Genetic Modifiers of B-Thalassemia

Thalassemia Syndrome • Molecular Basis of Disease

Genetic modifiers of b-thalassemia

Swee Lay Thein As the defective genes for more and more genetic disorders become unravelled, it is clear that patients with apparently identical genotypes can have many different clinical conditions even in simple monogenic disorders. b thalassemia occurs when there is a deficiency in the synthesis of b globin chains. The clinical manifestations of b thalassemia are extremely diverse, spanning a broad spectrum from severe anemia and transfusion-dependency to the asymptomatic state of thalassemia trait. The remark- able phenotypic diversity of the b thalassemias is prototypical of how a wide spectrum of disease severity can be generated in single gene disorders. The most reliable and predictive factor of disease phenotype is the nature of the mutation at the b globin locus itself. However, relating phenotype to genotype is complicated by the complex interaction of the environment and other genetic factors at the secondary and tertiary levels, some implicated from family studies, and others, as yet unidentified. This arti- cle reviews the clinical and hematologic diversity encountered in b thalassemia with an overview of the modifier genes that moderate their disease expression. Key words: b thalassemia, modifier genes. Haematologica 2005; 90:649-660 ©2005 Ferrata Storti Foundation

From the Department of Haematological Medicine, he thalassemias refer to a diverse Mediterranean region suggests that the dis- Guy’s, King’s & St Thomas’ School group of hemoglobin disorders char- ease was present as far back as the Neolithic of Medicine, King’s College London, acterized by a reduced synthesis of period.3 More than 200 mutations affecting UK. T one or more of the globin chains (a, b, g, db, the b globin gene are now known to result 1 Correspondence: gdb, d and egdb). b thalassemia occurs in a phenotype of b thalassemia. The most Professor Swee Lay Thein, when there is a deficiency of b globin; typ- common forms are those that are prevalent Department of Haematological ically, it is caused by a direct down-regula- in the malarial tropical and sub-tropical Medicine, Guy’s, King’s & tion in the synthesis of structurally normal regions where a few mutations have St. Thomas’ School of Medicine King’s College London, Denmark Hill b chains. However, a thalassemia pheno- reached high gene frequencies because of Campus, Bessemer Road type can also arise from structural b chain the protection they provide against malaria. London SE5 9PJ. variants if they are synthesized at a reduced In these countries in which b thalassemia is E-mail: [email protected] rate, e.g. Hb E (b26GluÆLys). Alternatively, prevalent, a limited number of alleles (4 to 5) the variants are produced at a normal rate account for 90% or more of the b tha- but are so unstable that they are rapidly lassemias, such that a focused molecular destroyed giving rise to a functional defi- diagnostic approach can be undertaken.4 ciency. The former group of variants is also The epidemiology of the disease, however, referred to as b thalassemic hemoglo- is changing due to a fall in total birth rate, binopathies. The hyperunstable b chain prevention programs and recent population variants act in a dominant negative fashion, movements. In Northern Europe, including causing a disease phenotype even when the UK, the US and Australasia, due to present in a single copy, and hence, have recent population migration, thalassemia been referred to as dominantly inherited b has become an important part of clinical 2 thalassemia. In contrast, typical b tha- practice, and in these regions a much wider lassemia is inherited as a haploinsufficient spectrum of b thalassemia mutations can be Mendelian recessive disease. The tha- encountered. Furthermore, with improved lassemias are the commonest monogenic clinical care and increasing survival of disorders in the world and globally it is esti- affected individuals, complications such as mated that there are 270 million carriers, of chronic liver disease, thrombosis and pul- which 80 million are carriers of b tha- monary hypertension are being increasingly lassemia. Archeological evidence in the recognized in the older patients.

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cluster A bLCR b 3’HSI 5 4321 e GgAg ybdb 5’ 3’ 10 kb Scale

B CCAAT CACCC CACCC 1 30 30 104 105 146 5’ UTR 5’ UTR 5' IVS 1 IVS 2 3' 1 2 3

C ATG GT AG GT AG TAA ATAAA AATAA Promoter 5’ and 3’ UTR

RNA + 0 processing b ,b ATG Nonsense Codons Frameshifts

G A Figure 1. A. The b globin gene cluster and its flanking regions on chromosome 11p. The e, g, g, d and b globin genes are indicated as gray boxes. The 5' hypersensitive sites (1 to 5) which comprise the bLCR and the 3' hypersensitive sites (3'HS1) are shown as vertical arrows. Hatched boxes represent the olfactory receptor genes. B. General structure of the b globin gene with the 3 exons (gray boxes) and the 2 intervening sequences (IVS1 and IVS2). Conserved sequences (detailed in text) are indicated. The different classes of point mutations causing b thalassemia are shown below the b globin genes.

The general structure of the b globin gene is typical 6,7 The b globin gene of the other globin loci (Figure 1B). The genomic sequence spans 1600 bp and codes for 146 amino acids; b globin is encoded by a structural gene found in a the transcribed region is contained in three exons sep- cluster with the other b-like genes spanning 70 Kb on arated by two introns or intervening sequences the short arm of chromosome 11 (11p15.4) (Figure 1). (IVS).The first exon encodes amino acids 1 to 29 G A The cluster contains five functional genes, 5e g- g-yb‚ together with the first two bases for codon 30, exon 2 d-b3’, which are arranged in the order of their develop- encodes part of residue 30 together with amino acids mental expression. Upstream of the entire b globin 31 to 104, and exon 3, amino acids 105 to 146. Exon 2 complex is the locus control region (LCR), which is encodes the residues involved in heme binding and ab essential for the expression of all the genes in the com- dimer formation, while exons 1 and 3 encode for the plex. This region consists of five DNase 1 hypersensi- non-heme-binding regions of the b globin chain. tive (HS) sites (designated HS1 5) distributed between Conserved sequences important for gene function are 6 and 20 Kb 5’ of the e gene. There is one other hyper- found in the 5’ promoter region, at the exon-intron sensitive site ~20 Kb 3’ to the b gene. The two extreme junctions, and in the 3’ untranslated region (3’-UTR) at HS sites flanking the b complex have been suggested to the end of the mRNA sequences. The b globin gene mark the boundaries of the b globin gene domain. The promoter includes 3 positive cis-acting elements: TATA b globin complex is embedded in a cluster of olfactory box (positions –28 to –31), a CCAAT box (positions receptor genes (ORG), part of the family of ~1000 –72 to –76) and duplicated CACCC motifs (proximal genes that are widely distributed throughout the at positions –86 to –90, and distal at position –101 to genome, and expressed in the olfactory epithelium.5 –105). While the CCAAT and TATA elements are

| 650 | haematologica/the hematology journal | 2005; 90(5) Genetic modifiers of b-thalassemia found in many eukaryotic promoters, the CACCC silencing and gene competition, mediated by the dif- sequence is found predominantly in erythroid cell-spe- ferent transcription factors in embryonic, fetal and cific promoters. Binding of the Erythroid Krüppel Like adult cells. While the e- and g globin genes are auto- Factor (EKLF) to the CACCC motif appears to be cru- nomously silenced at the appropriate developmental 8,9 cial for normal adult b globin expression. In addition stage, expression of the adult b globin gene depends on to these motifs, the region upstream of the b globin lack of competition from the g gene for the LCR promoter contains two binding motifs for the ery- sequences. This is supported by the down-regulation throid transcription factor GATA 1. The importance of of the cis b gene when g gene is up regulated by point these various 5’ flanking sequences for normal gene mutations in their promoters as illustrated by the non- expression is underscored by b thalassemia arising deletional hereditary persistence of fetal hemoglobin 13 from point mutations in these sequences specifically in (HPFH). Mutations which affect the b promoter, and around the TATA box and the CACCC motifs in which remove competition for the bLCR also, tend to the –80 to –100 region. An enhancer is also found in be associated with variable increases in g and d gene intron 2 and 3’ of the globin gene, 600 to 900 bp down- expression (see later). stream of the poly (A) site.10 The 5’ untranslated region (UTR) occupies a region Molecular basis of b thalassemia of 50 nucleotides between the CAP site, the start of There are two main varieties of b thalassemia alleles; 0 + transcription, and the initiation (ATG) codon. There b thalassemia in which no b globin is produced, and b are two prominently conserved sequences in the 5’ thalassemia in which some b globin is produced, but UTR of the various globin genes (both a and b). One is less than normal. Less severe forms are sometimes des- ++ the CTTCTG hexanucleotide found 8 to 13 ignated b to reflect the minimal deficit in b chain pro- nucleotides downstream from the CAP site, i.e. at posi- duction. Although more than 200 b thalassemia alleles tions +8 to +13. The second conserved sequence is have been characterized (HbVar, available at URL CACCATG, in which the last three nucleotides form http://globin.cse.psu.edu/globin/hbvar/), population stud- the initiation codon (ATG). Again, the importance of ies indicate that about 40 account for 90% or more of 4 these sequences in the regulation of b gene expression the b thalassemias worldwide. This is because in the is exemplified by the several mutations in the 5’ UTR areas in which it is prevalent, only a few4 mutations are causing b thalassemia. common, with a varying number of rare ones, and The 3’ UTR constitutes the region between the ter- each of these populations has its own spectrum of b mination codon (TAA) and the poly (A) tail. It consists thalassemia alleles. of 132 nucleotides with one conserved sequence, In contrast to the a thalassemias, the b thalassemias AATAAA, located 20 nucleotides upstream of the poly are rarely caused by deletions.7,14 One group of dele- (A) tail. This consensus hexanucleotide serves as a sig- tions affects only the b globin gene and ranges in size nal for the cleavage of the 3’ end of the primary tran- from 290 bp to >60 Kb. Of these, only the 619 bp dele- script and addition of a poly (A) tract which confers tion at the 3’ end of the b gene is common, but even stability to the processed mRNA and enhances transla- that is restricted to the Sind and Punjab populations of tion. Several mutations affecting the AATAAA India and Pakistan where it accounts for ~20% of the 15,16 sequence and other sequences in the 3’ UTR causing b b thalassemia alleles. The other deletions, although thalassemia have been described. The b-like genes extremely rare, are of particular functional and pheno- undergo two switches (embryonic Æ fetal Æ adult). At typic interest because they are associated with unusu- 6 months after birth, Hb F comprises less than 5% of ally high levels of Hb A2 in heterozygotes. These dele- the total hemoglobin and continues to fall until reach- tions differ widely in size, but remove in common a ing the adult level of <1% at 2 years of age. It is at this region from positions –125 to +78 relative to the stage that mutations affecting the b gene become clin- mRNA cap site in the b promoter which includes the ically apparent. The switch from fetal (g) to adult (b) CACCC, CCAAT and TATA elements. The mecha- hemoglobin production is not complete since small nism underlying the markedly elevated levels of Hb A2 amounts of b expression persist in adult life. The resid- appears to be related to the removal of the 5’ promot- ual amount of fetal hemoglobin (a2g2) is present in a er region of the b gene. This may remove competition sub-set of erythrocytes called F cells which also contain for the upstream LCR leading to its increased interac- adult (a2b2) hemoglobin. The tissue- and developmen- tion with the g and d genes in cis, enhancing their tal- specific expression of the individual globin genes is expression. governed by the direct physical interactions between Indeed, studies of an individual heterozygous for the 11,12 the globin promoters and the bLCR, the interaction 1.39 Kb deletion and a d chain variant showed that is mediated through binding of tissue-restricted and there is a disproportionate increase of variant Hb A2 ubiquitous transcription factors. This developmental (a2d2) derived from the d globin gene cis to the b globin expression is thought to rely on two mechanisms, gene gene deletion.17 This mechanism may also explain the

haematologica/the hematology journal | 2005; 90(5) | 651 | S.L. Thein et al. moderate increases in Hb F which characterize this mutants affect the polyadenylation signal (AATAAA) + group of deletions and those due to point mutations and the 3’UTR. These are generally mild b tha- affecting the promoter region. Although the increases lassemia alleles. in Hb F are variable, and modest in heterozygotes, they Approximately half of the b thalassemia alleles affect are adequate to compensate for the complete absence the different stages of RNA translation and in all 18,19 0 of b globin in homozygotes. Two homozygotes for instances, no b globin is produced resulting in b tha- different deletions of this kind have a mild disease lassemia. Most of these defects result from the intro- despite the complete absence of Hb A2 (a2b2). duction of premature termination codons due to Expression of the b globin gene can also be silenced frameshifts or nonsense mutations and nearly all ter- 1,20 0 by deletions of the b globin complex as part of (egdb) minate within exon 1 and 2. Mutations that result in thalassemia. At a molecular level, the deletions causing premature termination early in the sequence (in exons 0 (egdb) thalassemia fall into two sub-groups; one group 1 and 2) are associated with minimal steady-state lev- removes all or a greater part of the b globin gene com- els of b mRNA in erythroid cells, due to an accelerated plex including the b gene itself. The other sub-group of decay of the abnormal mRNA referred to as nonsense- deletions removes extensive upstream regions leaving mediated mRNA decay (NMD).21 In heterozygotes for the b globin gene intact, although its expression is such cases, no b chain is produced from the mutant silenced because of inactivation of the bLCR. allele and only half the normal b globin is present, 0 Heterozygotes for such (egdb) thalassemia have a resulting in a typical asymptomatic phenotype. By blood picture typical of b thalassemia trait but with a contrast, mutations that produce in-phase termination normal Hb A2 level. later in the b sequence, in exon 3 are not subjected to The vast majority of b thalassemias are caused by NMD, resulting in substantial amounts of abnormal b point mutations within the gene or its immediate mRNA comparable to that of the normal allele.22,23 flanking sequences. These single base substitutions, Heterozygotes for such late termination mutations minor insertions or deletions of a few bases are classi- tend to have a much more severe phenotype, and the fied according to the mechanism by which they affect mutations are termed dominantly inherited (see below). gene regulation: transcription, RNA processing or RNA Other mutations of RNA translation affect the initia- 14 0 translation. Mutations affecting transcription can tion (ATG) codon and again these result in b tha- involve either the conserved DNA sequences that form lassemia. the b globin promoter or the stretch of 50 nucleotides in the 5’UTR. Generally they result in a mild to minimal deficit of b globin output and can be silent in carri- Variants of b thalassemia ers (see below). Mutations that affect RNA processing can involve Dominantly inherited b thalassemia either of the invariant dinucleotides (GT at 5’ and AG In contrast to the common b thalassemia alleles that at 3’) in the splice junction in which case normal splic- are prevalent in malarial regions and inherited typically ing is completely abolished with the resulting pheno- as Mendelian recessives, some forms of b thalassemia 0 type of b thalassemia. Mutations within the consensus are dominantly inherited, in that inheritance of a single sequences at the splice junctions reduce the efficiency b thalassemia allele results in a clinically detectable dis- + 2,24 of normal splicing to varying degrees and produce a b ease despite a normal a globin genotype. Hetero- phenotype that ranges from mild to severe. Mutations zygotes have a thalassemia intermedia phenotype with within introns or exons might also affect the splicing moderate anemia, splenomegaly and a thalassemic pattern of the pre-mRNA. For example, a cryptic splice blood picture. Apart from the usual features of het- site which contains the sequence GT GGT GAG G has erozygous b thalassemia, such as increased levels of been found in exon 1 of the b globin gene, spanning HbA2 and the imbalanced a/b globin biosynthesis, large codons 24 to 27. Three mutation within this region inclusion bodies similar to those seen in thalassemia activate this cryptic site which acts as an alternative major are often observed in the red cell precursors, donor site in RNA processing. The mutation in codon hence the original term of inclusion body thalassemia.25 26 (GACÆAAE) that gives rise to Hb E (b26 GlnÆLys) More than 30 dominantly inherited b thalassemia alle- is one such mutation that activates this cryptic splice les have now been described;2,24 they include a spectrum site, with a reduction of the normal splicing that pro- of molecular lesions from missense mutations to trun- duces the Hb E variant. Since Hb E production is also cated b variants resulting from nonsense mutations. quantitatively reduced, the compound heterozygous The common denominator of these mutations is the state, Hb E/b thalassemia, results in a clinical picture predicted synthesis of highly unstable b chain variants, closely resembling homozygous b thalassemia ranging so unstable that in many cases, they are not detectable from severe anemia and transfusion-dependency to and only implied from the DNA sequence. The predict- thalassemia intermedia. Other RNA processing ed synthesis is supported by the presence of substantial

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0 amounts of abnormal b mRNA in the peripheral reticu- mal levels of Hb A2 is a d thalassemia (Cd59-A) cis to 23 30 locytes, in amounts comparable to that produced from the b27 AlaÆSer mutation. b haplotype analysis sug- 0 Knossos the normal b allele. Indeed, the large intra-erythroblas- gests that the d 59/b allele is relatively common in tic inclusions, that are so characteristic of this form of b the Middle East and Mediterranean. thalassemia, have subsequently been shown to be com- Another fairly common cause of normal Hb A2 b tha- 26 posed of both a and b globin chains. In contrast, the lassemia phenotype in the Greek population is the inclusion bodies in homozygous b thalassemia consist Corfu form of db thalassemia, a 7.2 Kb deletion that 31,32 only of precipitated a globin. The differential effects of includes the 5' part of the d gene. Although the cis b these in-phase termination mutants on the accumula- globin gene is intact, it is down-regulated by a GÆA tion of mutant mRNA exemplify how shifting the posi- mutation in position 5 of the IVS1. Heterozygotes for tion of a nonsense codon can alter the phenotype of the Corfu deletion have a db thalassemia phenotype recessive inheritance caused by haplo-insufficiency, to a characterized by a variable increase in Hb F and low to dominant negative effect due to the synthesis of an normal Hb A2 levels, while homozygotes have almost abnormal and deleterious protein. 100% Hb F with no Hb A2 and trace levels of Hb A. Again, a spectrum in phenotypic severity in this class The mutation has been described as separate lesions in of b thalassemia variants is observed, which can be two different populations. The normal b gene in cis to related to a variation in the degree of instability of the the 7.2 Kb deletion in an Italian individual is expressed 33 b globin products. The phenotype resembles the inter- at normal levels while Algerian homozygotes for the mediate forms of b thalassemia by virtue of the ineffec- bIVS1 5 GÆA mutation have a severe transfusion- 34 tive erythropoiesis, but there is also a variable degree of dependent anemia. The phenotype of normal Hb A2b peripheral hemolysis. Unlike recessive b thalassemia thalassemia is also seen in heterozygotes for egdb tha- which is prevalent in malaria-endemic regions, domi- lassemia and overlaps the phenotypes encountered in nant b thalassemias are rare, occurring in dispersed geo- carriers of a thalassemia. graphical regions where the gene frequency for b thalassemia is very low. The vast majority of the dominant Silent b thalassaemia b thalassemia alleles have been described in single fam- The silent b thalassemias cause only a minimal deficit ilies, many as de novo events. It is likely that the low fre- of b globin production. Heterozygotes do not have any quency of the dominant b thalassemia alleles is due to evident hematologic phenotype; the only abnormality the lack of positive selection that occurs in the recessive being a mild imbalance of globin chain synthesis. It is forms. Clinically, since spontaneous mutations are not surprising therefore, that these mutations have common in dominant b thalassemia, it is important been identified in homozygotes who have a typical b that the disorder should be suspected in any patient thalassemia trait phenotype35 or in the compound het- with a thalassemia intermedia phenotype even if both erozygous state with a severe b thalassemia allele 36 parents are hematologically normal and the patient is where they cause thalassemia intermedia. Silent b tha- from an ethnic background where b thalassemia is rare. lassemia alleles are not common except for the CÆT 37 mutation at position –101 of the b globin gene which Normal Hb A2 thalassemias b accounts for most of the milder forms of b thalassemia 38 The diagnostic feature of b thalassemia is the in the Mediterranean. Recently, a novel mutation, a hypochromic microcytic red cells and an elevated level CÆG transversion, has been reported in the same posi- + 0 39 of Hb A2 in heterozygotes, whether b or b . Normal tion. Some of the promoter mutations in the 5' and 3' Hb A2 b thalassemias (previously referred to as type 2) UTRs are also silent. It has been suggested that the refer to the forms in which the blood picture is typical [TA]x[T]y sequence variation at position –530 of the b of heterozygous b thalassemia except for the normal globin gene may be responsible for some silent b tha- 40 levels of Hb A2. Most cases result from co-inheritance lassemia carriers. The reduced b globin expression has 0 + of d thalassemia (d or d ) in cis or trans to the b tha- been related to increased binding of a repressor protein 0 + 41 lassemia gene, which can be of the b or b type. The (BP1) isolated from K562 cells. However, population 0 d 59 (59 A) mutation has been reported to occur in cis to surveys and clinical studies do not show a consistent 0 + 27 + the b 39 and b IVS1-110 mutations; and d 27 (GÆT) correlation between the +ATA, T variant and a b tha- has been reported to occur in cis and in trans to the lassemia phenotype, suggesting that it is a neutral poly- IVS2-745 mutations.28,29 One relatively common form of morphism.42 normal Hb A2 thalassemia is that associated with Knossos thalassemia trait with unusually high Hb A2 Hb (b27 AlaÆSer). Like Hb E, the mutation b b27(GCCÆTCC) activates an alternative splice site Despite the vast heterogeneity of mutations, the reducing the amount of normal transcript that contains increased levels of Hb A2 observed in heterozygotes for the variant. Unlike Hb E, the Hb A2 level is not elevat- the different b thalassemia alleles in different ethnic ed in heterozygotes. The molecular basis for the nor- groups are remarkably uniform, usually 3.5-5.5% and

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48 rarely exceeding 6%. Unusually high levels of Hb A2 lation of hematopoietic cells. These unusual cases over 6.5% seem to characterize the sub-group of b tha- once again illustrate that the severity of anemia of b lassemias caused by deletions that remove the regula- thalassemia reflects the quantitative deficiency of b tory elements in the b promoter. As discussed earlier, globin chain production. Furthermore, with respect to the unusually high Hb A2, often accompanied by mod- potential gene therapy, expression of a single b globin est increases in Hb F, may be related to the removal of gene in a proportion of the red blood cells appears to competition for the upstream LCR, allowing an be sufficient to redress the chain imbalance to produce increased interaction with the cis d and g genes. a condition mild enough not to need major medical intervention. b thalassemia due to insertion of a transposable element Transposable elements may occasionally disrupt Clinical and hematologic phenotypes, human genes and result in their inactivation. The inser- pathophysiology and modulating factors tion of such an element, a retrotransposon of the fam- of b thalassemia ily called L1 has been reported with the phenotype of + b thalassemia. Despite the insertion of 6-7 Kb DNA The clinical manifestations of b thalassemia are into its IVS2, the affected gene expresses full length b extremely diverse, spanning a broad spectrum from globin transcripts at a level corresponding to about the transfusion-dependent state of thalassemia major 43 15% of normal b globin mRNA. to the asymptomatic state of thalassemia trait. The most severe end of the spectrum is characterized by thalassemia due to trans-acting determinants b the complete absence of b globin production and Population studies have shown that ~1% of the b results from the inheritance of two b0 thalassemia alle- thalassemias remain uncharacterized despite extensive les, homozygous or compound heterozygous states. sequence analysis, including the flanking regions of the This condition is referred to as b thalassemia major b globin genes. In several families, linkage studies and, at their worst, the patients present within 6 demonstrated that the b thalassemia phenotype aggre- months of life with profound anemia, and if not treat- gates independently of the b globin complex implying ed with regular blood transfusions, die within their 14,44 that the genetic determinant is trans-acting. first two years. b thalassemia trait forms the other end Recently, it has been found that mutations in XPD that of the phenotypic spectrum of b thalassemia, and is cause trichothiodystrophy (TTD) are frequently asso- typically associated with the inheritance of a single b 45 ciated with a phenotype of b thalassemia trait, sup- thalassemia allele. Carriers for b thalassemia, whether 0 + ported by reduced levels of b globin synthesis and b or b , are clinically asymptomatic; they may have reduced b globin mRNA. The XPD protein is a subunit mild anemia with characteristic hypochromic micro- of the general transcription factor TF11H which is cytic red blood cells, elevated levels of Hb A2 and vari- involved in basal transcription and DNA repair. able increases of Hb F (up to 2%). However, as seen Mutations in the transcription factor GATA 1 on the X earlier, the heterozygous state for b thalassemia can chromosome have also been reported to cause b tha- also be extremely diverse, ranging from one that is lassaemia in association with thrombocytopenia.46 completely phenotypically silent, to one with severe This was the first example of b thalassemia in humans anemia (dominantly inherited). caused by a mutation in the erythroid-specific tran- Between the two extremes of the heterozygous scription factor. states, severity of the different b thalassemia alleles is reflected in their hematologic phenotype, in the degree Somatic deletion of b globin gene of hypochromia and microcytosis as indicated by the This novel mechanism was recently described in an mean cell hemoglobin (MCH) and mean cell volume individual who had moderately severe thalassemia (MCV) values, respectively. Rund et al.49 showed that intermedia despite being constitutionally heterozygous the b0 thalassemia alleles which are associated with the 0 47 for b thalassemia with a normal a genotype. most severe phenotype demonstrated a fairly tight + Subsequent investigations revealed that he had a range of MCV (63.1 fL, SD = 3.4) while the b alleles somatic deletion of a region of chromosome 11p15 were associated with a wider range of MCV (69.3 fL, + including the b globin complex giving rise to a mosaic SD = 5.6). The cut-off point between the b0 and b tha- + of cells, 50% with one and 50% without any b globin lassemias was 67 fL. The broader range of MCV in b gene. The sum total of the b globin product is ~25% thalassemia, when compared to b0 thalassemia, is not less than the normally asymptomatic b thalassemia surprising given the broad range in the deficit of b glo- trait. Subsequently, two unrelated Italian patients were bin production, from barely detectable levels at the also reported to have thalassemia intermedia caused by severe end, to just a little less than normal in the very somatic deletions of chromosome 11p15 in a subpopu- mild or silent alleles.

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A more recent study has taken the correlation the underlying b thalassemia mutation itself (see between the severity of b thalassemia alleles with Dominantly inherited b thalassemia). Given the differ- hematologic parameters in heterozygotes to a finer ences in the spectrum of b thalassemia mutations and 50 level. Skarmoutsou et al. measured a series of hemato- differences in the frequency of a thalassemia, the rela- logic parameters, including reticulocyte hemoglobin tive importance of the different factors would vary content (CHr), soluble transferrin receptor (sTfR), retic- accordingly in different population groups. It is impor- ulocytes and Hb A2 and F levels in 57 iron-replete indi- tant to note that the genotypic factors are not mutual- viduals with heterozygous b thalassemia. There was a ly exclusive (Table 1). negative correlation between the values of sTfR, a reli- The underlying pathophysiology of b thalassemia able quantitative assessment of erythropoietic activity, relates to the degree of globin chain imbalance and the 1,55 and the severity of the b thalassemia alleles; the values excess of a globin chains. The latter aggregate in red were lowest in the very mild b thalassemia (b silent), cell precursors forming inclusion bodies, causing and highest in b0 thalassemia heterozygotes. CHr, a mechanical damage and premature destruction of the product of reticulocyte hemoglobin and volume, was cells in the bone marrow. The ensuing ineffective ery- significantly higher in the bsilent group (27.0–32.0 pg) thropoiesis results in anemia and intense proliferation than in severe groups (b+ and b0 thalassemia alleles at and expansion (10 to 30 times) of the bone marrow 19.5–25.3 pg). Furthermore, while sTfR values showed with resulting skeletal deformities. The red cells that a positive correlation with Hb A2, there was a signifi- survive to reach the peripheral circulation are also pre- cant negative correlation between CHr and Hb A2 lev- maturely destroyed in the spleen which becomes els. This study confirms that all heterozygous b tha- enlarged, eventually leading to hypersplenism. Anemia lassemias have some degree of ineffective erythro- in b thalassemia thus results from a combination of poiesis that varies with the severity of the b tha- ineffective erythropoiesis, peripheral hemolysis, and lassemia mutation. an overall reduction in hemoglobin synthesis. Factors Between the two clinical extremes of thalassemia which reduce the degree of chain imbalance and the major and trait, lies the clinical syndrome of tha- magnitude of a chain excess in the red cell precursors, lassemia intermedia which comprises a diverse spec- will have an impact on the phenotype. At the primary trum of phenotypes, from a condition that is slightly level, this is related directly to the nature of the b tha- less severe than transfusion-dependence to one that is lassemia mutation itself. At the secondary level, the asymptomatic and often identified through a routine severity of globin chain imbalance is influenced by 56 blood test. Dissecting the mechanisms generating the variability at two loci:- a globin and g globin genes. intermediate status has provided tremendous insights In many populations in which b thalassemia is into the genetic basis for the phenotypic diversity of prevalent, a thalassemia also occurs at a high frequen- 51-54 the b thalassemias. Although definition of the two cy and hence it is not uncommon to co-inherit both extremes of the clinical spectrum of b thalassemia is conditions. As with b thalassemia, the different a tha- easy, assigning the severity of the intermediate form lassemias which predominate in different racial groups can be problematic. Criteria such as age and level of display a wide range of severity. This interaction alone hemoglobin at presentation, transfusion history, and provides the basis for considerable clinical heterogene- the requirements for intermittent blood transfusion ity; the degree of amelioration depends on the severity have been used, but these have their inherent limita- of the b thalassemia alleles and the number of func- 51,53 tions and are highly clinician-dependent. Thalassemia tional a globin genes. Co-inheritance of a single a 0 intermedia can result from the inheritance of one or gene deletion has very little effect on b thalassemia + two b thalassemia alleles. In a large number of patients, while individuals with two a gene deletions and b the reduced disease severity can be explained by the thalassemia may have milder anemia. At the other ++ inheritance of the milder b thalassemia alleles (b and extreme, patients who have co-inherited Hb H disease silent) that allow the production of a significant propor- (equivalent to only one functioning a gene) and tion of b globin chains. A substantial number, howev- homozygous b thalassemia, also have moderately 0 57 er, have b thalassemia, and in such cases, the absence severe anemia. of b globin chains is compensated by an inherent abil- In b thalassemia heterozygotes, co-inheritance of a 2 2 ity to produce fetal hemoglobin (Hb F, a g ). Co-inher- thalassemia normalizes the hypochromic microcyto- 0 58 itance of a thalassemia has very little effect on b tha- sis, while the presence of increased a globin product lassemia while individuals with a gene deletions and in b thalassemia heterozygotes tips the globin chain + b thalassemia may have milder disease. Yet other tha- imbalance further, converting a typically clinically lassemia patients have inherited only one b tha- asymptomatic state into that of thalassemia interme- lassemia allele. Most cases of unusually severe het- dia. In the majority of cases, this is related to the co- erozygous b thalassemia are due to the co-inheritance inheritance of triplicated a globin genes. Triplicated a of extra a globin while others are due to the nature of genes (aaa/) occur in most populations at a low fre-

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Table 1. Molecular basis of thalassemia intermedia. Although the increases in Hb F and F cells are minimal in normal people, clinical studies have shown that, under conditions of hematopoietic stress, for example Homozygous or compound heterozygous state for thalassemia b in homozygous thalassemia and sickle cell disease, Inheritance of mild b thalassemia alleles b Co-inheritance of a thalassemia the presence of the Xmn1-Gg site favors a higher Hb F Increased Hb F response response.64,65 This could explain why the same muta- Xmn1 G polymorphism g tions on different chromosomal backgrounds (some b globin gene promoter mutations b Trans-acting HPFH genetic determinants with and others without the Xmn1-Gg site ) are associ- Heterozygous state for thalassemia ated with disease of different clinical severity. b Other determinants within the cluster are related to Co-inheritance of extra a globin genes (aaa/aa, aaa/aaa, aaaa/aa) the mutation itself. The increased Hb F output Dominantly inherited b thalassemia observed in deletions or mutations that involve the (Hyperunstable b globin chain variants) promoter sequence of the b globin gene reflect the Compound heterozygotes for b thalassemia and b chain variants competition between the g and b globin gene promot- e.g. Hb E/b thalassemia ers for interaction with the LCR. Hence, although such deletions cause a complete absence of globin prod- Compound heterozygotes for b thalassemia and HPFH or db thalassemia b uct, the severity of the phenotype is offset by the con- comitant increase in hemoglobin F.18 Although the presence of the cis Xmn1-Gg site is a quency. The co-inheritance of two extra a globin genes modulating factor, clearly there are some patients who (aaa/aaa) or aaaa/aa) with heterozygous b tha- have an enhanced Hb F response despite being Xmn1- 59,60 51,54 lassemia results in thalassemia intermedia. Gg -/-. In many cases, family studies have shown However, the phenotype of a single extra a gene this inherent capacity for producing Hb F is due to a (aaa/aa) with heterozygous b thalassemia is more genetic determinant that is not linked to the b globin variable and depends on the severity of the b tha- cluster. This is in keeping with our sib-pair studies in lassemia allele.61,62 There appears to be a critical thresh- normal adults which showed that >50% of the F cell old of globin chain imbalance in each individual above variance in the general population is accounted for by which clinical symptoms appear. trans-acting factors.63 Indeed, analysis of a group of tha- Globin chain imbalance can also be reduced if there lassemia intermedia patients revealed seven sib-ships is an inherent capacity for producing g chain which with discordant phenotypes despite identical a and b combines with the excess a to form fetal hemoglobin genotypes. The steady state Hb F values between the (Hb F, a2g2). Although the production of Hb F is almost siblings ranged from 1 g/dL to as much as 8/9 g/dL and switched off at birth, all adults continue to produce was ascribed to genetic determinants not linked to the 51 residual amounts of Hb F. In all b thalassemias, Hb F b globin complex. Trans-acting loci controlling Hb F levels are relatively increased due to the selective sur- and F cell levels have now been mapped to three vival of the erythroid precursors that synthesize rela- regions of the genome – chromosomes 6q23, Xp22 and 66-68 tively more g chains. However, patients with b tha- 8q. As the genetic basis of the propensity to pro- lassemia differ considerably in their ability to synthe- duce Hb F becomes unravelled it is becoming clear that size g chains and their Hb F response. This becomes the conglomeration of the Xmn1-Gg polymorphism, 0 evident in the group of homozygous b thalassemia the QTL on 6q, Xp and 8q and others, linked and patients who have a mild disease despite the absence unlinked to the b globin complex, constitute the loose- of Hb A.51,54 These patients appear to have an inherited ly defined syndrome of heterocellular HPFH.13 These ability to produce Hb F and are able to maintain a rea- trans-acting factors presumably play an important role sonable level of hemoglobin, all of which is Hb F. in the fine tuning of g globin production in normal Hence, against this background of an increase from the adults, in the response to erythropoietic stress and possi- expanded erythroid mass and the selective survival of bly, in the capacity to respond to pharmacologic induc- F cells, are genetic factors which account for the indi- ers of Hb F synthesis. But until the molecular basis of vidual Hb F response to the stress of b thalassemia. these different entities is characterized, detection of an Genetic determinants influencing Hb F response can inherent capacity for increased Hb F production is, at be within the b globin complex or trans-acting. The present, difficult and usually inferred from family stud- CÆT substitution at position –158 of the Gg globin ies. Apart from the number of a globin genes and an gene, referred to as the Xmn1-Gg polymorphism, is a inherent capacity to produce Hb F, the proteolytic common sequence variant in all population groups, capacity of the erythroid precursors in catabolising the 63 present at a frequency of 0.32 to 0.35. Our studies excess a globin chains has often been suggested as show that this genetic variant accounts for about one another factor but this effect is difficult to define. 63 third of the variation in Hb F levels in normal adults. Recently, the newly discovered a hemoglobin stabiliz-

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Bone disease 69 ing protein (AHSP), a chaperone of a globin, has been suggested as another genetic modifier, but so far stud- Progressive osteoporosis and osteopenia is another ies have been inconclusive.70,71 increasingly common complication encountered in 84,85 young adults with b thalassemia. Several studies suggest that the prevalence of bone disease in b tha- Secondary complications of b thalassemia lassemia is higher in men than in women, and that it is and genetic modifiers more severe in the spine than in the femoral neck.86-88 It is manifested by diffuse bone pain, particularly in the With the increasing lifespan of the b thalassemia lower back, vertebral fractures, cord compression, patients, subtle variations in the phenotype with spontaneous fractures and femoral head necrosis. Bone regard to some of the complications in the older mass is determined by a combination of genetic and patients have become apparent and evidence suggests environmental factors; anemia and bone marrow that they may be affected by genetic variants. expansion which are prevalent in b thalassemia are major contributors in inadequately treated patients. Bilirubin levels and gallstones The osteoporosis that occurs in other patients who are Hyperbilirubinemia and a propensity to gallstone reasonably well transfused but in whom there is severe 89 formation is a common complication of b thalassemia iron loading, may be related to hypogonadism. and is attributed to the rapid turn-over of the red blood However, it has become apparent from recent studies cells, bilirubin being a break-down product of hemo- that bone disease is increasingly common, with a fre- globin. Studies have shown that the levels of bilirubin quency of 40-50%,84 even among the well-transfused and the incidence of gallstones in b thalassemia, from and iron-chelated patients which may be related to the trait to major,72-74 is related to a polymorphic variant prolonged use of desferrioxamine. Further, some (seven TA repeats) in the promoter of the uridine patients appear to be more susceptible to bone disease diphosphate-glucoronyltransferase IA (UGTIA) gene, than others. Bone mass is a quantitative trait influ- also referred to as Gilbert’s syndrome. In vitro studies enced by several quantitative trait loci (QTL)90-92 includ- indicate that the variant reduces expression of the ing the estrogen receptor gene, the vitamin D receptor UGTIA gene.75 Normal individuals who are homozy- (VDR), COL1A1 and COL1A2 genes, and transforming gous for the [TA]7 variant instead of the usual six, tend growth factor b1 gene (TGFB1). Polymorphism in 75 to have higher levels of bilirubin. The [TA]7 variant TGFB1 has been associated with severe osteoporosis has also been shown to be associated with increased and increased bone turnover in women93 but a study of bilirubin levels in sickle cell disease76,77 and other TGFB1 polymorphism failed to demonstrate a statisti- hemolytic anemias. cal difference in b thalassemia patients with different bone mineral densities. The VDR gene polymorphism Iron loading in intron 8 (involving the Bsm1 site) was associated 88 A common complication of b thalassemia involves with osteopenia in thalassemia. A GÆT polymor- organ damage from iron overload, not just from blood phism involving an Sp1 binding site in the collagen transfusions but also from increased absorption. In a type a1 gene (COL1A1) is strongly associated with patient with thalassemia intermedia, co-inheritance of reduced bone mass and osteoporosis.94 This same poly- a single copy of the C282Y mutation in the HFE gene morphism in the COL1A1 gene has also been shown was associated with hemochromatosis and diabetes,78 to be strongly associated with reduced bone mineral 87 while the co-existence of b thalassemia trait aggravates density and osteoporosis in b thalassemia. and accentuates iron loading in C282Y HFE homozygotes.79 Since the C282Y mutation is rare in popula- Cardiac disease tions in which b thalassemia is common it has a limit- Cardiac complications are the main cause of death in 80 ed role in iron loading amongst these patients. Much b thalassemia. Many aspects of cardiac complications more common is the H63D polymorphism in the HFE are still poorly understood, and again it is clear that car- gene, whose functional role is still being investigated. diac disease in b thalassemia is multifactorial reflecting A recent study showed that b thalassemia carriers who the chronic anemia, iron overload and pulmonary are homozygous for H63D in the HFE gene have high- hypertension. The situation is further confounded by er serum ferritin levels than carriers without the poly- the unpredictability of the severity of cardiac iron dep- morphism, suggesting that the H63D polymorphism osition. A risk factor for left ventricular failure in tha- may have a modulating effect on iron absorption.81 As lassemia is decreased anti-oxidant activity of other genes in iron homeostasis become uncovered, it apolipoprotein (APOE) e4 related to the frequency of 95 is likely that genetic variants will be found in these loci the apolipoprotein e4 allele. More recently, the APOE that influence the different degrees of iron loading in b e4 allele has also been shown to be an independent risk thalassemia.82,83 factor for cardiac dysfunction in elderly people.96 The

haematologica/the hematology journal | 2005; 90(5) | 657 | S.L. Thein et al.

risk of cardiac dysfunction was increased 3-fold in individuals homozygous for APOE e4. Conclusions

Other complications The genetic heterogeneity underlying the phenotyp- Genetic variants implicated in other complications of ic diversity of the b thalassemias is prototypical of how b thalassemia include: specific HLA alleles in the ten- the wide spectrum in disease severity of a monogenic dency to develop hepatitis and liver cirrhosis; genetic disorder can be generated at different levels – severity variants in factor V, prothrombin and MTHFR, and the of anemia at the primary level and severity of compli- tendency to develop thrombosis. Eldor and cations related to the anemia and to treatment at the Rachmilewitz97 presented compelling evidence for an secondary level. An overview of the molecular basis of increased risk of thrombosis in the different tha- b thalassemia has been presented followed by a short lassemias and a thalassemia syndromes. A retrospec- description of the clinical and hematologic diversity, tive review by Capellini et al.98 showed that tha- and the underlying pathophysiology. With the increas- lassemia intermedia patients who had been splenec- ing lifespan of patients with b thalassemia, it seems tomized are particularly at risk of developing venous likely that an increasing number of complications sec- thromboembolic events. Chronic lung disease and pul- ondary to this condition will be encountered in the monary hypertension are other complications that are older patients. An outline of the various genetic loci being increasingly recognized in older b thalassemic (modifier genes) that modulate the secondary compli- patients, and are thought to be related to the increased cations affecting various organs has been presented. tendency to thrombosis97 and small pulmonary emboli.99 A mechanism related to the scavenging of I thank Claire Steward for secretarial help in the preparation of this manuscript. nitric oxide by free plasma hemoglobin implicated in Manuscript received January 20, 2005. Accepted April 1, 2005. sickle chronic lung disease may also underlie the pathogenesis of pulmonary hypertension in b thalassemia.100

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