BETA THALLASSEMIA :CURRENT CONCEPTS IN MANAGEMENT

MODERATOR PRESENTER DR. K. K. GUPTA VIVEK KUMAR

HISTORICAL PERSPECTIVE AND PREVALENCE : The Thalassemias are a group of congenital anemia that have in common deficient synthesis of one or more of the globin subunits of the normal human hemoglobin , particularly common in the Mediterranean region and south east Asia .Over 180 million people in the world and 20 million in India carry the gene for β-thalassemia.

Fig : distribution of beta thalassemia

There is very little evidence that it was recognized as a specific clinical entity before its first clinical description in 1925 . But by the start of the 20th century clinician were becoming aware of the syndrome

of splenic anemia of infancy, which was first described by Rudolf Von Jaksch – Warterhorst and it became a common practice to describe any unusual anemia in infancy as “Von Jaksch’s anemia” , particularly if the spleen was enlarged. The credit for the first clinical description of the condition which later became known as thalassemia is given to the American pediatrician Thomas B. Cooley , in 1925. The term ‘thalassemia’ was introduced by Whipple and Bradford in 1932.The word is taken from the Greek word ‘thalassa’, meaning the sea , indicating its Mediterranean connection.

GENETIC MECHANISM AND MOLECUAR PATHOLOGY: β-thalassemia is an autosomal recessively inherited disorder. The β-globin gene is located in the short arm of chromosome 11 in a region containing the δ-gene, the embryonic ε-gene , the fetal Gγ and Aγ genes, β-gene and the pseudogene ψβ1 . The five functional globin genes are arranged in the order of their developmental expression .

The appropriate expression of the different β-like globin genes in erythroid tissues during development depends on a major regulatory region named the Locus Control region (LCR) , located 5 to 25 kb upstream from globin gene . More than 200 different mutations producing β-thalassemia have been so far described ; the large majority are point mutations in functionally important sequences , where as in contrast of alpha thalassemia , gene deletion is rare cause of beta thalassemia . Beta thalassemia mutations result in either a complete absence of globin chain (β0-thalassemia ) or a largely variable reduction of globin output (β+-thalassemia ) .

Point mutations resulting in beta thalassemia are single nucleotide substitution or oligonucleotide insertions/deletions that affect the β-gene expression by a variety of mechanism.

β -globin gene complex (chromosome 11p)

CLASSIFICATION OF THALASSEMIA

α-Thalassemia

silent thalassemia(- α/αα)

Thalassemia trait(- α/- α, --/αα)

Hemoglobin H disease(--/-α)

Hydrops fetalis(--/--)

β-Thalassemia

Major

Intermedia

Minor

β-Thalassemia hemoglobinopathies

δ-thalassemia

δβ-thalassemia

Heriditary persistence of Fetal Hemoglobin(HPFH)

γδβ-thalassemia

Hb Lepore

Hyperunstable globins

Transcription Mutation :

Promoter mutation: several mutations have been described in or around the conserved motifs in the 5’ – flanking sequence of β– globin genes ( TATA box and the proximal and distal CACCC box) . This reduce binding of RNA polymerase, there by reduce the rate of mRNA transcription to 20 – 30 % of normal . Hence result in a mild phenotype.

5’ Untranslated region mutation : several mutations also have been reported in this 50- nucleotide region ; all have a mild effect on gene transcription. Heterozygote have a normal or borderline red cell indices and HbA2, and compound heterozygotes , with severe β-thalassemia alleles ,usually have mild phenotype.

Mutations affecting mRNA processing :

RNA processing essentially consists of removal of intervening sequences and the splicing of the coding regions to produce functional mRNA . The precision of this process relies on critical sequences present at intron/exon boundries ; the invariant dinucleotide –GT- at the 5’ (donor ) and –AT- at the 3’ ( acceptor ) splice junction and the flanking sequences ( consensus sequences )that are rather well conserved .

Splice junction and consensus sequence mutation : Mutations of the invariants 5’ –GT- and 3’ –AG- dinuleotide completely abolish normal splicing and result in βo–thalassemia. The efficiency of normal splicing may be decreased by mutations within the consensus sequences immediately adjacent to the splice junction . The reduction of beta globin production is quite variable and the resulting phenotypes range from mild to severe.

Cryptic site mutations in Introns and Exons : Along introns and exons there are sequences simlar to those found at the intron/exon boundaries, which normally are not used for splicing (“cryptic” splice sites) . A number of nucleotide substitutions involving these sequences transform a cryptic site into a legitimate one , resulting in a severe βo– or β+– thalassemia phenotyope.

Poly (A) and other 3’ untranslated region mutation : Downstream of the mRNA terminal codon , there is highly conserved AAUAAA sequences , which represent a signal for the cleavage and polyadenylation reaction , as a part of the RNA transcript processing . Because polyadenylation is important in determing the stability of mRNA, mutations at the AAUAAA sequences affect the efficiency of translation ,resulting in β+– thalassemia of variable , but usually mild severity . Other mutations in 3’ UTR also produce β silent thalassemia.

Mutations Affecting mRNA translation :

A large group of mutations alter the different steps of mRNA translation . Three categories of mRNA translation mutation can be identified : Initiation codon mutations , nonsense mutations and frameshift mutation . Initiation codon mutation : The initiator codon ATG, which encodes for methionine , is a critical signal for starting translation . Seven different point mutations of the initiation codon have been reported as cause of β - thalassemia.

Nonsense mutation : Single nucleotide substitution may change a codon for a given amino acid to one of the three possible chain termination codons :TAA,TAG or TGA .The result is a premature interruption of mRNA translation with absence of β-globin production (βo - thalassemia ).

Frameshift mutation : Insertion or deletion of one or a few nucleotides (other than three or multiples of three ) alters the reading frame of the encoded mRNA starting at the site of the mutation . The new reading frame usually results in a novel abnormal amino acid sequence and in a premature termination further downstream . The mutant globin chain is rapidly degraded , and the final result is a βo- thalassemia .

In addition to above mentioned mutations , several deletions , affecting only the β- globin gene and ranging size from 290 bp to approximately 67 kb , have been reported .

β – Thalassemia Hemoglibinopathies

This group includes some structurally abnormal hemoglobin with a thalassemia phenotype , they can be classified according to the molecular mechanism .

δβ- Hybrid Gene : Unequal crossing over between the homologous δ- and β- globin genes results in the formation of hybrid δβ and βδ genes ,referred to as Lepore and anti-Lepore genes. The Lepore hemoglobin contain the N- terminal sequence of the normal δ – chain and C- terminal sequences of the normal β- chain. The pathophysiology is similar to that of β- thalassemia .This abnormal hybrid globin chains are relatively instable and are also in-effectively synthesized , leading to an excess of α- chains , leading to ineffective erythropoiesis and anemia . The homozygous and the heterozygous states of Hb – Lepore are clinically similar to those of β- thalassemia .

Hyper unstable Globins : This is singular group of β- globin gene mutants that are characterized by amino acid substitution , additions or deletions in the β- globin chain associated with a thalassemia phenotype in heteroygous state . For this reason , these forms are also referred to as dominantly inherited β- thalassemia . In contrast to the typical recessively inherited forms of β- thalassemia , which lead to a reduced synthesis of normal β-globin chain. This group of mutations results in the production of β – globin variants that are extremely unstable. These hyperunstable globins fail to form functional tetramers and precipitate in the erythroid precursors , leading to ineffective erythropoiesis, which is exacerbated by the concomitant relative excess of α – chain . Most patients present with clinical phenotype of thalassemia intermedia.

δ- Thalassemia : Several mutations of the δ- globin gene results in reduced (δ+- thalassemia ) or absent ( δ0- thalassemia ) production of δ- globin chains . These conditions do not have clinical relevance but the co-inheritance with β-thalassemia mutations may create problems in β-carrier identification ,because the HbA2 may be normal or borderline.

δβ – Thalassemia : This includes a group of disorders characterized by reduced or absent production of both δ- and β – globin chains and by a variable increase in γ-chain synthesis that only partially balances the δ- and β-chain deficiency . The most common molecular mechanism consists of deletion of variable extent of β-globin gene cluster , which involve the δ- and β-genes . The thalassemia due to homozygous mutation is frequently confused with HPFH because both disorders have 100 % HbF .However, unlike HPFH , increased γ- chain production fails to fully compensate for the loss of β- chain production . It appears that in δβ-thalassemia , there is less compensation by γ –chain synthesis than in HPFH but more than in homozygous β-thalassemia . Thus most patients with δβ-thalassemia have a mild anemia with slight hepatoslenomegaly and some bone changes associated with chronic erythroid hyperplasia . The heterozygous form of δβ-thalassemia is not identified with any specific clinical finding .

Hereditary Persistence of Fetal Hemoglobin (HPFH) : HPFH is characterized by absence of relevant hematologic abnormalities. The amount of HbF is variable , ranging in heterozygotes from 2 to 30 % , to 100 % in homozygous subject . The condition is asymptomatic . In homozygous HPHF , there is erythrocytosis as a result of the high oxygen affinity of HbF. High Hb levels from 14.8 to 18.2 g/dl are typical . HPHF is characterized by either deletion or inactivity of the β- and δ- structural gene complex . In the deletion variants both Gγ and Aγ are synthesized in increased quantities . In the non deletion variants , only one of the two γ- chains are over expressed. The γ- chain continues to be produced in increased amount throughout life , compensating for the lack of β- and δ- chains , consequently there is no accumulation and no precipitation of excess α- chains . A typical finding in HPHF is the “cell wide” uniform distribution of HbF in erythrocytes , a feature the helps to distinguish this disorder from other disorders associated with an increase in HbF .

γδβ- thalassemia : This rare form of thalassemia has several variants and is characterized by deletion or inactivation of the entire β- gene complex . Only the heterozygous state has been encountered , although neonates have severe hemolytic anemia but as the child grows , the disease evolves to a mild form of β-thalassemia .

PATHOPHYSIOLOGY :

Normally equal quantities of α- and β- chains are synthesized by the maturing erythrocytes resulting in a β- to α- chain ratio of 1.0 . the basic defect in β- thalassemia is a reduced or absent production of β-globin chains with relative excess of α- chain . The direct consequences is a net decrease in the hemoglobin production and an imbalance of globin chain sythesis . In heterozygous thalassemia , the β- to α- chain ratio is decreased to 0.5 – 0.7 , where as in homozygous thalassemia the ratio is less than 0.25 . This imbalance has a dramatic effects on the red cell precursors , resulting in their premature destruction in the bone marrow and extramdullary sites . This process is referred to as ineffective erythropiesis and is the hallmark of β- thalassemia . Using ferrokinetic analysis , it has been shown that in β- thalassemia patients only 15 % of Fe59 is incorporated in circulating erythrocytes , indicating that ineffective eryhtropoiesis could account for as much as 60 to 75 % of the erythropoiesis . Hemolysis of the erythrocytes containing inclusion that reach peripheral blood is a minor cause of anemia , particularly in thalassemia major . The excess α- chain lies at the center in the pathophysiology of β-thalassemia and thus the main determinant of the clinical severity is the degree of α/non-α gene imbalance.

The free excess α-chains are unstable and precipitate within the cell. There is further oxidation of excess α-chains which result in the formation of hemichromes . Irreversible hemichromes and denatured α-chains precipitate as inclusionbodies early during differentiation and throughout erythroid maturation . α- chain precipitation in the red cell membrane causes structural and functional alterations with changes in deformability , stability and red cell hydration . A further consequence of the membrane bound hemichromes is their association with the cytoplasmic domain protein band 3 , creating a neoantigen that is subjected to opsonization with autologous immunoglobulin G and compliments , and later removal of the cell by macrophages . Besides oxidation , free α – chain are subjected to degradation resulting in the formation of denatured α- globin protein , heme and free iron . These degradation products play a role in damaging erythroid precursors and red cell membranes. Free iron via the “Fenton reaction” generates reactive oxygen species , which cause lipid and protein peroxidation with consequent damage to red cell membrane and intracellular organelles . Also heme and its oxidized form hemin produce oxidative damage to the different components of the red cell membrane with consequent structural and fuctional alterations.These alterations of erythroid precursors result in an enhanced rate of apoptosis . Apoptosis contributes significantly to ineffective erythropoiesis and occurs primarily at the polychromatophilic erythroblast stage. However it is not clear how α-globin precipitation causes apoptosis .

Ineffective erythropoiesis and anemia have several consequences . The first response to anemia is an increased production of erythropoietin , causing marked erythroid hyperplasia , which may range between 10 to 30 times normal . Anemia may produce cardiac enlargement and may contribute to cardiac failure .Erythroid expansion produces skeletal deformities , osteoporosis and occasionally , extramedullary masses . Untreated and undertreated individuals with thalassemia major have retarted growth as a result of anemia and an excessive metabolic burden imposed by erythroid expansion . Ineffective erythropoiesis is associated with increased iron absorption that further contributes to the iron burden imposed by blood transfusions. Iron overload damages several organs , including the myocardium , liver and endocrine glands . Removal of the abnormal RBCs by the reticuloendothelial elements of the sleen results in splenomegaly and hypersplenism . A further consequence of the RBC membrane damage is the increased surface exposure of the procoagulants , negatively charged phospholipids phosphatidyl serine and phosphatidylethanolamine , the anionic phospholipids increases

thrombin generation.

CLINICAL AND LABORATORY FEATURES

The designation commonly used to describe the β- thalassemia syndromes are based on clinical severity The clinical severity is determined by the genotype of the individual.

The most severe form is defined β-thalassemia major and is characterized by transfusion dependent anemia . Thalassemia intermedia is the term used to designate a form of anemia, independent of the genotype , that does not require transfusions. Thalassemia minor indicates the heterozygous state that is usually asymptomatic.

Thalassemia major presents in early infancy ( 6 – 18 months ) with progressive pallor, hepatosplenomegaly and bony changes ,and if left untreated , is invariably fatal during the first few years of life . At the other end of the spectra is a heterogenous form ( thalassemia minor ) in which patient can lead practically normal life except for a mild persistent anemia and have a normal life span . In between these two extremes is a form with varying degrees of clinical manifestations of anemia , splenohepatomegaly and bony changes and who maintain their life fairly comfortably, and are usually not dependent on regular blood transfusion .

TABLE :CHARACTERISTICS OF β- thalassemia

Genotype Hemoglobin pattern Clinical severity

β+/ β ↑HbA2,slight ↑HbF Mild

β+/β+ ↓HbA,↑ HbF , variable HbA2 Variable but usually severe

β0/β ↑ HbA2 , ↓HbA, ↑HbF Mild

β0/β0 No HbA, , variable HbA2, Severe remainder HbF

(δβ)0/δβ HbA2 normal or slight ↓ Mild 5-20 % HbF, ↓HbA

(δβ)0/ (δβ)0 No HbA or HbA2,100 % HbF Mild

δ0β0/δβ,HPHF(heterozygous) ↓HbA, ↓HbA2 , 10-30% HbF Normal

δ0β0/δ0β0 100% Normal

Diagnosis :

CBC is frequently sufficient to postulate a diagnosis of thalassemia . Hb is very low < 6.75 g/dl in thalassemia major , may be variable in thalassemia intermedia and near normal to mildly decreased in thalassemia minor . MCV and MCH is reduced .

Thrombocytopenia or neutropenia may present due to hypersplenism .

Peripheral blood smears are diagnostic with microcytic , hypochromic , poikilocytic and polychromatic red cells . There is also moderate basophilic stippling with fragmented erythrocytes , target cells and large numbers of normoblasts . Reticulocyte count is low usually below 1 % in thalassemia major , but it is generally increased to twice than normal in thalassemia minor and have been found to correlate with Hb level .

Osmotic fragility reveals reduced fragility .

Bone marrow examination shows normoblastic erythroid hyperplasia with increased stained iron .

Red cell survival measured by Cr51 shows ineffective erythropoiesis rather than peripheral hemolysis .

Hb electrophoresis is diagnostic . HbF is increased , HbA2 is usually over 3.5 % , but may be normal . HbA is reduced to absent depending upon the genotype .

Radiological findings include widening of medulla due to bonemarrow hyperplasia , thinning of cortex and trabeculation in the long bone metacarpals and metatarsals . Skull X-ray shows hair-on-end appearance .

Serum iron , ferritin and iron saturation is increased.

GENERAL TREATMENT SRATEGIES

1. RBC TRANSFUSION

2. IRON CHELATION

3. SPLENECTOMY

4. VITAMIN SUPPLEMENTATION

VITAMIN C, FOLIC ACID

5. MANAGEMENT OF COMPLICATIONS

OSTEOPENIA AND OSTEOPOROSIS

ENDORINOPATHIES

HEART FAILURE

Red Blood Cell Transfusions:

For severe anemia as in beta thalassemia major , usually defined as Hg levels of < 7 g/dl regular transfusions are absolutely life saving . And as for now regular blood transfusions are the mainstay of the treatment of .

Transfusion therapy in thalassemia has two goals1. To prevent anemia ,2. To suppress endogenous erythropoiesis to avoid in-effective erythropoiesis.

Transfusion may also be required for those children with thalassemia intermedia who cannot maintain Hb above 7 g/dl or for those who show evidence of growth retardation , severe bony changes or hypersplenism.

Transfusion regimen may be (1) low transfusion regimen where Hb is maintained around 6 – 10 g/dl , (2) hypertransfusion : Hb level 10 – 12 g/dl and (3) supertransfusion, where Hb is maintained at 12 – 14 g/dl or PCV maintained above 35 % .The most widely accepted regimen of today is a hypertransfusion regimen . To reduce the volume transfused and to avoid the administration of foreign protein packed RBC must be transfused and to avoid transfusion reaction the blood product should be rendered WBC free. This can be achieved by using frozen blood but it is expensive . At present leucocyte depletion is achieved by filtering donor blood.Transfusion of neocytes obtained by centrifugation has been proposed in the attempt to reduce the blood requirement , but the results were not cost effective .In general , the amount transfused should not exceed 15 to 20 ml/kg/day at a maximum rate of 5ml/kg/hr to avoid rapid increase of blood volume . The recommended interval between transfusion is usually 2 to 3 weeks.

Complication of transfusions :

1.Febrile reactions : it is seen in 3 – 20 % of patients and may be due to leucocyte or platelet antibodies, antibodies against RBC antigens, allergic reactions to other plasma or due to pyrogens present in transfused blood . Febrile reactions usually respond to antipyretic and antihistaminic .

2. Hemolytic transfusion reactions : it occurs in 5 – 15 % of cases. These are due to major or minor blood group mismatch and are characterized by fever , chills, tachycardia, restlessness, flushing of face ,chest pain, dyspnoea ,cynosis and collapse. Most feared complications are acute renal failure, DIC and anaphylactic shock .

3. Transfusion transmitted diseases like malaria , syphilis, hepatitis B, hepatitis C, CMV and HIV infection can occur. All thalassemics who are negative for the hepatitis B surface antigen should receive hepatitis B vaccine.

4. Iron overload : Two factors contribute to iron overload in a thalassemic child :a) Enhanced gastrointestinal absorption of iron ,b) Transfusional siderosis .

In normal individual 1 mg of iron/day is absorbed from the gut , while in a thalassemic child it may be as high as 10 mg/day .Each unit of blood contains approximately 200mg of iron (1 mg/ml) ,a patient who receives 25 to 30 units of blood /year, in the absence of chelation, accumulates more than 70 g of iron by the third decad of life . The accumulation of iron in tissues causes cellular damage presumably by accelerating the generation of reactive oxygen species (ROS), which then overwhelm the cell’s protective ability to reduce them . These uncontained ROS then attack and oxidatively alter cellular proteins and lipids. Some authors believe that the level of nontransferrin bound iron (NTBI) in the blood is a major cause of oxidative damage . Iron accumulates in those tissues that have the highest levels of transferrin receptor , which are primarily the liver , heart , and endocrine organs . Iron accumulation is documented by measuring levels of ferritn, serum iron, transferrin and calculating the percent of iron saturation.Though helpful , neither the ferritin level nor the percent iron saturation provides sufficient definitive information to guide therapy . The gold standard for assessing accumulation of iron stores is an adequate liver biopsy , followed by measurement of the liver iron content by histochemical staining or more accurately by atomic absorption spectrometry . An accurate and noninvasive method for measuring liver iron is SQUID (superconducting quantum interference device). Unfortunately none of the methods listed above accurately records the extent of cardiac iron loading and there is a poor correlation between ferritin, liver iron content, and cardiac iron .Even studies using endomyocardial biopsies have yielded variable results .The traditional diagnostic tools ( i.e. ECG, holter tracing , echocardiography , nuclear studies ) ,although routinely used , are not predictive of subsequent cardiac dysfunction. The prognosis for patients with heart failure has always been poor.

CHELATION THERAPY :

To prevent hemosiderosis , iron administered as blood must be chelated and excreted . Two main sources of chelatable iron are (a) the intracellular pool derived from lysosomal catabolism of ferritin and from transferrin and NTBI and (b) the iron derived from red cell catabolism in macrophages . The first contributes to the hepatocellular load and is excreted as fecal iron , where as the second is the ,major source of urinary iron. Ferric iron has six coordination sites , which need to be chelated completely to prevent free radical generation .The iron chelators are :

Desferrioxamine (DFO) Desferrioxamine is a hydroxylamine compound produced by streptomyces pyloses. It is a hexadentate agent that effectively chelates iron , masking all six coordination sites so that the complex cannot generate ROS . A single gram of DFO is

able to bind 85 mg of iron. It does not enter cells, so the iron is removed from transferrin or the NTBI in blood and bile. The chelated iron is excreted equally in urine and stool. DFO must be given parenterally , either by bolus plus intravenous continuous infusion or via prolonged subcutaneous infusion. For best results the infusion needs to be continued for 8 – 12 hrs using a constant infusion pump and must be given on daily basis for a minimum of 5-6 times per week. The daily dose is about 20 – 60 mg/kg and should be tailored according to the need of the patient.To avoid severe effects on growth and bone metabolism DFO should not be started before a significant amount of chelatable iron is present . It is usually advised that chelation be started when ferritin levels reach 1000 ng/ml or after 10 – 15 units of blood have given. In general , the goal is to keep the serum ferritin level below 1000 ng/ml.Side effects include pain , induration , irritability and redness locally at the site of injection , when given parenterally there may be liberation of histamine leading to rigors , headache , photophobia and hypotension. Visual abnormalities and high incidence of high frequency sensorineural hearing loss may occur which are reversible ,yearly slit lamp examination and audiometry are mandatory. Stunted growth and rickets like bone abnormalities have been reported when treatment was initiated early and at dose >40 mg/kg. Pulmonary toxicity has been observed in patient treated intensively with rescue doses of DFO (10-20 mg/kg/hr).

Deferiprone :

Deferiprone is an orally effective bidentate iron chelator .Three molecules of deferiprone are required to effectively chelate one iron molecule and prevent it from generating ROS. Deferiprone can enter cells and bind iron there , and the iron thus chelated is primarily excreted in the urine . Deferiprone in customary doses is less effective than DFO in preventing liver iron accumulation . In addition , it causes arthralgia and leucopenia in 2 % of patients and neutropenia in 0.5 % .Deferiprone may be able to remove iron from from the heart , as measured by the T2*method and there seems to be parallel improvement in clinical measures of cardiac disease. Defepriprone being able to pass through membranes , could ‘shuttle’ tissue iron to DFO in the blood and then be removed .Based on this investigators are testing a ‘shuttle’ method to treat cardiac hemochromatosis . An initial dose of deferiprone is given orally presumbly to enter cardiac cells , bind iron there, and then transfer the chelated iron to the blood. After precisely timed interval , parenteral DFO is given to bind this iron in the blood and exrete this iron in urine and stool.

ICL670 (EXJADE)

ICL670 is an orally active tridentate chelator that has shown good pharmacokinetics in trials in several sites . It seems to be relatively nontoxic and removes iron from the liver about as well as DFO , but no data on its ability to enter cells or remove cardiac iron. This drug has recently been approved by FDA.

SPLENECTOMY

The introduction of regular transfusion has significantly delayed appearance of splenomegaly The splenectomy should be performed when transfusion requirements exceeds 50 % above that of the average of the splenectomized population, this occurs when more than 200 – 250 ml/kg/yr of pure RBC are required to maintain a pretransfusional Hb of 9.0 -9.5 g/dl.Patient undergoing slplenectomy should

receive pneumococcal vaccine , H. influenza vaccine and meningococcalvaccine 4 weeks prior to surgery . Prophylactic penicillin therapy must be continued life-long

Management of Specific Conditions :

Osteopenia and Osteoporosis :In addition to problems with height and overall stature, patients with osteopenia or osteoporosis have bone pain, which is occasionally very severe.The causes are complex and include associated endocrinopathies , nutritional deficiencies, increased levels of osteoclastic activity , increased osteoid thickness associated with delayed mineralization in areas adjacent to foci of iron deposition , reduced levels of IGF-1, inadequacy of transfusion and chelation and medullary erythroid expansion . Treatment with Ca, vit D and biphosphates reverses some of the indices of osteoclastic activity and increase BMD.

Endocrinopathies : Iron deposition can damagethe endocrineglands,either ditrectly or throughthe hypothalamic-pituitary axis. Endocrinopathies are quite common in thalassemic patients . One study found hypogonadism in 22.9% of boys and 12.2% of girls , hypoparathyroidism in 7.6%, short stature in 39.3%. Fertility is impaired in the more severe forms of thalassemia.

Cardiac Problems :Cardiac complications are the major cause of death in well-transfused patients with β-thalassemia major. The more important complications include congestive heart failure and arrhythmias. The underlying causes are chronic anemia , iron overload , myositis. Cardiomyopathy and pulmonary hypertension. Monitoring of left ventricular ejection fraction (LVEF) is the most sensitive method for detecting evolving problems. Recent data indicate that in case of a drop in LVEF to < 45% ,instituting a very aggressive chelation program can markedly improve cardiac function and survival. Pulmonary hypertension is now recognized as a major issue, particularly in splenectomized patients. Anecdotal studies suggest the sildanfil may be beneficial in reducing pulmonary hypertension.

Role of VITAMIN C Hemosiderotic parients are often found to be vit C deficient .At one hand Vit C enhances DFO induced iron excretion by helping in conversion of homosiderin to ferritin , but on the other hand it may potentiate cardiotoxicity .So vitamin C supplementation is therefore recommended only in patients not affected by myocardiopathy,unsatisfactory iron excretion and demonstrated vitamin C deficiency in usual dose of 50 – 100 mg/day.

Molecular Therapies in β-thalassemia : NEW CONCEPTS

So far only curative therapy in β-thalassemia available is allogeneic haemopoitic stem cell transplantation but this is successful in only 65-70% of cases and is not available to most patients. Therefore the mainstay treatment for the majority remains life long blood transfusion in combination

with a rigorous regime of iron chelation . Improved understanding of the pathophysiology and molecular basis of the disease has provided clues for more effective strategies that aim to correct the defect in β-globin chain synthesis at the primary level or redress the α/β-globin chain imbalance at the secondary level. Improved understanding of the , molecular basis of the disease complications, such as iron overloading, has also provided clues for potential molecular targets at the tertiary level.

PRIMARY TARGET –gene correction therapy

Targeting the primary causes of reduced β-globin production involves several strategies including haemopoietic stem cell transplantation (HSCT), gene therapy using viral vectors and antisense mRNA .

HSCTHaemopoietic stem cell transplantation is conceptually the simplest and the only approach so far , that may lead to a definitive cure for β-thalassemia. Patients who stand to derive most benefit from such treatment are those with early but severe transfusion dependent disease and have few co-morbities. But the major limitations of HSCT are-1. Shortage of suitable donors2. Transplant related mortality (TRM)3. Graft failure4. Graft versus host disease (GVHT)

Recent evidence suggests that a cohort of patients who have stable Mixed Chimerism express sufficient functional β–globin to be transfusion independent. This has led to trials with non-myeloablative (Mini-) Bonemarrow translplantaion with some encouraging results particularlly in high risk group (Andreani et al, 2000).

The use of related or non-related umbilical cord blood transplants has extended the donor pool ,but cord blood transplantations have limited success because of the large numbers of cells that are required to sustain haemopoiesis

GENE THERAPYThe genetic correction of autologous haematopoitic stem cell overcomes the dual dis- advantages of donor shortage and GVHD in allogenic HSCT. Gene therapy in thalassemia has to meet following criteria.

1. Safe and efficient viral / non-viral transfection2. Erythroid lineage specificity and 3. Therapeutic levels of β-globin expression

Table : potential targets of molecular therapies in β-thalassemia.TARGET LEVEL MOLECULR TARGETS POTENTIALTHERAPIES

Primary :β-globin expression All genetic defects Haematopoietic stem cell transplant gene therapy using lentivirus

Mutation causing aberrant Antisense/small interfering mRNA splicing

Secondary :Reducing α/β-globin Increased γ-globin expression Gene therapy using lentivirus vectors Imbalance Synthetic transcription factors

Pharmacoinduction of HbF hydroxyurea 5-azacytidine butyrates, arginine butyrate Chaperone excess α-globin α-haemoglobin stabilizing protein mimic

α-chain promoting proteolysis Ubiquitin aldehyde

Tertiary :Promoting erythroid Supplementing inappropriately Human EPO Survival low EPODecreasing iron overload supplementing inappropriately intraperitoneal injections of synthetic Low hepcidin hepcidin Antioxidants Reducing oxidative stress vitamins C and E

In recent studies HIV based lentiviral (LV) vectors were shown to stably transmit the human β-globin gene and a large LCR element , resulting in correction of beta thalassemia intermedia in mice , with similar reports by several other groups , however the level of expression were insufficient to fully correct the anemia in thalassemia major mouse model .Insertion of chicken hypersensitive site -4, chicken insulator element (cHS4) in self in activating (SIN) LV vector resulted in higher and less variable expression of human β-globin . The levels of β-globin expression achieved from insulated SIN – LV vector were sufficient phenotypically to correct the thalassemia phenotype in vitro study in four patients and this correction persisted long term up to 4 month in xenotransplated mice in vivo . In summary LV vectors have paved the way for clinical gene therapy trial.

Therapeutic antisense mRNAAbout half of the β-thalassemia mutations are caused by aberrent RNA splicing. Studies from a decade ago have shownthat antisense oligonucleotides targeted at aberrant splice sitescan restore correct

splicing in erythroleukaemic cell lines(Sierakowska et al, 1996).More recently, the use of so-called morpholino oligonucleotides has enabled high level correction of transcribed mutant β -globin mRNA by free uptake of oligonucleotides through prolonged exposure to cultured erythroid precursors, where they have been shown to achieve up to 80% correction in human erythroid cells with the splice site mutation IVS2–654 (Suwanmanee et al, 2002a). However, the ability of erythroid precursor cells, modified ex vivo and re-infused, to express sufficiently high levels of functional β -globin remains untested.To date there are no published studies of successful in vivo use of therapeutic antisense mRNA.

Secondary targets – modifiers of chain imbalance in β-thalassaemia

The central mechanism underlying the pathophysiology of β-thalassaemia relates to the degree of the α/β-chain imbalance and deleterious effect of the free α-globin chains, as clearly demonstrated by several genotype/phenotype correlation studies (Camaschella et al, 1995; Rund et al, 1997; Ho et al, 1998). The α/β imbalance may be partially redressed by co-inheritance of α- thalassaemia , suggesting that downregulation of the α-globin genes may be a potential molecular target. The other measures that are being explored are-

HbF Augmentation : The other object of therapy in the severe beta thalassemias is to increase the synthesis of gamma globin chains, thereby compensating for the deficit of beta globin. These gamma chains combine with the excess unmatched accumulating alpha globin chains. The combination produces a useful Hb, namely HbF (fetal hemoglobin), but more importantly it reduces the burden of the alpha chains that produce most of the pathophysiology.Apart from gene therapy using lentivirus vectors in murine models being explored , several classes of agents have been tested, but none has been approved for regular clinical use, like-

Hydroxyurea Studies in sickle disease have shown that hydroxyurea, an inhibitor of ribonucleotide reductase, can result in an increase in HbF. The mechanism is not entirely clear, but hydroxyurea has been given to patients with beta thalassemia major and intermedia, and to patients with beta thalassemia/HbE disease, with varying degrees of success . Some patients have become independent of RBC transfusion. In some cases the Hb level has increased 1–3 g/dl, though in other reports there has been no increase in Hb. The underlying reasons for this variability of results remain obscure. Hydroxyurea has potentially severe side effects. It is myelosuppressive, and there is theoretical concern about the dangers of late-onset acute leukemia or myelodysplastic disease although so far these have not occurred.

Hypomethylating Agents Inhibitors of methyl-transferase can result in hypomethylation of DNA, a mechanism that overcomes the normal downregulation of gamma globin synthesis. These inhibitors, 5 Azacytidine and its analog 5-aza-2'-deoxycytidine (decitabine), have been given to beta thalassemic patients and have produced variable

increases in HbF along with variable increases in Hb levels. Again the lack of predictability has remained puzzling. Both agents must be given parenterally and both cause myelosuppression.

Histone Deacetylase Inhibitors Acetylation of histones opens up a gap between the covering chromatin and underlying DNA, thereby allowing that DNA to be transcribed. Histone deacetylases remove these acetyls, resulting in downregulation of that segment of DNA. Thus, inhibitors of histone deacetylases would allow otherwise suppressed gamma globin transcription to proceed. Examples of histone deacetylase inhibitors include short-chain fatty acids such as butyrates, which can cause variable increases in HbF in beta thalassemic patients. The agents that have been tested include arginine butyrate, which is given intravenously, and sodium phenylbutyrate, which can be given orally. Side effects include gastrointestinal discomfort.

Combinations of hydroxyurea, hypomethylating agents, and histone deacetylase inhibitors have been tested in isolated case reports and may be more effective in combination in increasing HbF synthesis.

Upregulation of a chaperone for α-globin : α -haemoglobin stabilising protein (AHSP)In addition to γ-globin chains, other α-binding proteins could in theory prevent b-chain precipitation and subsequent cell damage. Such a protein, is the AHSP. AHSP is an abundant erythroid-specific protein that specifically binds various forms of α -globin subunits, but not β-haemoglobin or HbA (Gell et al, 2002; Kihm et al, 2002). AHSP limits the oxidative effects of α-chain and prevents its precipitation, in solution and cells.Clinical studies suggest that AHSP mRNA is unusually low in some patients with severe β- thalassaemia (Galanello et al,2003), but studies in patients with β -thalassaemia/HbE have failed to identify mutations or variations in the AHSP gene which could correlate with disease severity (Viprakasit et al, 2004). Recently, however, it has been showed that AHSP expression is variable in healthy adults, and suggest that variable AHSP expression may be associated with certain polymorphic variants accounting for some of the phenotypic heterogeneity in β -thalassemia (Lai et al, 2006). A potential molecular therapy could thus involve upregulation of AHSP protein or the synthesis of an AHSP mimic to chaperone the free redundant α -globin in β -thalassaemia (Luzzatto & Notaro,2002).

Ubiquitin-dependent α -chain proteolysis There is evidence that excess α -globin chains in β -thalassemic erythrocytes are degraded by ATP and ubiquitin-dependent mechanisms. The promotion of efficient proteolysis may reduce the cytosolic α-chain precipitation and subsequent cellular damage and haemolysis. Ubiquitin aldehyde, a chemical that inhibits isopeptidases, which hydrolyses the bond between ubiquitin and its adduct protein, has been demonstrated to increase degradation of radiolabelled α -chains in haemolysates from β-thalassemia patients (Shaeffer & Cohen,1997). Unfortunately, there are as yet no studies to demonstrate efficacy of this strategy in either cell lines or animal models.

Tertiary targets – addressing complications of β -thalassaemia

Potential hepcidin agonists in iron overload Hepcidin is a peptide hormone synthesised by the liver. It is a key regulator of iron homeostasis (Hentze et al, 2004). It inhibits iron absorption from the gut and studies indicate that it is the negative regulator of iron absorption, recycling, and release from stores. In thalassemic patients, despite the iron overload, hepcidin levels are inappropriately severely decreased presumably because of the over-riding effect of anemia on hepcidin production (Kattamis et al, 2006; Nemeth & Ganz, 2006) Furthermore, intraperitoneal injections of synthetic hepcidin leads to dose-dependent hypoferraemia in murine models, suggesting a potential clinical use of hepcidin agonists or mimics to reduce iron overload in β-thalassemia (Rivera et al, 2005).

Potential role of antioxidants in cellular damageSeveral studies have demonstrated that reactive oxygen species play an important role in the pathophysiology of β -thalassemia (Rund & Rachmilewitz, 2001; Amer et al, 2004).Although treatment with vitamins C and E (Dissayabutra et al, 2005) is safe and has been shown to improve the oxidative profile in β -thalassaemia patients, there is as yet no evidence to suggest that such treatments improve anemia. Plant flavenoids and tea polyphenols are other groups of antioxidants with therapeutic potential in thalassemia (Grinberg et al, 1994, 1997; Grinberg et al, 1996).

ConclusionThe β- thalassemias were among the first human diseases to be delineated at the molecular level. Many clues toward molecular therapy have been derived from the extensive pathophysiological studies, and treatment is evolving from the management of symptoms and complications to more effective curative strategies that aim to correct the β-globin chain deficit, such as gene therapy using lentiviral vectors, or redress the α/β-globin chain imbalance, such as induction of γ-globin chain synthesis by gene transduction or pharmacological agents . These potential treatement options offer proof of principle of the molecular mechanism underlying the disease but many are still in evolution.However,three treatment options are nearing maturity : pharmacological induction of HbF , gene therapy using LV vectors and mini-(or non-myeloablative -) bonemarrow transplantation.

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