Genotype-phenotype correlations in β-thalassemias

Genotype-phenotype P-thalassemias

A. Cao, R. Galanello,

Correlations in

M. C. Rosatelli

S UMMA R Y. In this paper we review the molecular basis of the marked heterogeneity of the tWassemia syndromes as well as the relative implications for carrier screening and prenatal diagnosis. The classical phenotype of heterozygous &thalassemia may be mod&d by a number of envirooIllental and genetic interacthtg factors-among which the most relevant are: (1) coinheritance ofu-~wsichmaynonnalizetheredbloodcelliodices;(2)thepresenceofamild &thahxux&a mutation; (3) cotrasmission of &thalassemia which may reduce the increase of HbA2 typical of heterozyg~ &thp1pssemiato normal vahtes and (4) the presence of a silent mutation which can be defined only by imbahut& &globm chain synthesis. AmnnberofmoJeadarmedtanisms are able to produce the non transfusion dependent attenuated forms of thalassemia syndromes referred to as thalassemia intermedia. The most common are homozygosity for mild g&alassemia mutations, cohtheritance with homozygous B_thalassemiaof a-thalassemia or genetic determinants able to s&stain a contimmus production of HbF in adult life or the presence of heterozygosity for hyperunstable globin variants.

/3-thalassemias are a heterogeneous group of autosoma1 recessive disorders characterized by reduced (p’) or absent (PO) production of the P-chains of the hemoglobin tetramer. This results in unbalanced u/norm globin chain synthesis, which is the major determinant of the clinical and hematological severity. In both heterozygous and homozygous S-thalassemia, overall ol/nono! chain synthesis varies widely and depend either on the residual output of P-chains from the affected P-globin locus or on a number of genetic and environmental modifying factors.‘-’ Herein we review the molecular bases of this marked heterogeneity of the thalassemia syndromes as well as the relative implications for carrier screening and genetic counselling.

A. Cao, R. GahneBo, M. C. RoWdli,

Istituto di Clinica e Biologia dell’Etri Evolutiva, Universiti Studi Cagliari, via Jenner s/n-121 Cagliari, Italy. Tel: 39-70-503341, Fax: 39-70-503696. Correspondence to Prof. A. Cao. Blood Reviews (1994) 8, 1-12 0 1994 Longman Group UK Ltd

Molecular Basis for &thalassemia The P-thalassemias are very heterogeneous at the molecular level. So far, more than 120 different molecular defects have been defined.3 The large majority of these defects are single nucleotide substitutions affecting critical areas for the function of the P-globin gene; only a minority are produced by the mechanism of gene deletion (Fig. 1). Deletion of the S-globin gene, initiation codon mutations, frameshift mutations resulting from the insertion or deletion of single nucleotide or oligonucleotides in the coding region of the globin gene, nonsense mutations and mutations affecting the invariant dinucleotides GT at the 5’ donor site and AG at the 3’ acceptor site thus abolishing the normal splicing process, produce the hematological phenotype of p”-thalassemia. On the other hand, mutations affecting the conserved sequences in the 5’ promoter (TATA box, proximal and distal CACCC elements at - 30, -90 and - 105 position upstream to the Cap site respectively) and the 3’ polyadenylation

2

GENOTYPE-PHENOTYPE

--.

CORRELATIONS IN (&THALASSEMIAS

WP

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4.1-10.6

6.4-6.1

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Fig. 1 Deletions of the P-globin gene.

signal sequence result in the production of p’thalassemia. This phenotype is also produced by a number of mutations affecting the consensus sequences flanking the invariant dinucleotide GT and AG at the donor or acceptor splice site, as well as by mutations within either introns or exons leading to the creation of new splice sites which can partially or completely eliminate normal splicing (Fig. 2) (for review see Kazazian 1990).4 Molecular Basis of Complex pthalassemias In addition to the simple /3-thalassemias above described, the P-globin gene is also silenced by a number of gross deletions involving the P-globin

CCAAT box

gene cluster (Fig. 3). These deletions, in relation to their extent, produce different overlapping hematological phenotypes which are referred to as hereditary persistence of fetal hemoglobin, GP-thalassemias, or syGP-thalassemias.5 HPFH in the heterozygous state is characterized by level of HbF of 15 to 25%, normal or reduced HbA,, normal red cell indices and balanced u/nona globin chain synthesis, whereas heterozygous GP-thalassemias have HbF in the range of 5 to 15%, normal or reduced HbAz levels, hypochromic microcytic red blood cells with detectable globin chain imbalance. Deletion of all the P-globin gene cluster leads to the hematological phenotype of y6gthalassemia, which is characterized by microcytosis, hypochromia,

POLY A

InitiationCodon

region

region

I

5’DFCETIDN

NUMBER OF BETA-THALASSEMIA Promoter Mutation ( *) CACCC box No. 1 -proximal CACCC box No. 9 -TATA box No. 7

-distal

CAP Site No. 2 initiation Codon No. 5 Fig. 2 The spectrum of P-thalassemia mutations.

I

I

3’DELETlON

MUTATIONS

RNA PROCESSING MUTATIONS -Splice junction (#) No. 13 -Consensussequence ( I) No. 14 -1VSchanges (e) No. 7

-Cryptic splicing activation (0 ) No. 4

Nonsense Mutations ( n) No. 12 Frameshift Mutations ( 0) No. 32 RNA Cleavage/POLYADENYLATlON No. 7

1

BLOOD REVIEWS

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Fig. 3 Deletion of the P-globin gene cluster.

normal HbF, normal or reduced HbA, levels, and unbalanced ol/nona chain synthesis. This phenotype may also be produced by the deletion of the hypersensitive sites HS-2, HS-3 and HS-4 of the major regulatory element for the function of the P-globin gene, denominated Locus Control Region (0 LCR)6 (Fig. 4). The b LCR consists of a set of developmentally stable DNase I hypersensitive sites (HS-I-HS4), distributed over approximately 15 kb upstream of the s-globin gene. ‘-’ Transgenic experiments have shown that LCR sequences are absolutely required for high-level position-independent globin gene expression of LCR P-globin constructs. Of the different HS sites, however, only HS-2 can function as a classic enhancer. lo Each P-LCR element consists of 200 to 300 bp segments of DNA containing

4

3

2

binding sites for a variety of erythroid and ubiquitous transcription factors.

Phenotype of Heterozygous fl-thalassemia Heterozygous P-thalassemia is characterized by hypochromic, microcytic red blood cells, increased HbAz level, inconstant and limited increase of HbF (1.5-&O%) and imbalanced u/nonu globin chain synthesis (Table 1) . This hematological phenotype, however, may be modified by a number of environmental and genetic interacting factors, which led to the production of atypical forms of heterozygous P-thalassemia. Iron deficiency may decrease the high HbA, levels, typical of the P-thalassemia carrier state. At least in our experience, however, in this 3’ HS-1

1

100 Kb 100 Kb *_1111

30 Kb -

3 Kb

Dutch

English

Hispanic Italian

Fig. 4 Deletion of the p-globin gene cluster involving the LCR and leaving the P-globin gene intact.

4

GENOTYPE-PHENOTYPE

CORRELATIONS IN B-THALASSEMIAS

Table 1 Hematological characteristics of heterozygous CL,j3, S/3and y@+thalassemia Type

MCV (fl)

MCH (pg)

HbA2(%)

HbF(%)

u/B ratio

B-thal. u-j3 thal. GB-thal. p-101 S IVS-1-6 /3CAP site 6 and g

Reduced Reduced Reduced Normal Reduced Normal Reduced

Reduced Reduced Reduced Normal Reduced

Increased Increased Normal or reduced Normal or borderline Increased or borderline Borderline Normal or borderline

Normal or slight increment Normal or slight increment Marked increased Normal Normal

Increased Increased or normal Increased Normal or increased Increased

Normal or slight increment

Increased

Reduced

condition the HbA, level remains in the B-thalassemia carrier range, unless severe anemia is present.” Heterozygous /3-thalassemia with Normal Red Cell Indices

The co-inheritance with heterozygous j3-thalassemia of a-thalassemia in the form of the deletion of two ol-globin structural genes, or non deletion defects affecting the cl,-globin gene may raise the mean corpuscular volume and the hemoglobin content per cell enough to bring those values in a proportion of double heterozygotes for a- and B-thalassemia, into the normal range.“-13 (Table 1). By contrast, the co-inheritance of the triplicated a-globin gene with heterozygous B-thalassemia may lead to marked thalassemia-like hematological manifestations intermediate from those of the P-thalassemia carrier state and those of thalassemia intermedia.14 In the Sardinian population, because of the high frequency of a-thalassemia, approximately 3.5% of the P-thalassemia heterozygotes have normal red cell indices. High frequencies of both c1-and B-thalassemia are relatively common in many at-risk populations in the Mediterranean basin, the Middle East and Far East (WHO, 1983).‘2-‘3 The practical implication of this finding is that carrier screening in populations with high frequences of both u- and B-thalassemia should include the quantitative determination of HbAz in the first set of examinations in order not to miss the double heterozygotes for a- and (3-thalassemia. Heterozygous P-thalassemia with Normal HbA,

Another group of atypical heterozygous Bthalassemias are characterized by thalassemic red cell indices but normal HbAz levels, and thus may be confused with heterozygous a-thalassemia. The molecular mechanisms underlying this form of Bthalassemia are quite heterogeneous. Recent studies have shown that the relative increase of HbA, levels is related to the type of B-thalassemia mutation.” Mutations of the P-globin gene leading to a high residual output of the B-globin chain from the affected locus may indeed result in minimal increases or completely normal levels of HbA, (Table 1). B-thalassemia mutations belonging to this category

are B-101 (C+T),16 BIVS l-6 (T-C),” p CAP site+ 1 (A+(Z)” (Table 1). However, many cases of normal HbAz heterozygous B-thalassemia result from the presence of double heterozygosity for 6- and B-thalassemia. The suspicion of the presence of interacting 6- and B-thalassemia may arise when borderline HbA, levels are found or if family studies show segregating 6-thalassemia (characterized by normal red cell indices and low HbA, levels) and B-thalassemia’%22 (Table 1). The identification of these double heterozygotes for 6- and B-thalassemia should, however, be based on globin chain synthesis analysis and/or a, 6 and B-globin gene analysis. Studies carried out in the last few years have defined at the molecular level a number of &thalassemia mutations (Fig. 5). Most of these &thalassemias are in trans to B-thalassemia, but some of them have also been detected in cis.2@22A relatively common normal HbA, /3-thalassemia is the B-chain variant Hb Knossos (B27 Ala-Ser) in Mediterranean populations, which in the heterozygous state is associated with minimal thalassemialike abnormalities of the red blood cells and increased @-chain synthesis ratio. 23 Normal HbA2 in this B-thalassemia determinant is the result of the combination of a mild B-thalassemia mutation such as Hb Knossos and a 6’-thalassemia determinant resulting from the deletion of a single nucleotide at codon 59 of the b-globin gene. As occurs for B-thalassemia, each at-risk population most likely has a limited number of &thalassemia mutations. Detection of &thalassemia may thus be carried out in each at-risk population by using a limited number of allelespecific primers or probes. In the Sardinian population, for instance, only three &thalassemia mutations have been detected so far, i.e. 6+27 (G-T),” Corfu GB-thalassemia (7.2 kb deletion)24 and nonsense codon 37.25 Normal HbA, Bthalassemia may also result from deletion of the complete B-globin gene cluster as well as upstream deletions which remove the HS-2-4 sites of the LCR but leaves the B-globin gene intact. Normal HbA, levels are found also in heterozygotes for Gj3-thalassemia,s who have, however, increased HbF levels, minimal thalassemia-like hematological abnormalities and unbalanced ol/nonol chain synthesis’ (Table 1).

BLOOD REVIEWS

Kb Fig. 5 6-thalassemia mutations.

Silent /I-thalassemias Silent P-thalassemias are thalassemic disorders characterized by normal red cell indices, normal HbAz and F levels and are defined only by unbalanced globin chain synthesis. Because of these characteristics this determinant may be missed by the procedure commonly in use for carrier identification. The compound heterozygous state for typical and silent j3-thalassemia result in the clinical phenotype of thalassemia intermedia (see later). The most common silent P-thalassemia is the C+T substitution at position - 101 from the Cap site within the distal CACCC box.“j This mutation is completely silent in adulthood. In the fetal and infant period (till 2 years) this mutation is, however, expressed as a severe mutation, being associated with absent P-chain synthesis in the fetal and newborn period and with high HbAz in infant age. ” It is interesting to note that mutations in the distal CACCC box produce silent P-thalassemia, while mutations in the proximal CACCC box produce a mild but otherwise typical P-thalassemia, indicating that the distal CACCC box is less crucial for the function of the fi-globin gene, as compared to the proximal one. The suspicion of the presence of silent P-thalassemia may arise because of borderline HbAz levels associated with high MCV values. Less common types of silent P-thalassemia are the A-4 mutation at Cap site + 118 and the C+G mutation at nt6 3’ to the terminating codon (Fig. 6). Homozygosity for the Cap site mutation results in a typical P-thalassemia carrier phenotype. It has been

suggested that sequence variation [(AT),(T),] at position -530 upstream of the P-globin gene may be responsible for silent fi-thalassemia.28 However this sequence variation has been detected also in completely normal individuals, even at globin chain synthesis levels.29

fi-thalassemia with Unusually High HbA, Levels In spite of the vast heterogeneity of P-thalassemia mutations, heterozygotes for P-thalassemia usually show a remarkably similar increase of HbA,, usually between 3.5 and 5.5%, rarely exceeding 6%. Recent studies have identified a group of l3-thalassemia heterozygotes with unusually high (>6.5) levels of HbA2, frequently associated with a variable increase of HbF levels.30 The molecular basis for these very high HbA, /3-thalassemia determinants are deletions in the 5’ part of the fl-globin gene, removing the p promoter, including the TATA and CACCC boxes. These mutations remove competition of the P-globin gene for the LCR, allowing better interaction of the LCR with the 6 and y genes in cis, and thus enhancing their expression.31

fl-thalassemias Caused by Unknown Mutations In a limited number of otherwise typical pthalassemia carriers, the j3-thalassemia mutation remains uncharacterized in spite of extensive sequence analysis of the P-globin gene. It has been postulated that the causal mutations may be found in the

3 CAP SITE PROMOl‘ER

-101 C+T Fig. 6 Silent &thalassemia

mutations.

STOP CODON

I

+1 A4

+6

C-*G

3

6

GENOTYPE-PHENOTYPE

CORRELATIONS IN PTHALASSEMIAS

upstream LCR or in the 3’ enhancer region, which is located 700 to 1000 bp 3’ of the P-globin gene.32-34 In some of these cases the P-thalassemia phenotype was not linked to the j3-globin gene haplotype and may therefore reside in the genes coding for erythroidspecific transcription factors. On the other hand it should be noted that a genetic trait characterized by isolated increase of HbAz level not linked to the P-globin cluster and transmitted in a dominant fashion has recently been reported.35 Implication for Carrier Screening

Carrier detection procedures should be able to identify heterozygous P-thalassemia as well as GP-thalassemia, y&Sthalassemia and the sickle trait which, by interacting with P-thalassemia, may result in the production of thalassemia syndromes. For the reasons discussed in the previous paragraphs, the first group of tests should include red cell indices determination, HbAz quantitation and Hb electrophoresis or Hb chromatography by high pressure liquid chromatography (HPLC). We recommend HPLC because it can, indeed, detect the most common clinically relevant Hb variants such as HbS, HbC, HbD Punjab, HbO Arab and HbE, which in homozygosity or compound heterozygosity may result in a thalassemic disorder. In addition, HPLC may also allow detection of Hb Knossos, a mild P-thalassemia allele, which is not identified by the commonly used electrophoretic procedures. Low MCV-MCH values, associated with elevated HbAz levels, indicate without any doubt heterozygous P-thalassemia. In individuals with hypochromia, microcytosis and normal HbA, levels it is necessary to exclude iron deficiency by appropriate studies. Normal iron values indicate the presence of a thalassemia determinant (a-thalassemia, r@3thalassemia, mild P-thalassemia, double heterozygosity for 6 and P-thalassemia). Discrimination between these genotypes is carried out by globin chain synthesis and ~1,p and 6-globin gene analysis by one of the many polymerase chain reaction-based methods. G/Sthalassemia is suspected in the presence of normal-low MCV-MCH, normal-reduced HbA, and high HbF. A similar phenotype results, however, also from HPFH. The distinction between these two conditions is carried out by the analysis of the distribution of HbF in peripheral red blood cells, which is heterogeneous in GP-thalassemia and homogeneous in HPFH, by globin synthesis analysis, which results in normal values in HPFH and in u/nona imbalance in Q3-thalassemia and by DNA analysis.

major. In a proportion of the cases, however, this genotype led to the production of milder clinical forms, which are clinically defined as thalassemia intermedia. These intermediate forms of thalassemia show a markedly heterogeneous clinical picture ranging in severity from the asymptomatic carrier state to transfusion-dependent thalassemia major. Studies carried out in the last few years have led to a coherent understanding of the molecular basis of this clinical condition. The degree of globin chain unbalance is the major determinant of the severity of thalassemia syndromes. In P-thalassemia, indeed, the absent or reduced output of P-globin chains leads to a great excess of a-chains which precipitate causing damage to the cell membrane. Any inherited or acquired factor able to reduce the globin chain imbalance may thereby result in a milder form of thalassemia. Three principal factors which are able to reduce the extent of the globin chain unbalance and thereby ameliorate the clinical picture of homozygous P-thalassemia are at present time recognized (Fig. 7). These factors are the presence of a P-thalassemia mutation associated with a high residual output of P-globin chains from the affected locus either in homozygosity or compound heterozygosity with a severe mutation; the coinheritance of u-thalassemia, by reducing the a-chain excess, and the coinheritance of genetic factors which increase the y-chain production, since the y-chains will combine with excess cl-chains to produce HbF.3G37 Less frequent mechanisms resulting in thalassemia intermedia are double heterozygosity for P-thalassemia and triplicated a genes and the presence of a hyperunstable Hb variant (dominantly inherited P-thalassemia) in the heterozygous state’4.3a3g (Table 2). Mild #l-thalassemia Mutations

A consistent group of P-thalassemia mutations are associated with a high residual output of P-globin chains and are indicated as mild P-thalassemia

MILD P-THALASSEMIA ALLELES

REDUCED GLOBIN CHAIN IMBALANCE

Clinical Phenotype of Homozygous &thalassemia Homozygosity or compound heterozygosity for P-thalassemia most commonly result in the severe phenotype of transfusion-dependent thalassemia

a-THALASSEMIA

HPFH tip-THALASSEMIA

Fig. 7 Molecular mechanisms for thalassemia intermedia.

BLOOD REVIEWS Table 2

Molecular bases of thalassemia intermedia

chain production A. Mild defects of w 1. Homozygosity for mild &thalassemia 2. Compound heterozygosity for mild and severe P-thalassemia B. Homozygosity or compoood heterozygosity for severe ~thalasemia associated with: 1. a-thalassemia 2. Genetic factors enhancing y-chain production l Gy or Ay promoter mutation l Heterocellular HPFH C. Inheritance of deletion ~thalassemia removing the promoter region D. Homozygosity for Gfi-thalassemiaor compoundheterozygosity for this determinantand severe &thalasemia E. Double heterozygosity for fl-tbalassemia and triplicated a-globin gene F. Heterozygosity for bypenmstable Hb variants

mutations (Fig. 8). Homozygotes for these mutations produce a mild form of thalassemia, usually not requiring regular transfusions. Compound heterozygotes for one of these mutations and a severe defect of P-globin chain production, however, result in a markedly heterogeneous clinical picture, ranging in severity from thalassemia intermedia to late presenting forms of thalassemia major. The first group of mild P-thalassemia mutations to be considered, involve the promoter area of the P-globin gene.j Mutations in the promoter sequence result in a decreased production of /3-globin mRNA. Of the mutations occurring in the TATA box, definite evidence demonstrating a mild defect of S-chain production is available for the -29 A to G substitution in the black population, the - 3 1 A to G in the Japanese population and the -28 A to G in the Chinese population. The - 29 A to G mutation is a very common form of mild l3+thalassemia in the black population. Homozygotes for this mutation have a mild form of thalassemia intermedia. whereas

compound heterozygotes for this mutation and a severe defect were shown to have a disease of modest severity. 4o All mutations involving the proximal CACCC box have mild phenotypic manifestations. The most common of proximal CACCC box mutations is the C+G substitution at position -87, which is relatively frequent in Mediterranean populations. The only homozygote for this mutation so far described showed a very mild clinical picture.41 A thalassemia inter-media-like clinical picture is also observed in compound heterozygotes for the -87 (C-G) mutation and codon 39 nonsense mutation4’ The mildest /3-thalassemia mutation so far characterized is a A to C transversion at position + 1 from the Cap site (Cap site mutation) which in the homozygous state produces the hematological phenotype of P-thalassemia trait.‘* Unexpectedly the compound heterozygous state for this mutation and a typical high HbA, P-thalassemia mutation led to the production of a thalassemia major phenotype. The C-+T substitution at position - 101, within the distal CACCC box, in the heterozygous state is associated with the silent carrier phenotype, as previously discussed.r6 Compound heterozygotes for the - 101 (C-+T) mutation and a severe mutation result in attenuated forms of thalassemia intermedia not requiring transfusions.43-44*26 A common mild form of P-thalassemia in the Mediterranean populations is the T+C substitution at position 6 within the consensus region of the first intervening sequence of the P-globin gene.45 This mutation activates a cryptic splice site and reduces the amount of normally spliced P-globin mRNA. Homozygotes for this IVS-I pos. 6 (T-C) mutation have a consistently mild disease. On the other hand compound heterozygotes for this mutation and a severe mutation showed a wide range of clinical manifestations ranging from mild thalassemia inter-media to thalassemia major. 6 5

_iYA

I

6’

TRANscRwrloNAL

3’

MUTATIONS

1 [-lo1 c+q 2 [-es C+r: -67 C+G; -66 C-A] 3 [-31 A+G; -36 T+A; -26 A+G] MUTATIONS ACTIYATING ALTERNATE SPUCE SITES 4 [codon 19 A+G; codon 24 T+A; codon 26 G+A Hb E; codon 27 G-T MUTATIONS IN coNsENSUS SEQUENCE 6@VS4nt6T+C;fVS4lnt644C+G] POLYADENYLATION SITE MUTATIONS 6 [AAlAAA+AAWW Fig. 8 Mild Pthalassemia

mutations,

7

MTAAB- MTAqol

(Hb houo~)]

8

GENOTYPE-PHENOTYPE

CORRELATIONS IN g-THALASSEMIAS

A group of mutations residing in the 3’ part of the first exon may also be considered as mild l.Sthalassemia mutations. These mutations include the T+A substitution at codon 24,46 common in the black population, the G+A at position 26 resulting in HbE @26 Glu to LYS),~~which is very frequent in South-East Asia and the G+T at position 27, which produces Hb Knossos (p27 Ala+Ser).48 These mutations result in the activation of a cryptic donor splice site in the first exon, thereby resulting in a reduced amount of correctly spliced mRNA. Compound heterozygotes for Hb Knossos and severe P-thalassemia result consistently in thalassemia intermedia. HbE seems to be a more severe mutation compared with Hb Knossos. Compound heterozygotes for HbE and P-thalassemia produce a wide spectrum of clinical severity with a number of patients developing thalassemia major. Homozygotes for HbE, however, are clinically asymptomatic. Co-inherited a-thalassemia The influence of a-thalassemia

on the clinical phenotype of homozygous P-thalassemia has been investigated by a-globin gene analysis in Greek Cypriots, Asian Indian and Sardinian populations.‘2-‘3,4M7 The main conclusions from these studies may be summarized as follows (Table 3): homozygotes for PO-thalassemia who coinherited the deletion of two a-globin genes (-W/-W) or a non-deletion lesion silencing the function of one of the a2-globin genes develop more frequently the clinical phenotype of thalassemia intermedia than thalassemia major. On the other hand p” homozygotes with a single a-globin deletion usually develop thalassemia major, albeit with a late clinical presentation. In the case of homozygous l3’ thalassemia even a single a-globin gene deletion seems to be able to produce a mild phenotype. The great P-globin chain output in p’ thalassemia explains the more consistent clinical effect of the single a-globin gene deletion in this condition than in Do-thalassemia. The contrasting effects of the single ol-globin gene deletion (3.7 Table 3

Criteria for early diagnosis of g-thalassemia intermedia

Clinical l Late presentation (2-5 years) l Relatively high Hb levels (8-10 g/dl) l Moderate bone changes l Normal growth Hematological l HbF (- lo-15%) l highHbA2 Gt3M.?tiC l

One _ -

or both parent with: Silent b-thalassemia High HbF g-thalassemia Normal

Molecular l Mild g-thalassemia mutation l Co-inheritance of u-thalassemia l Co-inheritance of nondeletion HPFH

deletion) with those resulting from the non deletion defect affecting the a,-gene are related to the fact that: a) the a,-gene produces 2 to 3 times more a-globin mRNA than the cl,-gene and b) the 3.7 deletion is associated with a compensatory increase of a-globin mRNA from the unaffected in cis cl-globin gene. 48 Coinheritance of three a-globin gene deletions with homozygous P-thalassemia does not modify consistently the severe clinical picture. It is worth noting that because coinherited cl-thalassemia seems to be associated with a wide range of clinical outcomes, the identification of the a-globin gene arrangement in any single patient does not allow accurate prediction of the clinical phenotype. Enhancement of y-chain Production

Determinants for enhancement of y-chain production in adult life are obviously able to reduce the overall u/nonu globin chain imbalance thereby resulting in the production of a mild phenotype when co-inherited with homozygous severe P-thalassemia. A group of complex P-thalassemias, referred to as @3-thalassemia, result from a deletion of variable extent of the P-globin gene cluster. These mutations are characterized by absent /3 and Z-chain production and increased y-chain production. Homozygotes for GP-thalassemia and less consistently compound heterozygotes for this defect and typical fi-thalassemia most commonly result in the production of thalassemia intermedia. The relatively benign nature of GP-thalassemia is obviously related to the relatively high level of y-chain synthesis which seems to result from an enhancer effect on y-chain production caused by remote sequences brought into the vicinity of the y-gene by the deletion. Another type of GP-thalassemia is the Hb Lepore, a Sp fusion chain having the amino acid sequence of the S-chain at its N terminus and that of the /3-chain at its C terminus. The Lepore globin gene results from non homologous crossing over between the 6 and P-globin genes. 49 Homozygotes for Hb Lepore and compound heterozygotes for this mutation and severe P-thalassemia result in a markedly heterogeneous clinical picture ranging from a mild form of thalassemia major to thalassemia inter-media. The milder clinical picture resulting from the presence of Hb Lepore is related to the fact that the Lepore determinant is usually associated with higher HbF levels as compared to the severe l3-thalassemia. Homozygotes for those P-thalassemia deletions removing the 5’ promoter region of the P-globin gene show 13°-thalassemia.3 However, because of the high levels of HbF, the resulting clinical phenotype is usually mild. The mild phenotype resulting from these mutations is most likely related to the fact that the removal of the P-globin promoter abolishes l3-globin gene interaction with the LCR, thereby favouring y promoter interaction. The coinheritance of deletion HPFH with hetero-

BLOOD REVIEWS

zygous 0’ thalassemia usually results in a clinically asymptomatic condition, the red cell indices being similar to those of heterozygous kthalassemia, the HbF level greatly increased (60-80%) and HbAz normal or elevated. Coinheritance of non-deletion HPFH with homozygous 8-thalassemia produces mild clinical manifestations. The best known example is the so called Sardinian GP-thalassemia in which the Gb-thalassemia chromosome contains two mutations, i.e. the codon 39 nonsense mutation silencing the b-globin gene and the C-+T mutation at position - 196 5’ to the Ay globin gene which results in elevated Ay chain production, thereby partially compensating for the absent b chain production.50 Compound heterozygotes for this mutation and codon 39 nonsense mutation produce a mild thalassemia clinical picture. Another very common non-deletion HPFH determinant, the - 158 Gy A to T substitution is also able to ameliorate the severe clinical picture of homozygous P-thalassemia. By contrast to the other non-deletion HPFH mutation, however, the - 158Gy (A+T) mutation in otherwise normal individuals or in P-thalassemia heterozygotes is completely silent.51 To explain these findings it has been assumed that the - 158 Gy mutation is able to enhance the y chain production only in conditions of erythropoietic stress.52 The - 158 Gy mutation creates a cleavage site for the restriction enzyme Xmnl and thus may be easily detected by restriction endonuclease analysis. The - 158 Gy mutation is present in the b-globin haplotypes III, IV and IX which are in linkage disequilibrium with the IVS-II nt 1 (G-A), frameshift 8 (-AA) and frameshift 6 (-A) 8-thalassemia mutations respectively. Normal adults have HbF levels of < 1.0% and F-cells percentage ~4.0%. However, some normal individuals have increased HbF levels in the range of 1 to 3%. This HbF is heterocellulary distributed among 15 to 20% of the red cells that are referred to as F cells. The co-inheritance of this form of ‘heterocellular’ HPFH is able to ameliorate the clinical picture of homozygous P-thalassemia as it has been clearly demonstrated in two families of Sardinian and Asian descent respectively.5s54 The presence of segregating heterocellular HPFH is demonstrated by the presence of increased levels of HbF in the normal members of these families. Heterocellular HPFH is most likely quite heterogeneous, some forms being linked to the b-globin gene cluster, some x-linked whereas for some no linkage has so far been established. In very rare cases the cause of the mild phenotype is due to the presence of quadriplicated y genes, which is associated with a high y chain output. Coinheritance of Heterozygous P-thalassemia and the Triple a-globin Gene Arrangement

A thalassemia intermedia phenotype may very rarely result from the combination of heterozygous

9

b-thalassemia with two extra cl-globm genes due to the homozygous state for the triplicated a-globin gene complex (aclc+xa) .I4 The association of a single extra a-globin gene (~a/a~ol) with heterozygous b-thalassemia produces a variable clinical outcome, some patients being clinically asymptomatic and some presenting with the clinical picture of thalassemia intermedia. Hyperunstable Hb Variants

Hyperunstable Hb variants produce a clinically detectable phenotype when present in a single copy. By contrast, heterozygotes for the common forms of P-thalassemia are clinically asymptomatic. For this reason hyperunstable Hb variants are also referred to as dominant P-thalassemia. An increasing number of hyperunstable Hb variants have been defined in the last few years3’ (Table 4). At the molecular level, four distinct categories may be recognized, i.e. single base substitutions, deletion of intact codons, premature terminations resulting in truncated j3-globin chains and frameshift mutations producing elongated b-chains. It is interesting to note that hyperunstable Hb variants resulting from premature termination of translation have the disease-causing mutation in the 3rd exon and result in the production of a truncated b-globin chain. By contrast, the mutations causing premature termination localized in the 1st and 2nd exon, do not result in the production of b-globin chain fragments and are associated with typical heterozygous b-thalassemia. Analysis of the nucleotide sequence predicts the existence of a b-globin chain variant. However, only in rare instances has the abnormal hemoglobin been demonstrated by chromatographic or electrophoretic procedures. Short-time incubation globin chain synthesis studies and pulse-chains experiments have clearly demonstrated that these variants are highly unstable and very rapidly catabolized. The continuous degradation of those hyperunstable Hb variants add an additional burden to the proteolytic activity of the red blood cells in such a way that the proteolysis of free a-chains is curtailed. This in turn results in accumulation and precipitation of a-chains to a greater extent as compared to that observed in b-thalassemia heterozygotes. In this context it should be noted that the inclusion bodies present in the peripheral red blood cells of patients affected by this disorder were shown to contain both c1and P-globin chains. The clinical phenotype presented by patients with hyperunstable Hb variant is markedly heterogeneous varying from that of a severe carrier state to occasional transfusion dependency. The anemia results both from ineffective erythropoiesis and peripheral haemolysis. Dominant P-thalassemias are rare disorders, which have been detected in individuals belonging to many racial groups. The presence of such thalassemic haemoglobinopathies should be suspected in any patient with a P-thalassemia

10

GENOTYPE-PHENOTYPE Table 4

CORRELATIONS IN fl-THALASSEMIAS

Hyperunstable globin: inclusion body B-thalassemia

B-variant

Mutation

Ethnic group

I. Single base substitutions Hb Chester-held Hb Cagliari Hb Terra Haute Hb Showa-Yakushiji Hb Houston Hb Brescia

828 [CTG-CGG]Leu+Arg 860 [GTG-GAG]Val-+Glu 8106 [CTG-CGG]Leu+Arg 8110 [CTG-CCG]Leu+Pro 8127 Gln+Pro 8114 Leu+Pro

English Italian N. European Japanese N. Italian N. Italian

II. Deletion of intact codons+destabilization Hb Korea Hb Gunma p 134-137

B32-34 [-GGT] Val-Val+Val B127/128 [AAG] Gin-Ala+Pro [-12. + 6]Val-Ala-Gly-Val+Gly-Arg

Korean Japanese Portuguese

III. Premature termination+truncated 8” 121 GAA+TAA 8” 127 CAG+TAG

8121 [Glu+Term] 8127 [Gln+Tenn]

N. European English

894 [+TG]+156aa 8109 [-G]+156aa 8114 [-CT.+G]+156aa 8123 [-A]+156aa B126 [-T]-+156aa 8128 [-4. +5. -11]+153aa 8123-125 [-81

S. Italian Askenazi Jew Swiss-French Japanese N. Italian Irish Thai

IV. Frameshift mutations+elongated Hb Agnana Hb Manhattan Hb Geneva Hb Makabe Hb Vercelli 8 F/S 128 Hb Kohn Kaen

8

8

intermedia-like phenotype when both parents are hematologically normal or when the disease is transmitted in an autosomal dominant manner. Diagnosis of Thalassemia Intermedia

The differentiation of thalassemia major from thalassemia intermedia at presentation is crucial for the subsequent management. The correct diagnosis allows an early start to the transfusion program in thalassemia major, thereby preventing the development of hypersplenism or red cell sensitization, which occurs more commonly when transfusions are started late. On the other hand those patients with thalassemia intermedia may avoid unnecessary transfusions and their attendant complications. Early differentiation of these two phenotypes is however very difficult. The use of clinical, genetic, haematological and molecular criteria in combination may allow reasonable decisions to be made. Patients who develop thalassemia intermedia usually present from 2 to 5 years whereas those with thalassemia major manifest the first symptoms within the first year. Furthermore patients with the mild variety of thalassemia show relatively high Hb levels in the order of 8-10 g/dl. The extent of bone change in thalassemia intermedia is usually of modest severity. Iron kinetics studies may also be useful because thalassemia intermedia patients have a higher radioactive iron medullary uptake, less ineffective erythropoiesis and greater peripheral hemolysis compared to patients who develop thalassemia major. Hematologically, relatively low levels of HbF in the patients may suggest the presence of a mild g-thalassemia mutation, while high HbAz may be a clue for the presence of ol-thalassemia. Genetic clues for the thalassemia intermedia pheno-

type are the presence in one or both parents of the silent carrier phenotype. The occurrence of a thalassemia-like phenotype in a propositus born from normal parents also represents suggestive evidence for the development of a thalassemia intermedia phenotype. One or both parents with high HbF may be an indication of a genetic determinant able to sustain high y-chain production which in combination with homozygous P-thalassemia may ameliorate the clinical picture. As we have reviewed herein, from the molecular aspects, a-globin gene analysis, definition of the l%thalassemia mutation, q and Ay gene analysis to detect non-deletion HPFH may identify genetic combinations which more frequently result in thalassemia intermedia than thalassemia major. However, because each genotype is associated with a variable clinical outcome, none of the various genetic determinants analyzed above may be used by itself to predict the clinical phenotype. The only exception to this rule is the homozygosity for a mild fI-thalassemia mutation, which is consistently associated with thalassemia intermedia. However, combining all the criteria mentioned above, a tentative diagnosis may be made in the large majority of the cases. Conclusions The genotype-phenotype correlations discussed in this article have marked implications for carrier and detection methods prenatal diagnosis. Understanding of the atypical forms of heterozygous g-thalassemia, that may be missed by commonly used procedures for screening the P-thalassemia trait, allows the design of appropriate methods for their detection. The presence of atypical l3-thalassemia should always be taken into account especially in the

BLOOD REVIEWS

mate of a P-thalassemia carrier with an apparently normal person. Appropriate analysis of the a and y globin genes, in addition to the definition of the P-thalassemia mutations in prospective parents may allow prediction of the possible development of the b-thalassemia intermedia phenotype thereby improving the accuracy of genetic counselling. As discussed above, however, the capacity to predict the clinical phenotype on the basis of an extensive molecular analysis is still very inaccurate. Further studies are indeed necessary to uncover genetic modifying factors able to ameliorate the clinical picture of thalassemia major. Acknowledgements We thank Francesca Fodde, Daniela Desogus and Rita Loi for editorial assistance. The research reported in this article was sponsored by the World Health Organization and was supported in part by grants from Assessorato Igiene e Sanita Regione Sardegna (Legge Regionale n. 11 30 Aprile 1990), National Research Council (CNR) Target project ‘Prevention and Control Disease Factors’ (FATMA) contract n. 92.00041.PF41, CNR Target project ‘Ingegneria Genetica’ contract n. 92.00431.PF99. CNR Target project ‘Diagnostica delle Talassemie: Organizzazione e Standardizzazione de1 depistage dei portatori e della Diagnosi Prenatale’ contract n. 91.04193.ST75 and CNR Istituto di Ricerca sulle Talassemie e Anemie Mediterranee. Cagliari.

16.

17.

18.

19. 20.

21.

22.

23.

24.

25.

26.

References 1. Weatherall D J, Clegg J B. The thalassaemia syndromes. 3rd ed. Oxford: Blackwell Scientific Publications, 1981. 2. Bunn H F, Forget B G. Hemoglobin: molecular, genetic and clinical asnects. Phlladelnhia. W B Saunders. 1986. 3. Huisman T H J. The g and 6:thalassemia repository. Hemoglobin 1992; 16: 237-258. 4. Kazazian H H Jr. The thalassemia syndromes: molecular basis and prenatal diagnosis in 1990. Sem Hematol 1990; 27: 209-228. 5. William G W. Increased HbF in adult life. Baillitre’s Clin Haematol 1993; 6: 177-213. 6. Townes T M, Behringer R R. Human globin locus activation region (LAR): role in temporal control. Trends Genet 1990; 6: 219-223. 7. Tuan D, Solomon W, Li Q, London I M. The ‘B-like-globin’ gene domain in human erythroid cells. Proc Nat1 Acad Sci USA 1985; 82: 6384. 8. Forrester W C, Thompson C, Elder J T, Groudin M. A developmentally stable chromatin structure in the human S-globin gene cluster. Proc Natl Acad Sci USA 1986; 83: 1359. 9. Grosveld F. Van Assendelft G B. Greaves D R. Kollias G. Position-indipendent, high-level expression of the human /3-globin gene in transgenic mice. Cell 1987; 51: 975. 10. Tuan D Y H, Solomon W B, London I M, Lee D P. An erythroid-specific, developmental-stage-independent enhancer far upstream of the human B-like globin genes. Proc Nat1 Acad Sci USA 1989; 86: 2554. 11. Galanello R, Ruggeri R, Addis M, Paglietti E, Cao A. Hemoglobin A, in iron deficiency (.l-thalassemia heterozygotes. Hemoglobin 5: 613-618. 12. Melis M A, Pirastu M, Galanello R, et al. Phenotypic effect of heterozygous CLand go-thalassemia interaction. Blood 1983; 62: 226-229. 13. Rosatelli C, Falchi A M, Scalas M T, et al. Hematological phenotype of the double heterozygous state for a and g-thalassemia. Hemoglobin 1984; 8: 25-35. 14. Galanello R, Ruggeri R, Paglietti E, Addis M, Melis M A, Cao A. A family with segregatin triplicated alpha globin loci and beta thalassemia. Blood 1983; 62: 1035. 15. Rosatelli C, Leoni G B, Tuveri T, et al. Heterozygous g-Thalassemia: Relationship between the hematological

27.

28.

29.

30.

31.

32.

33.

34.

35. 36. 37.

38.

39. 40.

11

phenotype and the type of S-thalassemia mutation. Am J Hematol 1992; 39: 14. Gonzales-Redondo J M, Stoming T A, Kutlar A, et al. A C-+T Substitution at nt-101 in a conserved DNA sequence of the promotor region of the Bglobin gene is associated with ‘silent’ g-thalassemia. Blood 1989; 73: 1705-1711. Orkin S H, Kazazian H H Jr, Antonarakis S E, et al. Linkage of g-thalassemia mutations and g-globin gene polymorphisms with DNA polymorphisms in human g-globin gene cluster. Nature 1982; 296: 627. Wong C, Dowlin C E, Saiki R K, Higuchi R G, Erlich H A, Kazazian H H Jr. Characterization of g-thalassemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 1987; 330: 384. Pirastu M, Galanello R, Melis M A, et al. a 6 + thalassemia in Sardinia. Blood 1983; 62: 341-345. Paglietti E, Galanello R, Addis M, Cao A. Genetic counselling and genetic heterogeneity in the thalassemias. Clin Genet 1985; 28: l-7. Trifillis P, Ioannou P, Schwartz E, Surrey S. Identification of four novel &globin gene mutations in Greek Cypriots using polymerase chain reaction and automated fluorescence-based DNA sequence analysis. Blood 1991; 78: 3298-3305. Loudianos G, Cao A, Ristaldi M S, et al. Molecular basis of Gb-thalassemia with normal fetal hemoglobin level. Blood 75: 526-528. Loudianos G, Cao A, Pirastu M, et al. Molecular basis of the &thalassemia in cis to hemoglobin Knossos variant. Blood 1991; 77: 2087-2088. Galanello R, Melis M A, Podda A, et al. Deletion S-thalassemia; the 7.2 kb deletion of Corfu GS-thalassemia in a non-S-thalassemic chromosome. Blood 1990; 75: 1447-1449. Gasperini D, Perseu L, Cossu P, Podda R, Cao A, Galanello R. A novel 6”-thalassemia mutation: TGG+TAG (TRP+STOP) at codon 37. Hum Mutation (in press). Murru S, Pischedda M C, Cao A, et al. A promoter mutation of the II-globin gene (- 101 C+T) has an age-related expression pattern. Blood 1993; 81: 2818-2819. Huisman T H J. A C+G mutation at nt position 6 3’ to the terminating codon may be the cause of a silent b-thalassemia. Int J Hematol 1991; 54: 289. Semenza G L, Delgrosso K, Poncz M, Malladi P, Schwartz E, Surrey S. The silent carrier allele: S thalassemia without a mutation in the g-globin gene or its immediate flanking regions. Cell 1984; 39: 123-128. Galanello R, Meloni A, Gasperini D, et al. The repeated sequence (AT)x(T)y upstream to the S-globin gene is a simple polymorphism. Blood 1983; 81: 1974-1975. Craig J E, Kelly S J, Bametson R, Thein S L. Molecular characterization of a novel 10.3 kb deletion causing g-thalassemia with unusually high Hb A,. Br J Haematol 1992; 82: 735-744. Hanscombe 0, Whyatt D, Fraser P, et al. Importance of globin gene order for correct developmental expression. Genes Dev 1991; 5: 1387-1394. Behringer R R, Hammer R E, Brinster R L, Pahniter R D, Townes T M. Two 3’ sequences direct adult erythroid-specific expression of human g globin genes in transgenic mice. Proc Nat1 Acad Sci USA 1987; 84: 7056. Kollias G, Hurst J, Deboer E, Grosveld F. The human /3 globin gene contains downstream developmental-specific enhancers. Nucleic Acids Res 1987; 15: 5739. Trudel M, Costantini F. A 3’ enhancer contributes to the stage-specific expression of the human 8 globin gene. Genes Dev 1987; 1: 954. Gasperini D, Cao A, Paderi L, et al. Normal individuals with high HbAZ Levels. Br J Haematol 1993; 84: 166-168. Wainscoat J S, Thein S L, Weatherall D J. Thalassaemia Intermedia. Blood Rev 1987; 1: 273-279. Cao A, Gasperini D, Podda A, Galanello R. Molecular pathology of thalassemia intermedia. Euro J Int Med 1990; 1: 227-236. Kanavakis E, Metaxotou-Mavromati A, Kattamis C, Wainscoat J S, Wood W G. The triplicated a-gene locus and S-thalassemia. Br J Haematol 1983; 54: 201. Thein S L. Dominanat S thalassemia: molecular basis and pathophysiology. Br J Haematol 1992; 80: 273-277. Antonarakis S E, Orkin S H, Cheng T C, et al. 8-thalassemia in American Blacks: Novel mutations in the TATA box and

12

41.

42.

43.

44

45.

46.

47.

GENOTYPE-PHENOTYPE

CORRELATIONS IN B-THALASSEMLAS

IVS-2 acceptor splice site. Proc Nat1 Acad Sci USA 1984; 81: 1154. Camaschella C, Alfarano A, Gottardi E, et al. The homozygous state for the - 87 C+G 8 + thalassemia. Br J Haematoll990; 75: 132-138. Rosatelli M C, Oggiano L, Leoni G B, et al. Thalassemia intermedia resulting from a mild B-thalassemia mutation. Blood 1989; 73: 601-605. Ristaldi M S, Murru S, Loudianos G, et al. The C-T substitution in the distal CACCC box is a common cause of silent 8-thalassemia in the Italian population. Br J Haematol 1990; 74: 480. Agosti S, Parodi M I, Cerruti P, Cao A, Pirastu M. Molecular characterization of B-thalassemia intermedia in patients of Italian descent and identification of three novel B-thalassemia mutations. Blood 1991; 77: 1342. Tamagnini G P, Lopes M C, Castanheira M E, Wainscoat J S. 8 + thalassemia-Portuguese type: clinical haematological and molecular studies of a newly defined form of B thalassemia. Br J Haematol 1983; 54: 189-200. Wainscoat J S, Kanavakis E, Wood W G, et al. Thalassaemia intermedia-the interaction of CLand B thalassemia. Br J Haemato153: 411-416. Thein S L, Hesketh C, Wallace R B, Weatherall D J. The molecular basis of thalassemia major and thalassemia intermedia in Asian Indians: application to prenatal diagnosis. Br J Haematoll988; 70: 225-231.

48. Liebhaber S A. a-thalassemia. Hemoglobin 1989; 13: 685-731. 49. Flavell R A, Kooter J M, De Boer E, Little P F, Williamson R. Analysis of the &&globin gene loci and Hb Lepore DNA: direct determination of gene linkage and intergene distance. Cell 1978; 15: 25-41. 50. Pirastu M, Kan Y W, Galanello R, Cao A. Multiple mutations produce 88°-thalassemia in Sardmia. Science 223: 924-930. 51. Gihnan J G, Huisman T H J. DNA sequence variation associated with elevated fetal Gy globin production. Blood 1985; 66: 183-787. 52. Miller B A, Olivieri N, Salameh M, et al. Molecular analysis of the High-Hemoglobin F-Phenotype in Saudi Arabian sickle cell anemia. New Engl J Med 1987; 316: 244246. 53. Cappellini M D, Fiorelli G, Bernini L F. Interaction between homozygous B”thalassemia and the Swiss type of hereditary persistence of fetal haemoglobin. Br J Haematol 1981; 48: 561-572. 54. Thein S L. Weatherall D J. A non-deletion hereditary persistence of fetal hemoglobin (HPFH) determinant not linked to the B-globin gene complex. In: Stamatoyannopoulos G, Nienhuis A W, eds. Hemoglobin switching, Part b: cellular and molecular mechanisms, New York: Alan R Liss. 1989: 97-l 11.