2 affinity 119 * The major clinical features associated with each variant hemoglobin are listed. "Unstable" refers to hemolytic anemia characteristic of unstable hemoglobins and not associated with features of thalassemia, such as microcytosis and unbalanced globin chain synthesis. The symbols t and 402 affinity refer to increased and decreased oxygen affinity and the presence of erythrocytosis in the case of high-affinity variants. "Thalassemia" refers to the phenotype of heterozygous P3-thalassemia. are no additional thalassemia-like variants associated Two other a-variants that have thalassemia-like ex- with P-IVS-1 and no candidates clustered about pression are associated with very GC-rich sequences P-IVS-2. In addition, the expected nucleotide se- not found in other regions of the a-globin gene."1 Hb quence changes associated with these hemoglobins do Fort Worth (a27 glu--gly) appears unstable but is asso- not cause recognizable 5' or 3' consensus sequences ciated with an MCV of 68.122 The sequence around for splicing (Table 4). the A -> G transition is 5'-GCGGGGGCCC-3' (the There are a number of a-globin variants located altered nucleotide is underlined). Hb Suan-Dok near both IVS-1 and IVS-2 which are possibly as- (a1o0 leuarg) is also unstable and when present with sociated with a thalassemic phenotype (Figure 3 and a-thal-1 leads to HbH disease.141 The nucleotide Table 5). Instability of some of these variants may ac- transversion causing this variant (T -- G) leads to the count for their diminished proportion and could con- sequence 5'-CCCGGGCCGCCC-3'. We have ex- ceivably play a role in their association with HbH dis- amined the mRNA for Hbs Fort Worth,122 Suan- ease. Hb Queens (a34 leu arg) seems the best candidate Dokl41 and Evanston143 (vide infra) and other vari- for an a-globin variant leading to an alternative splice ants using a computer program that generates a site (Table 3). This stable variant is present at a level secondary structure for these nucleic acids (PROPH- of about 12% and is associated with mild microcy- ET computer system). While these variant mRNAs tosis.128-129 The usual a34 CTG (leu) is changed to are associated with changes in secondary structure, CGG (arg), generating the sequence 5'-TTCCGG-3', compared with normal mRNA, similar changes can which bears some resemblance to the 3' acceptor at times be found in non-thalassemia-producing site.14 Proof that this sequence is indeed associated structural variants, and the significance of these ob- with a splicing defect has not been obtained. servations is not yet clear. Additional work is needed Vol. 113 * No. 3 THALASSEMIC HEMOGLOBINOPATHIES 403

Table 5-a-IVS-1 and a-IVS-2 Acceptor and Donor Site Variants Clinical Name Residue Substitution Codon % Variant features Reference a-IVS-1 Chad 23 GLU - LYS GAG - AAG 16 Normal 120 G-Audhali 23 GLU - VAL GAG - GTG 25 Normal 121 Fort Worth 27 GLU - GLY GAG - GGG 5 a-Thalassemia-2 122 Unstable Spanish Town 27 GLU - VAL GAG - GTG 11 Normal 123 Shuang Feng 27 GLU - LYS GAG - AAG 13 Unstable 124 O-Padova 30 GLU - LYS GAG - AAG 25 Normal 125 G-Honolulu 30 GLU - GLN GAG - CAG ?50 Uncertain 126 Prato 31 ARG - SER AGG - AG? 28 Normal 127 Queens 34 LEU - ARG CTG - CCG 12 a-Thalassemia-2 128, 129 a-IVS-2 J-Cape Town 92 ARG - GLN CGG - CAG 6-8 Normal 130 Chesapeake 92 ARG - LEU CGG - CTG 25-30 tO2 affinity 131 Setif 94 ASP - TYR GAC - TAC 12-15 Uncertain 132 Titusville 94 ASP - ASN GAC - AAC 35 + 02 affinity 133 G-Georgia 95 PRO - LEU CCG - CTG 23 Uncertain 134, 135 Rampa 95 PRO - SER CCG - TCG 25 45 Uncertain 134,136 Denmark Hill 95 PRO - ALA CCG - GCG 19 tO2 affinity 137 St. Lukes 95 PRO - ARG CCG - CGG 10 Unstable 138 Dallas 97 ASN - LYS AAC - AAA 25 tO2 affinity 139 Manitoba 102 ER - ARG AGC - AGA 5 Uncertain 140 CGC Suan-Dok 109 LEU - ARG CTG - CGG 8 a-Thalassemia-2 141 Unstable Petah-Tikva 110 ALA - ASP GCC - GAC 33 Unstable 142 a-Thalassemia-2 The major clinical features associated with each variant hemoglobin are listed. "Unstable" refers to hemolytic anemia characteristic of unstable hemoglobins and not associated with features of thalassemia, such as microcytosis and unbalanced globin chain synthesis. The symbols t and +O2 affinity refer to increased and decreased oxygen affinity and the presence of erythrocytosis in the case of high-affinity variants. "Thalassemia" refers to the phenotype of heterozygous ,l-thalassemia. The phenotype of a-thalassemia-2 is assumed from the presence of HbH disease in individuals with the variant hemoglobin and a-thalassemia-1. for us to see how and whether these nucleotide that acted to suppress the synthesis of new P-chains, changes alter the secondary structure of mRNA, leading to an elevated a/p ratio. It is possible that a whether tertiary structure is changed, and how the similar mechanism is responsible for the elevated a/l stability of mRNA is affected. ratios in heterozygotes for Hb Petah Tikva and Hb Hb Petah Tikva 42 is an unstable a-chain variant Constant Spring. that produces the phenotype of a-thalassemia. The The discussion of Hbs Petah Tikva, Constant thalassemia phenotype seems to be due to instability Spring, and Ann Arbor also emphasizes an interest- of the variant chain as well as from the premature ing point alluded to earlier. All three of these variants termination of its synthesis during erythroid matura- are unstable and are present in very small amounts; tion. An interesting observation in the Hb Petah yet Hb Constant Spring and Petah Tikva produce the Tikva study is that there are significantly elevated a/p phenotype of a-thalassemia, while Hb Ann Arbor synthesis ratios in two simple heterozygotes for this does not. In searching all of the known hemoglobin variant. 142 These findings appear to be paradoxical in variants for characteristics of thalassemia (microcy- view of the fact that Hb Petah Tikva produces a-tha- tosis, increased HbA2, and the classic thalassemia lassemia. Similar findings were reported in simple phenotype), we found that the distribution of these heterozygotes for the Constant Spring,19 another a- variants does not appear to be random (Figure 3). It chain variant producing the phenotype of a-thalas- can be seen that most variants with thalassemic char- semia, and in simple heterozygotes for Hb Ann acteristics seem to be located in areas of the protein Arbor,"44 an unstable a-variant. Although Hb Ann that are near where the intervening sequences of the Arbor does not produce the phenotype of a-thalas- gene are known to occur. This is especially true of the semia, it is extremely unstable and constitutes only P-chain IVS-1. For some of these variants, the prox- 1-2% of the hemolysate in simple heterozygotes. It imity to an intervening sequence most likely promotes was thought in the case of Hb Ann Arbor that the alternate splicing. The mechanism by which most of degradation of Hb Ann Arbor released free P-chains the other variants produce thalassemic stigmata re- 404 STEINBERG AND ADAMS AJP - December 1983 mains unclear. Further study of these variants may this does not appear unique in any way. At our present elucidate other mechanisms that lead to faulty ex- level of understanding, it seems likely that Hb Vicks- cision of the intervening sequences during mRNA burg is associated with a P3-thalassemia-producing processing. lesion on the same chromosome, although this has yet to be proven. The Hb Vicksburg-pI-thalassemia gene have arisen mutation of a with one of Within Protein could by gene Silent Nucleotide Mutations these defects or by homologous crossing over between Coding Regions of the Gene (3-Vicksburg and Pf-thalassemia genes.73145 To date, only a single example of P(-thalassemia Hb North Shore ((I334val - glu) a very mildly un- produced by this mechanism has been described.'03 In stable variant, resembles HbE hematologically, being a black patient with (3-thalassemia, three indepen- associated with mild microcytosis, a mild deficit of P- dently cloned P-globin genes had a single nucleotide globin synthesis, and a reduced proportion of Hb change in codon 24 (GGT -- GGA). Both triplets North Shore.147-149 When present with , encode for glycine, so that the amino acid sequence it produces the phenotype of HbS-P(-thalassemia.147 of the P-chain remains normal. Yet the concentration Examination of the nucleotide sequences about the of mRNA transcribed from this gene was over four- Hb North Shore mutations does not reveal an obvious fold less than that transcribed from the normal donor or acceptor splice signal; and, in addition, the P-globin gene when cloned genes were placed in altered codon is remote from splice junctions10 (Fig- plasmid vectors and introduced into monkey kidney ure 3). However, preliminary Si nuclease mapping of cells. An aberrantly spliced mRNA which utilized a Hb North Shore IVS-2 suggests that some splicing splice site within codon 25 was detected in the mRNA defect may be present.157 transcribed from this (3+-thalassemia gene. Thus, a Hb Evanston (a14 trp arg) a stable a-globin vari- silent nucleotide change activates a nearby, hitherto ant, was found in a black child with HbH disease.143 cryptic, site for mRNA splicing and induces the Of the two possible nucleotide changes that would P(-thalassemia phenotype. change the normal TGG codon (tryptophan) to arginine, AGG or CGG, the latter would introduce a run of 12 Gs and Cs (5'-GGCCGCCCGGGG-3'), Hemoglobins Possibly Associated With a in Hbs Fort in Cis recalling the changes introduced Worth116 Thalassemia Mutation and Suan Dok.141 There is little clinical information In the previously described instances, thalassemia available about Hb Evanston; however, it does appear was induced by the coding region mutation either by to confer the phenotype of a-thalassemia-2. Further virtue of hyper-instability or by the unveiling of al- studies will be required to define the mechanism ternative splice sites. whereby this effect occurs; however, it is curious and Hb Vicksburg (375 leu o) was found in a 7-year-old unlikely to be a result of chance, that three a-globin black boy.'45 Hematologic and genetic data indicated variants with thalassemia-like expression all have (or that he was a mixed heterozygote for Hb Vicksburg potentially have) long stretches of G and C nucleo- and PO-thalassemia. Hb Vicksburg was a stable vari- tides. ant but constituted only 8% of the hemolysate. Other stable (3-variants present with PO-thalassemia usually constitute the major portion of the hemolysate.19 A Conclusions profound deficit of P-mRNA was found, further sub- The association of the thalassemia phenotype with stantiating that the paucity of Hb Vicksburg resulted hyper-unstable hemoglobins, hemoglobin variants from a primary defect in synthesis rather than from which create alternative opportunities for mRNA posttranslational events. Whether the deleted codon splicing, and silent coding region mutations tends to altered the secondary structure of P-Vicksburg mRNA blur any major distinctions between the pathogenesis and reduced its stability was unknown, but the ex- of these disorders. In addition, it is very likely that ample of an unstable variant, Hb St. Antoine, caused both structural and thalassemia-producing mutations by deletion of P-74 and 75, yet unassociated with can occur within the same globin gene. Silent nucleo- thalassemic features make this possibly less likely.146 tide substitutions can produce thalassemia or be The Hb Vicksburg deletion is remote from splice totally innocuous, depending upon the site and type junctions10 (Figure 3) and does not create an iden- of mutation. Coding region amino acid substitutions tifiable alternative splice site.14 The loss of codon 75 can cause thalassemia, any of a wide variety of does permit the sequence GGCGC to appear; but, pathologic hemoglobin variants, or entirely harmless unlike the longer GC sequences described previously, variant hemoglobins. Which occurs depends on the Vol. 113 * No. 3 THALASSEMIC HEMOGLOBINOPATHIES 405 site in the gene where the nucleotide change takes 20. Benz EJ Jr, Forget BG: The molecular genetics of the thalassemia syndromes, Progress in . Vol place and which new base sequence is produced. IX. Edited by EB Brown. New York, Grune and Strat- ton, 1975, pp 107-155 References 21. 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Niazi GA, Efremov GD, Nicklov N, Hunter E Jr, M, Steinberg, MH, Adams JG: Hemoglobin North Huisman THJ: Hemoglobin Strumica or 2 12(G19) Shore: A variant hemoglobin associated with the his-'oarg P2. (with an addendum: Hemoglobin-J-Paris phenotype of P-thalassemia. Blood 1983, 61:378-383 1, a22 (A10) ala - asp (2. Biochim Biophys Acta 1975, 150. Kennedy CC, Blundell G, Lorkin PA, Lang AG, 412:181-186 Lehmann H: Haemoglobin Belfast 15(A12) tryp- 156. Blake C: Exons and the evolution of proteins. TIBS tophan- arginine: A new unstable haemoglobin vari- 1983; 8:11-13 ant. Br Med J 1974, 4:324-326 157. Benz EJ Jr, Adams JG, Smith C, Steinberg MH: Un- 151. White JM, Brain MC, Lorkin PA, Lehmann H, Smith published data M: Mild "unstable Haemoglobin haemolytic anae- mia" caused by haemoglobin Shepherds Bush (Q374(E18) gly- asp). Nature 1970, 225:939-941 152. Finney R, Casey R, Lehmann H, Walker W: Hb New- castle: (392(F8)His- Pro. FEBS Letters 1975, 60:435- 438 Acknowledgments 153. Outeirino J, Casey R, White JM, Lehmann H: Haemoglobin Madrid (3115(G17) alanine- proline: An We thank Connie Palmer for secretarial assistance and unstable variant associated with haemolytic anaemia. W. Tilly Morrison for help with the computer analysis of Acta Haematol 1975, 52:53-60 mRNA structure.