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Thalasemia Hemoglobinopati Review Article

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  REVIEW ARTICLE Thalassemic Hemoglobinopathies MARTIN H. STEINBERG, MD, and JUNIUS G. ADAMS, PhD Hemoglobinopathies are due to changes in the normalamino acid sequence of globin. Thalassemias result fromimbalance in the normal coordinated synthesis of the globin subunits that make up the hemoglobin tet- ramex It is now apparent that a single globin gene can havecoding regionmutations which simultaneously produce a structuraldefect (hemoglobinopathy)and a biosynthetic defect (thalassemia). It is likelythat two distinct mutations within the samegene can occur and THE GENES that specify the structure and direct the synthesis ofglobin have been assigned to specific chromosomal regions, 2 mapped by restriction en- donuclease analysis,3-6 and completely sequenced.'- The a and a-like embryonic C genes are located on the short arm of Chromosome 161 as shown in Figure la. The a-globin genes are duplicated, as are most globin genes.3 The short arm of Chromosome 11 contains the P-like globingene cluster2 thatincludes the em- bryonic E-gene, the paired y-globin genes and adult 6- and P-globin genes (Figure lb). Several varieties of thalassemia involvedeletion ofglobin genesor ofportionsof these genes. The extent of deletion in these varieties of thalassemia is illustrated beneath each gene cluster. A more detailedpicture of the structure of aglobin gene is shown in Figure 2, along with theprocess of gene transcription, mRNA processing, and mRNA translation. Of special note for this review is the process of RNA splicing wherein theintervening sequences (introns) are cleanly excised from precursor mRNA and the codingsegments (exons) areligated to form a continuous stretch of mRNA that codes for globin. This process is complex,not yetfully under- stood, and may takeplace by more than one mech- anism. 14-16Itis generally believed that specific nucleo- tide sequences are required at the 5 exon-intron (donor)junction and at the 3 intron-exon (acceptor) From theVeterans Administration MedicalCenter and Department of Medicine, University of Mississippi School of Medicine, Jackson, Mississippi produce a hemoglobinopathy with features of thalasse- mia. In this review theauthors discuss such disorders and include the Hb Leporeand Constant Spring vari- ants, hyper-unstable globins, mutations which create alternative sites for mRNA splicing, and amino acid substitutionslikely to be associated withan additional thalassemia lesion within the same gene. (Am JPathol 1983, 113:396-409) junction forsplicing to cleanly and faithfully take place. I Hemoglobinopathies are disorders of the primary structure ofglobin and are most often due to the substitution of a single amino acid.18 The biosyn- thesis of structural variants is generally normal except for hemoglobinopathies caused by 6(3-fusion genes (Lepore hemoglobins)18 9 or by mutations in the a-globin chain termination codon (ConstantSpring hemoglobins).'18 9 In distinction, the thalassemias result from disruption of the usual coordinated syn- thesis of the globin chains that comprise tetrameric hemoglobin.'9 With the above exceptions, the pri- mary structure of globin in thalassemiawas con- sidered to be normal,20 the clinical manifestations of the disease a result of suboptimal synthesis of the in- volvedglobin chain. The thalassemia phenotype includes combinations and varying degrees of hypochromiaand microcy- tosis, anemia, reticulocytosis, splenomegaly, and erythroid bone marrow hyperplasia. The most prev- alent and clinically important thalassemias are the a- Supported by Research Funds of the Veterans Adminis- tration and the Mississippi Affiliate of the American Heart Association. Address reprint requests to Martin H. Steinberg, MD (151), VA Medical Center, Jackson, MS 39216. 396  THALASSEMIC HEMOGLOBINOPATHIES Kbp O 5' C2 I1 CHROMOSOME 16 20 *i   Et I -ML *c' lfa, a2 a, M-ED- 30 3' _ ,NErASIAN, MEE= MED MED CHROMOSOME II K b 0 10 20 30 40 50 60 5' 3 _  o   se alu_ GyAy */1 8 R CLEPORE1 Gd-- AY (,§0)0-THAO _ y HPFI AlHPF±~~ (61)0 THA~ (Y,13)0 THA L7 (Y613n0 THAL~~ (6) THAL~ Figure la-The a-like gene complex showing the duplicated a-genes, a2 and a, and thedeletionsdescribed in a-thalassemia. The open boxes indicatethe possible limits of deletion, whilethe hatchedboxes 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 Asianpeoples. b-The p-like gene complexshowing 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-likecondition. Wa, Pp,, wPP2 and yC, refer to pseudogenes whichhave basesequences homologous to expressed genes but due toa varietyof sequencechanges are incapable of producing arecognizable globin. (Modified from Weatherall and Clegg.'3) and P-thalassemias. Both groups encompass a large and still growing number of molecular defectsthat result in suboptimal globin synthesis. Thalassemia can result ineither no globin synthesis  31 or ao) or diminished globin synthesis  f3 or a+) directed by the defective gene(s).19 As mentioned previously, deletion of genes or portionsof genes is a mechanism of pro- ducing thalassemia. Gene deletion is the major cause of a-thalassemia. Deletion of one of the four a-glo- bingenes resultsin a clinically normalphenotype referred to as the  silent carrier or heterozygous a-thalassemia-2. Deletion of two a-globin genes by either loss ofone gene from each chromosome (ho- mozygous a-thalassemia-2) or both genes from a single chromosome (heterozygous a-thalassemia-1) produces a clinically mild phenotype withmicrocy- tosis and minimal anemia. Deletion of three a-globin genes resultsin HbH disease, characterized by a clini- cally significant hemolyticanemia. HbH, atetramer of four P-chains, is present in thered cells of individ- uals with this disorder. Deletion of all four a-globingenes resultsin intrauterine or neonatal death (hy- drops fetalis). In addition to deletion of genetic material,21-28 molecular lesions associated withthalassemia include nucleotide substitutions in conserved sequences 5 to the protein coding portionof -thegene, which may  down-regulate gene transcription2930; intervening sequence nucleotide substitutions or deletionsthat alter existing sites or create new sites for intron ex- cision and exon splicing, impairing mRNA process- ing31-36; and nonsense mutations which introduce stop codons into the coding portionof the gene.37-39 There have been a number of recent reviews of the molecularbiology of the thalassemias. 13.4044 For many years structural and thalassemic genes wereconsidered to be so closely linked that the pres- ence ofoneof these mutations on a particular chromosome precluded the presence of the other on the same chromosome. This concept began to erode with the findings of hemoglobin variants with two separate amino acidsubstitutions such as HbC Har- 397 ol.113 * No. 3 *12    398STEINBERG AND ADAMS DNA a. 5 1 t Transcription Nm RNA Processing CAP AAAA... mRNA CAP F AAAA...   Translation T Globin -Untranslated -Tronslated   ntrons Figure 2a-The nucleotide sequences that specifythe structureof globin are encoded in discontinuous blocks of DNA called exons. The threeglobin exons are interrupted by two intervening sequences, or introns; 5' and 3' to the coding sequences are untranslatedportions of DNA. The 5 sequences contain specific signals which in partdeter- mine the frequency and fidelity of transcriptional events and have beentermed  promoters. b-In an enzymatically governed pro- cess the entire gene is transcribed into a large nuclear RNA copy, which containsuntranslated sequences and introns. c-This large RNA is processed to yield a smaller mRNA, which can be trans- ported to the cytoplasm.Duringprocessing the introns are cleanlyexcised and the exons ligated in aprocess called splicing. A special cap nucleotide is added, and a poly(A) tail attached, which promotes the translational efficiency and stability of mRNA. The above events are all intranuclear. d-mRNA is translated into globin on the polyribosomes of the cell cytoplasm. This involves the interplay of a number of initiation and elongation factors and transfer RNAs that convey thespecified amino acid to the growing globinpolypeptide. The completed a and P-chains, which are made on separate ribo- somes, are released, heme groups inserted, and tetrameric hemo- globin formed. lem45 and Hb Arlington Park,46 thesuggestion that a-globin genes may be duplicated,47 and the demon- stration ofa-chain locus duplication by the presence of HbG Buda, HbJ Pest, and HbA in a single individ- ual.48 From our current, very complete, understand- ing of the location, organization, and nucleotide sequences of the globin genes and with our expanding knowledge of the molecular lesions of thalassemia, it can be predicted that mutations causing both a hemo- globinopathy and thalassemia might be present with- in the same gene. There is now a great deal of evi- dence that mutations in the protein coding portion of the gene may produce the thalassemia phenotype. The mechanisms appear varied. In addition, other variant globins appear to be associated with a sepa- rate thalassemiaproducing mutation on the same chromosome. Thus the distinction betweenhemoglo- binopathies and thalassemia becomes unclear and the particular clinical syndrome determinedby the type and place of the coding region mutation. In this review, we will discuss thalassemiaphe- notypes associated with alterations of the primary structure of globin. These alterations may be the primary cause of thalassemiaor be incidentalto an associated thalassemia lesion. We include in our discussion the Lepore and Constant Spring-like de- fects, hyper-unstable globins, coding region defects creating alternative sites for mRNA splicing with associated amino acid substitutions, silent coding region mutations creating alternativesplice sites, and amino acid substitutions which arelikelyto be as- sociated with an additional thalassemia mutation on the same chromosome. The Lepore and Constant Spring-like Hemoglobins These P-like and a-globin variants are considered together becauseboth have extensive differences be- tween the structure of the variant and normal globins. These differences are caused in the case ofLepore hemoglobinsby nonhomologous crossing over be- tween 6 and P genes and in the Constant Spring mu- tants by termination codon mutation and elongated a-globin chains. These variants have been recognized as being associated with the thalassemia phenotype for a number of years, have been thesubject of con- siderablestudy, and will not be covered in detail. The Lepore hemoglobins, the first structural vari- ants to be associated with the thalassemia phenotype,49 consist of 3 known different6(3-fusionchains,50-53 although additional examples are possible (Table 1). The crossing over event deletes the normal P-globin gene from the chromosome containing the fusion gene.5'54 This, in addition to the reduced synthesis of Lepore globin,leads in heterozygotes, to hematologic features of P-thalassemia trait.19 It is not clear what underlies the reduced synthesis ofLepore hemoglo- bins, which, in some respects, resembles the synthesis of the normal HbA2.5559 Since the amino terminal portion of Lepore globin is derived from the 6-globin gene which is inherently a poorly expressed gene when compared with the P-chain gene, from which it arose, it is possible thatthe 6-chain and Lepore hemo- globin genes both contain the same changes which impair the efficiency of transcription. The CCAAT box of the 6-globin gene, a region which affects the frequency of transcriptional events, contains a single nucleotide change (CCAAC) from that of the P-globin gene.9 Hb Parchman, a 6P6 variant which probably arose by a double nonhomologous crossover, is syn- thesized like normal 6-chain but probably contains a P first intervening sequence (IVS-1).60 Thus,changes in6 or Lepore IVS-1 are alone unlikelyto explain the reduced transcription of these globins. There is evi- dence suggesting that 6 and 6p mRNA is unstable.57 It has also been demonstrated thatthere is defective b. C. d. AJP - December 1983  THALASSEMICHEMOGLOBINOPATHIES 399 Table 1 - Features of the Lepore andConstant Spring-like Hemoglobins Siteof 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 10-12p-thalassemia trait Constant Springa142 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 primed with  Lepore genes constructed in vitro.6 Hb Constant Spring was first definitively described by Milner and his co-workers62,63 and was followed by the finding of several other variants with similar char- acteristics (Table 1). In this familyof a-globin mu- tants, a nucleotide change in thetermination codonwhich follows the last translated codon allows con- tinued translation of a-mRNA until a subsequent in- phase termination codon appears.63 Thisextends the a-chain by 31 amino acid residues. The structure ofthefour known termination codon variants differ at amino acid residue 142, the site of the expected ter- mination codon.64-66 The remaining 30 extra residues are identical and predicted by the sequence of the 3 non-coding mRNA and DNA. 67 An in-phase termi- nation codon occurs following the 31st additional amino acid.67 Hb Constant Spring mRNA may be unstable, leading to reduced globin synthesis.19 The presence of an Hb Constant Spring gene causes the hematologic features of heterozygous a-thalassemia- 2. When present with the a-thalassemia-1 chromo- some, which has deletion of both a-globin genes, HbH disease occurs. Messenger RNA studies demon- strated decreased amounts of a-mRNA which was probably unstable, because the amount of a-mRNA was greater in bone marrow cells than inreticulo- cytes. 68 70 The clinical and biochemical properties ofLepore and ConstantSpring hemoglobins have been reviewed indetail by Weatherall and Clegg.'9 The Hyper-unstable Hemoglobins Hb Indianapolis  3112 cys'arg) provided the first evidence that a single amino acid substitution could produce the findings of typicalthalassemia.71.72 This variant is synthesized at anear normal rate but is so rapidly catabolized that the half-life of the plndianapolis chain is less than 10 minutes.71 Thus, Hb Indianapolis is undetectable in the hemolysate by all the commonly used techniques,72 and it is not known whether the Plndianapolis chainbinds heme or participatesin tet- ramer formation. Furthersupporting the near normal synthesis of Hb Indianapolis with rapidposttransla- tional degradation is the observation that pIndianapolis made up 35 of non-a-radioactivity when reticulo- cyte mRNA was translated in a wheat germ cell-free system and that incubationof reticulocytes at 22 C led to a 30 increase in pIndianapolis radioactivity, when compared withincubation at 37 C.73 An in- triguing and still unsolvedquestion is why heterozy- gotes with Hb Indianapolis appear to have severe P-thalassemia, while individuals heterozygous for (O-thalassemia or otherunstable P-chain variants, having a similar net accumulation of P-chain, have minimal disease. It may be that the erythrocyte mem- brane is damaged by both the accumulationof exces- sive a-globin and the extremely rapid catabolism of pIndianaPolis chains.73 It is not unreasonable to assume that in other pa- tients with the phenotype of P-thalassemia the disease is produced by a similar mechanism. The detection of thesevariants could be extremely difficult if the muta- tion was chromatographically  silent. the dominant transmission of severe thalassemia, whichappeared to result from a new mutation, might provide a clue to the presence of this type of defect. Hemoglobin Quong Sze (a125 leu,pro), an a-chain variant that produces a thalassemic phenotype by a mechanism conceptually similar to Hb Indianapolis, was found in quite a different way.74 A patient with HbH disease was noted to have deletion ofonly two a-globin genes.75 HbH disease is typically caused by deletion of three of thefour a-genesor deletion of two a genes plus the presence of Hb Constant Spring. It was postulated that the chromosome having two a- globin genes contained one gene that was  dysfunc- Table 2-The Hyper-Unstable Variants Method ofClinical Variant Mutation detectionfeatures Indianapolis p112cys- arg RadiolabeledpeptideSevere p- and amino acid thalassemiaanalysis Quong Sze a125leu- pro Gene sequencing a-thalassemia-1 and radiolabeled protein Vol.113 * No. 3
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