Thalassemias

THALASSEMIA results from an imbalance in the synthesis of a- and ß-globin Chains. To date we know of > 1000 naturally occurring inherited mutations that affect either the structure or synthesis of the a- and ß-like globin chains. Thalassemia occurs when there is a deficiency in a globin expression and ß thalassemia occurs when ß globin synthesis is downregulated. Many different mutant alleles have been selected to reach very high frequencies in tropical and subtropical regions of the world because heterozygotes are, to some extent, protected from the effects of falciparum malaria. Carriers of a and ß thalassemia are clinically normal but the associated hypochromic microcytic blood picture requires careful diagnostic screening to differentiate it from other conditions with similar hematological pictures (e.g., iron deficiency and sideroblastic anemia). In areas where a and ß thalassemia commonly occur, compound heterozygotes and homozygotes for these mutations may suffer from the clinically severe forms of thalassemia (HbH disease and Hb Bart’s hydrops fetalis) and ß thalassemia (ß thalassemia major and the less severe ß thalassemia intermedia). A summary of the key clinical syndromes (a) and pathophysiology of a (b) and ß (c) thalassemia. The consequences of reduced a and ß globin synthesis are indicated by dotted lines. Note, a thalassemia affects both the fetus and adult whereas ß thalassemia only becomes apparent after birth. HbF may persist or become re-activated during adult hematopoiesis in ß thalassemia.

Epidemiology : thalassemia is the world’s most common monogenic disorder. In areas where thalassemia is prevalent, the carrier frequency of thalassemia varies from 1% (e.g., in Southern Spain) to 90% (e.g., the tribal populations of India). Similarly, the carrier frequency of ß thalassemia may vary from 1% (e.g., in Northern Italy) to 50–70% (e.g., ß-E in some regions of southeast Asia). Considerable variation occurs even within a single country. It has been estimated that worldwide there are 270 million carriers of mutant globin alleles which can potentially cause severe forms of thalassemia and hemoglobinopathy (including sickle cell disease), and every year some 300–400,000 severely affected infants are born^ref. > 95% of these affected births occur in Asia, India and the Middle East. However, over the past few decades there has been considerable mobility in the world’s populations so that now in Australasia, Europe and North America a significant proportion of recent immigrants originate from countries in which thalassemia commonly occurs. Therefore, the management of families from ethnic minorities, at risk of producing offspring with severe life-threatening forms of thalassemia, has become a new important aspect of hematologic practice. It has also been noted over the past 50 years that, as the world economy improves and the overall infant mortality rate falls, thalassemia is rapidly emerging as a major economic and health burden in developing countries since infants with thalassemia who would otherwise have died from infection and malnutrition now survive and require lifelong treatment. Therefore, worldwide, thalassemia is an enormous and ever-increasing hematologic problem in both developed and developing countries^ref. Today’s epidemiology of thalassemia is strikingly different from that of the past. Thalassemia is now a heterogeneous group of diseases with varied ethnicities, phenotypes and treatments. In the past, Hb E-ß-thalassemia and Hb H disease were rarely seen in North America and Europe. Now, as a result of changing demographics, these disorders have become more common than classical ß-thalassemia major in many regions^{ref1,
ref2,
ref3,
ref4,
ref5}. HbH, HbH-Constant Spring and homozygous a-thalassemia affect at least a million people worldwide^ref. The natural history of these disorders is highly variable and poorly studied. The phenotype, for patients with similar mutations, can range from asymptomatic to transfusions

dependency. Ethnic groups originating from regions with malaria have a high frequency of thalassemia mutations. There are almost 300 million carriers of hemoglobin disorders in the world, with the majority living in South East Asia^{ref1,
ref2,
ref3,
ref4}. Worldwide, the Asian, Indian and Middle Eastern regions account for 95% of thalassemia births. The frequency of a-thalassemia reaches 25% in Thailand, and HbE approaches 60% in many regions of Thailand, Laos and Cambodia. Hb Constant Spring is found in 1–10% of the population in these areas. The WHO estimates that Thailand will have over 250,000 symptomatic thalassemia patients diagnosed over the next few decades^{ref1,
ref2,
ref3,
ref4,
ref5}. At least 100,000 new cases of HbE-ß-thalassemia are expected. Similar estimates are predicted for India, Sri Lanka and Malaysia^ref. In southern China, with a population of over 350 million, approximately 5% are carriers for a-thalassemia and 4% for ß-thalassemia or HbE^ref. In the last decade, 75% of immigrants to North America came from areas where thalassemia mutations were prevalent. These demographic changes have resulted in thalassemia becoming a more significant health problem in North America. Hb E-ß-thalassemia and a-thalassemia disorders are now the most common thalassemias in California. In response to the high prevalence of -thalassemia and its associated morbidity, California has initiated universal newborn screening for HbH and HbH-Constant Spring^ref. Characterizing the molecular defects underlying and ß thalassemia has transformed the management of these conditions in many developed countries. Over the past 25 years, throughout Europe, Australasia and North America comprehensive control programs involving education, counselling and pre-natal diagnosis have succeeded in limiting the numbers of new births of affected individuals. One of the best-documented examples of this approach has been in Cyprus where thalassemia was first recognized in the 1940s following the eradication of malaria. With a population size of ~700,000 and a carrier frequency for ß thalassemia of 15–17%, in the early 1970s it was estimated that to treat all severely affected children would require 78,000 units of blood per year, 40% of the population would be donors and the total cost to the health service would exceed the island’s health budget^ref. With judicious application of a control program there are now fewer than 600 severely affected patients with thalassemia in Cyprus and only 2-3 new cases arising each year. A similar highly effective control program was also established by pioneering work in Sardinia^ref. In First World countries the outlook for the relatively small numbers of patients with ß thalassemia major and intermedia has also dramatically improved over the last 25 years.
Pathogenesis : uneffective erythropoiesis : decreased or no synthesis of one or more kinds of globin => precipitation of the remaining kinds => peripheral hemolysis / hemocatheresis. Complicated by parvovirus B₁₉

infection

a-thalassemias => HbH (b₄)

Epidemiology

^{ref1,
ref2,
ref3,
ref4}

^ref1,
ref2

Aetiology :

a⁰-thalassemia : a₂and a₁ deletion => -- locus

heterozygous : only 2 a genes (--/a₂a₁) : a-thalassemia minor
homozygous : no a gene (--/--) : hydrops foetalis ^ref

80% Hb Bart (g₄) in utero has an extremely high oxygen affinity and therefore delivers little oxygen to fetal tissues. The severe hypoxia results in cardiac failure, massive ascites and intrauterine death^ref. 5 cases survived beyond the newborn period, all with hypospadias secondary to the in utero edema leading to failure of fusion of urogenital folds or due to defect or deletion of another gene at 16p13.3^ref.
20% Hb Portland (z₂g₂)

Symptoms & signs

hypospadias

Laboratory examinations :

chorionic villus sampling (CVS)

Therapy

transfusions

transfusion

allogeneic hematopoietic stem cell transplantation

^ref1,
ref2

tea

a⁺-thalassemia : a₂ or a₁ down-regulation,

mutation

Hb Constant Spring (142 stop => Gln => 31 extra amino acid residues at the C terminus of the a chain). Hb CS^{ref1,
ref2,
ref3,
ref4,
ref5} is the most common nondeletional a-thalassemia mutation associated with Hb H disease. The gene frequency of Hb CS approaches 8% in Southeast Asia. Its mRNA is highly unstable, producing < 1% of the total protein output of a normal gene. Cells containing Hb CS become overhydrated, modifying the expected microcytosis. In comparison with cells from patients with deletional HbH disease, cells containing Hb CS have increased hemoglobin bound to the membrane, increasing the rate of hemolysis^ref (Styles LA. Hemoglobin H-Constant Spring disease: an under recognized, severe form of thalassemia. J Pediatr Hematol/Oncol. 1997;4:69–74). Hb H/CS disease has significantly more ineffective erythropoiesis and erythroid apoptosis than HbH disease^ref. The laboratory and clinical course of Hb H/CS disease is more severe than Hb H disease^{ref1,
ref2,
ref3,
ref4}.
hemoglobin G : any of various abnormal hemoglobins with an amino acid substitution on the a chain

the most common one is hemoglobin G Philadelphia, which causes a-thalassemia .

hemoglobin I : an abnormal hemoglobin resulting from an amino acid substitution in the a chain, causing a-thalassemia .

deletion of a₁ or a₂ genes => a₂- or a₁- locus

heterozygous : only 3 a genes (-a₁/a₂a₁ or a₂-/a₂a₁) : silent carrier (normal MCV)
homozygous : only 2 a genes (-a₁/-a₁ or a₂-/-a₁ or a₂-/a₂-) : a-thalassemia minor

Epidemiology

⁺

^ref

Symptoms & signs

splenomegaly

hydrops foetalis

^ref

Laboratory examinations

Therapy

splenectomy

portal vein thrombosis

transfusions

a⁰-a⁺-thalassemia : 1 only a gene (a₂-/-- or -a₁/--) => hemoglobin H disease (b₄)

Aetiology :

deletions on chromosome 16 (75%) cause a milder form of the disorder
2 deletions + a point mutation or insertion in the a globin gene (25%). Non-deletional HbH is often severe and likely to require RBC transfusions ^{ref1,
ref2,
ref3} (Styles LA. Hemoglobin H-Constant Spring disease: an under recognized, severe form of thalassemia. J Pediatr Hematol/Oncol. 1997;4:69–74)

Symptoms & signs :

splenomegaly

Laboratory examinations

hypochromia

Pathogenesis :

Symptoms & signs :

transfusions

Laboratory examinations

^ref

Therapy

b-thalassemias

Epidemiology

Aetiology :

Pathogenesis :

EPO

heterozygous (thalassemia minor) : increased levels of HbA₂ (5-7%), which is a result of increased binding of a-globin to d-globin, decreased levels of HbA (93-95%), and normal levels of HbF (< 2%)
homozygous (thalassemia maior or Cooley disease) : increase in [HbF]

Symptoms & signs

mongoloid facies

brush skull

b⁰-thalassemia(no expression of b-globin)

homozygous : b-thalassemia major or Cooley disease

associated with defects causing increased synthesis of g chains : intermediate b-thalassemia (Hb 6-9 g/dl)

heterozygous : b-thalassemia minor or minima (Hb > 9 g/dl)

b⁺-thalassemia(lower-than-normal expression of b-globin)

black type

homozygous : intermediate b-thalassemia (Hb 6-9 g/dl; 30÷50% HbA, 50÷70% HbF)
heterozygous : b-thalassemia minor or minima (Hb > 9 g/dl; HbA₂increase)

Mediterranean type

homozygous : b-thalassemia major or Cooley disease (10÷20% HbA)
heterozygous : b-thalassemia minor or minima or Rietti-Greppi-Micheli syndrome (Hb > 9 g/dl)

b-thalassemia with normal HbA₂

type 1

homozygous : intermediate b-thalassemia (Hb 6-9 g/dl)
heterozygous : b-thalassemia minima (Hb > 9 g/dl; normal HbA₂)

type 2

homozygous : unknown
heterozygous : thalassemia minor or Rietti-Greppi-Micheli syndrome (Hb > 9 g/dl; normal HbA₂)

^ref

cis

trans

^{ref1,
ref2,
ref3}

^ref1,
ref2

^ref

^ref

cis

^ref

In vitro

in vivo

^ref

cis

^{ref1,
ref2,
ref3,
ref4,
ref5}

in vivo

^ref

in vivo

^ref1,
ref2

^ref

cis

^ref

	a cluster 16p13.3 telomeric	ß cluster 11p15.5 interstitial
GC content	54%	39.5%
CpG islands	common	none
gene density	high	low
Alu family repeats	25%	5%
LINE repeats	rare	present
chromatin	open	closed => open
replication timing	early	late => early
matrix attachment sites	none detected	common
effect of ATRX mutations	present	absent
regulatory element	enhanced	LCR
effect of deletion of major regulatory element on long range chromatin structure	no	yes
predominant mutations	deletions	point mutations
evolution of intergenic regions	rapid	slow
expression in transient assays	enhancer independent	enhancer dependent
expression in hybrids	early	late

^ref

caused by mutations in trans-acting factors

in one family, a mutation affecting the key erythroid transcription factor GATA-1 has been shown to cause thrombocytopenia and ß thalassemia^ref. Subsequently, other families with similar mutations in GATA-1 causing ß thalassemia have been recognized. It has been suggested that differences in the types of GATA-1 binding sites present in the a and ß clusters may explain why this trans-acting mutation has different effects on expression of the 2 clusters^ref.
the second example of a trans-acting mutation causing ß thalassemia has been characterized in a group of 19 patients with a rare autosomal recessive disorder called trichothiodystrophy (TTD) characterized by dry photosensitive skin, brittle hair, short stature and variable degrees of mental retardation. These individuals inherit mutations in one protein (XPD helicase) of the multicomponent, general transcription factor TFIIH^ref (Viprakasit V. The regulation of a and ß globin gene expression. Weatherall Institute of Molecular Medicine. Oxford: Oxford University; 2002). To date, all of these patients have the hematologic features of ß thalassemia trait. The simplest explanation for this is that the and ß promoters have different requirements for the activity of TFIIH in initiating transcription during erythropoiesis but the details of this are currently unknown.
there are several other families with ß thalassemia in whom no mutations have been detected in the genes or their regulatory elements indicating that other trans-acting mutations remain to be found^ref1,
ref2.
in addition, it has been shown that there are genetic modifiers of a globin expression which are unlinked to the ß globin cluster. These modifiers have been linked to chromosomes 6, 8 and X but the identity of these genes and their function are unknown^ref1,
ref2 (Garner C, Silver N, Best S, et al. A quantitative trait locus on chromosome 8q influences the switch from fetal to adult hemoglobin. Blood. 2004).

cis

a-thalassemia/mental retardation syndrome, nondeletional type, X-linked (ATR-X)

a thalassemia myelodysplasia syndrome (ATMDS)

trans

^ref

a thalassemia/mental retardation syndrome, deletion-type on chromosome 16, ATR-16 syndrome

a-thalassemia/mental retardation syndrome, nondeletional type, X-linked (ATR-X)

trans

^ref1,
ref2

de novo

^ref1,
ref2

^ref

in vitro

^ref

^{ref1,
ref2,
ref3}

^ref

hematologic malignancy

^ref

₄

^ref1,
ref2

registry

myelodysplastic syndrome (MDS)

a thalassemia myelodysplasia syndrome (ATMDS)

^ref1,
ref2

trans

^ref

^ref

^{ref1,
ref2,
ref3}

per se

Prevention :

preimplantation genetic diagnosis (PGD)

Therapy

^ref1,
ref2

packed red cell transfusion (PRCT) & iron-chelating drugs ^ref1,
ref2 : in the last 30 years, conventional treatment of ß-thalassemia major, based primarily on regular blood transfusions and iron chelation therapy with desferrioxamine (DFO), has markedly improved the prognosis of the disease. Adequate administration of parenteral DFO reduces or prevents iron accumulation and iron-mediated organ damage, resulting in a consistent decrease of morbidity and mortality (Borgna-Pignatti C, Rugolotto S, De Stefano P, et al. Survival and disease complications in thalassemia major [abstract]. Cooley’s Anemia-Seventh Symposium. 1998;850:227). However, for several reasons chelation with DFO may be inadequate. In emerging Middle Eastern and Far Eastern countries, where thalassemia is a substantial public health problem, the high costs of the drug and of the supplies for its administration make it unavailable for most patients. Worldwide, it has been estimated that DFO is prescribed for only 25,000 of 72,000 patients with thalassemia major who are regularly transfused (Beutler E, Hoffbrand AV, Cook JD. Iron deficiency and overload. Hematology (Am Soc Hematol Educ Program). 2003:46–52). Despite wide availability of DFO in Western countries, some patients are unable to comply sufficiently with the prescribed treatment because the effective chelation regimen is unpleasant and cumbersome, requiring prolonged daily subcutaneous or intravenous administration. Moreover, some patients may develop side effects of variable severity that affect compliance or sometimes result in cessation of treatment. In a recent report, 105 of 328 patients from North America who had ever received chelation therapy with DFO reported complications requiring modifications of the dose or route of administration of the chelator and 20 reported stopping DFO^ref.3 The inability to cope with the rigorous and demanding long-term use of DFO may result in death from iron overload. The unavailability of DFO for most patients with thalassemia major and the failure of prescribed therapy to prevent complications in other patients have led to a search for alternative iron chelators. Over the last 25 years > 1000 molecules of synthetic, microbiological or plant origin have been tested in vitro and in vivo and several interesting compounds have been identified. One of them, deferiprone (DFP), has been commercially available for 9 years in India and 5 years in Europe, while others are under intensive clinical investigation. Iron accumulates at different rates in various organs, and these organs show a different susceptibility to the damage induced by reactive iron species such as nontransferrin bound iron (NTBI) and the intracellular labile iron pool (LIP). More work is needed to understand the relative accessibility of iron chelators to those different pools. Methods for measurement of these iron species are becoming available and more sensitive, and they could improve the understanding of the mechanism of action of the various chelators and facilitate the design of specific chelation strategies^ref. Based on these findings, targeted chelators that remove iron from specific organs could prove useful. Therefore, besides the overall reduction of iron, effective chelation should aim for a decrease of iron in specific organs, especially the heart, since cardiac disease is the most important life-limiting consequence of iron overload in thalassemia (Borgna-Pignatti C, Rugolotto S, De Stefano P, et al. Survival and disease complications in thalassemia major [abstract]. Cooley’s Anemia-Seventh Symposium. 1998;850:227). During the last 25 years, investigators have made great strides in developing new iron chelators for the treatment of iron overload in thalassemia. Many candidate drugs have been screened, but only a few have had the physicochemical and biological properties suitable for potential clinical application. Some of these are now under intensive investigation. Patients may ultimately benefit from having a choice between several chelators, including orally active drugs. New strategies of chelation, such as combination therapy and organ-targeted chelation, may soon have a considerable impact on the therapeutic outcome and quality of life of patients with thalassemia.

Complications

iron overload

^ref

clinical presentation : the cardiac complications of thalassemia major were first described prior to the introduction of chelation therapy. Engle described an increase in heart size after 10 years of age with first-degree heart block in a third of the patients. Episodes of pericarditis occurred in half of the patients after 11 years of age. Congestive cardiac failure developed at a mean age of 16 years. As heart failure progressed, atrioventricular (AV) block worsened and either complete heart block or right or left bundle branch block occurred in a third of patients. Atrial arrhythmias were noted in half and repetitive ventricular tachycardia was present in a minority. The duration of life after the onset of heart failure was < 3 months in over half of the patients. Currently, cardiac complications are reported to cause 71% of deaths in patients with thalassemia major. Therefore the heart is the target lethal organ in thalassemia. Once heart failure develops, the outlook is usually poor. The cardiomyopathy may be reversible if iron chelation treatment is intensified in time, but the diagnosis is often delayed by the unpredictability of cardiac iron deposition and the late development of symptoms and echocardiographic abnormalities. The early diagnosis of iron-induced cardiomyopathy with established clinical techniques such as echocardiography and stress radionuclide angiography has had limited success. In particular, overt dysfunction on echocardiography presents late in the disease process and the progression from mildly abnormal echocardiographic parameters to fulminant cardiac failure is often rapid and relentless.
pathophysiology : iron deposition is reported to be greatest in the ventricular walls with less in the atria and conduction system. There is also greater iron in the epicardial layer. The degree of cardiac dysfunction is considered to depend on the quantity of iron deposited in individual myocardial fibers and the number of fibers affected. In patients with only mild cardiac dysfunction, iron deposits are usually limited to the perinuclear areas, with only a few fibers involved, whereas in patients with significant cardiac dysfunction, iron deposits occupy large areas of the myocardial fibers. The relatively mild degree of fibrosis in most autopsy studies suggests that even when advanced, the cardiomyopathy is potentially reversible. The iron is stored in intracellular lysosomes in the relatively non-toxic forms of ferritin or hemosiderin but above a certain concentration, NTBI is released and the cell begins to fail. The toxicity of iron depends on intracellular iron concentration and its oxidative state. Reduction of ferric to ferrous iron promotes hydroxyl radical formation via the Haber-Weiss reaction.
iron chelation therapy and the reversibility of iron-induced cardiomyopathy : the iron chelator DFO was first introduced > 30 years ago. DFO reduces hepatic iron and the progression of hepatic fibrosis. Subsequent studies showed improved survival in thalassemia patients on long-term chelation therapy. However, many patients still die of iron overload, and this continued early mortality may be due to several factors: late presentation of cardiac disease, difficulties in assessing myocardial iron, and difficulties in compliance to long-term treatment with daily parenteral iron chelation regimens. The reversibility of iron-induced cardiomyopathy has been demonstrated in patients with hereditary hemochromatosis treated with recurrent venesection and in patients with thalassemia major who are treated with chelation therapy. In vitro, DFO removes iron from cardiac myocytes, reverses lipid peroxidation, and reverses the iron-induced abnormalities of cellular contractility and rhythmicity. However, the clinical reversibility of iron-induced cardiac failure has not been universally accepted, partly due to the high mortality of patients presenting with advanced cardiac failure despite chelation treatment, and more recently by alternative explanations for the heart failure such as myocarditis.
assessment of tissue iron stores : there is variability in iron deposition between and within different organs. Serum ferritin is the most commonly used indirect estimate of body iron stores, but this reflects only 1% of the total iron storage pool. In addition, interpretation of ferritin values may be complicated by a variety of conditions. Reliance on serum ferritin alone can lead to an inaccurate assessment of body iron stores in individual patients. The hepatic iron concentration by liver biopsy is considered the most accurate and sensitive method for determining the body iron burden. However, this is an invasive technique with a low but recognized complication rate; the result is affected by hepatic fibrosis (especially in small biopsies), which is common in thalassemia as a result of increased liver iron and HCV infection; and iron has been shown to be unevenly distributed in the thalassemic liver even in the noncirrhotic stages. Endomyocardial biopsy can evaluate iron deposition in the heart in thalassemia, but is not a routine part of patient management due to the invasiveness of the technique and the heterogeneous deposition of iron in the heart. In addition, iron has been reported to be absent from the right ventricular subendocardium in some patients with cardiac iron overload. The relation between myocardial iron and other measures such as serum ferritin and liver iron has been unknown until recently, and therefore assessing cardiac risk from these other measures has at best been uncertain. There are noninvasive techniques that can assess tissue iron. Both magnetic resonance (MR) and magnetic susceptometry are significantly affected by iron, and both show changes in iron overload. However, to date only MR can be applied to a moving organ such as the heart. Fortunately there has been substantial progress in the speed and image quality of cardiovascular MR in recent years, and therefore we investigated its use in assessing myocardial iron in thalassemia.

choice of MR technique for measuring myocardial iron :

signal intensity ratios : there are reports of the use of signal intensity ratios for the assessment of tissue iron levels, particularly the liver, but rather less so in the heart^ref. These measurements are semi-quantitative because while they rely on alterations in relaxation time caused by iron, they effectively relate the relaxation of one tissue of interest to another such as skeletal muscle, which it is assumed has a low propensity for iron uptake and therefore acts as a reference tissue. Attempts to derive absolute myocardial iron concentration using this approach have relied on tissue calibration curves from liver and the use of an empiric correction factor related to hearts with normal iron levels^ref. Reproducing the results between scanners is very difficult when imaging parameters vary even slightly, and this makes it difficult to compare results between centers. The multiple assumptions and problems associated with signal intensity ratio measurements limit their value and suggest that direct measurement of myocardial relaxation would have significant potential advantages.
T2 : on first exploring the optimal way to determine myocardial relaxation, we attempted to measure myocardial T2, a measure which in animals has been shown to reflect iron levels^ref. This requires the use of spin echo cardiac magnetic resonance (CMR) techniques, but in our experience with the techniques routinely available in the late 1990s for human in vivo use, these were not sufficiently reliable or reproducible. For example, there was marked motion artifact in noncompliant patients. Some groups have pursued myocardial T2 measurements^ref. Although there are now more advanced T2 imaging techniques, we have focused on assessing myocardial T2* measurement^ref. T2* is measured from gradient echo images, which are fast and easy to acquire, are robust to motion for CMR, and in principle are more sensitive to iron levels than T2. In addition, adjustment of the echo time within the sequence to build up a signal decay curve at multiple echo times that were short enough for moderate or severe iron overload was significantly easier using gradient echo techniques.
T2* : the principle of using T2* relaxation measurements to determine tissue iron is simple. As the echo time of the images is increased, the signal intensity of all tissues decreases and this rate of decay is strongly enhanced in the presence of iron deposition. Thus, increased myocardial iron reduces myocardial T2*. The key issue in evaluating myocardial T2* was to use echo times that allowed reproducible measurements. Initial experiments showed that echo times from approximately 4 to 20 ms separated by 2 ms increments allowed the best assessment of T2* in mild to severe iron overload. Patients with higher T2* values would have decreased measurement reproducibility, but we had already established that such patients had normal myocardial iron and therefore would not require intensive follow-up to monitor response to a change in treatment. In our first studies, where we were unable to control the sequence repeat time, we chose a low flip angle to minimize T1 effects that can cause errors. The scan resolution was fixed to allow typical breath-hold times per acquisition of 8–10 seconds, which is suitable for children as well as adults. In our early studies, one breath-hold acquisition was required per single image, which had a single echo time. We then acquired up to 9 images, each at a lengthening echo time. A fixed gating delay of 0 ms after the R-wave was chosen in order to obtain myocardial images in a consistent position in the cardiac cycle irrespective of the heart rate. The resulting image is therefore acquired in early systole. An end-systolic image would have offered the benefits of decreased wall motion and thicker ventricular myocardium in which to draw the region of interest, but the delay time would require alteration to end-systole for each patient and also between breath-holds if there was any variation in the patient’s heart rate. A short axis mid-ventricular slice was chosen to minimize artifacts from blood flow. The region of interest was drawn in the ventricular septum, encompassing all layers of the myocardium from the right ventricle through to the left ventricle (LV) endocardium. The region was drawn away from the cardiac veins and the lungs, which cause susceptibility artifacts. More recently, we have developed a single breath hold technique (15 seconds) that acquires all the images at lengthening echo times^ref. This has advantages of speed of acquisition and excellent registration, such that the heart position is constant between images of different echo times, which improves the ease of quantitative analysis. To calculate the myocardial T2*, the signal intensity of the myocardial region of interest was measured in each of the nine images using in-house software (CMRtools, Cardiovascular Imaging Solutions, London, UK). The value was plotted against the echo time for each image. A trendline was fitted to the resulting exponential decay curve, with an equation of the form y = Ke^–TE/T2* where K represents a constant, TE represents the echo time and y represents the image signal intensity.

normal ranges for T2* relaxation times : to determine normal ranges for T2* values in the heart we scanned 15 healthy volunteers. The normal value for the myocardium was 52 ± 16 ms. There is limited literature with which to compare these results. Previous reports of myocardial T2* values using a variety of echo times include values of 33 ± 6.5 ms, 48 ± 9 ms and 38 ± 6 ms. Importantly, however, the lower limit of normal (defined as the mean minus 2 standard deviations) with our technique was 20 ms, which is very similar to that of the other reports. Therefore, the lower boundary limit we have adopted for myocardial T2* is 20 ms, and values below this represent iron overload. No other clinical scenarios other than iron overload have been shown to cause myocardial T2* below this value.
calibration of the T2* technique : to investigate the relationship between tissue iron and T2* relaxation, we prospectively studied 30 patients undergoing liver biopsy. The biopsy iron concentrations were compared with the liver T2* measurements. We found a significant, curvilinear, inverse correlation between liver T2* and the liver iron content for all samples (r = 0.81, Figure 2). As predicted from the known variability of biopsy iron determination in the presence of hepatic fibrosis, there was a better correlation with the non-fibrotic liver samples (r = 0.93, P < 0.0001) than the fibrotic samples (r = 0.68, P < 0.0001). Therefore, we subsequently employed nonfibrotic samples to generate predictions of liver iron content from the measured T2* values. Log transformation for the nonparametric data showed a linear relation between liver iron and liver T2*, confirming that tissue T2* can be used as a quantitative measure of tissue iron. These data confirm that tissue T2* is sensitive to tissue iron levels, but they do not provide calibration for myocardium.
reproducibility of T2* measurements : in order for the T2* sequence to be suitable for the clinical assessment of tissue iron, good reproducibility of the technique is required. Therefore, patients with thalassemia major were scanned on two occasions to assess the interstudy reproducibility of the T2* technique. The coefficient of variation (SD of differences divided by the mean) was found to be 3.3% for the liver and 5.0% for the heart^ref. Reproducibility between scanners was also shown to be very good at 9.4%^ref. To assess intercenter reproducibility, patient scans were performed in 2 different countries and the patients rescanned in a center in London (Westwood MA. Cardiovascular magnetic resonance in myocardial siderosis. MD Thesis. Imperial College, London. 2004). The myocardial reproducibility in each country was 2.4% and 3.5%. Finally, myocardial reproducibility of the single breath-hold technique was 2.3% for abnormal T2* values^ref. All these reproducibility values were considered appropriate for clinical application. The improved reproducibility of these gradient-echo T2* techniques over the fast-spin-echo and spin-echo techniques results principally from improved quality of the original images from which T2* is measured.
myocardial T2*, cardiac function and normality : LV function was shown to fail in association with myocardial T2* values below the normal range (< 20 ms). As myocardial iron increased, the LV dilated, ejection fraction fell and mass increased^ref. Importantly, no patient was observed to have heart failure who did not have a low myocardial T2* unless there was an obvious alternative cause, such as congenital heart disease. Another notable finding in this cohort study was that in patients with normal myocardial T2* the ejection fraction was usually in the high range for normal subjects. Because of the anemia in these patients, it is possible that to compare their cardiac volumes and other parameters against normal ranges for non-anemic subjects could lead to a significant underestimation of the number of thalassemia patients recognized as having the early stages of LV impairment. We therefore studied a further cohort of thalassemia patients and sought to determine the normal ranges for LV function in those patients in whom myocardial iron loading could be confidently excluded by the presence of a myocardial T2* which was greater than 20 ms (Westwood MA. Cardiovascular magnetic resonance in myocardial siderosis. MD Thesis. Imperial College, London. 2004). This study showed a significant difference for normal LV parameters between the published normal ranges and "normal function" in thalassemia. The latter group had increased LV end-diastolic volume, ejection fraction and mass with reduced end-systolic volume in comparison with normal subjects. These findings confirm that great care must be taken to interpret LV function parameters in thalassemia if significant misdiagnosis of the presence of early cardiomyopathy is not to occur. Further analysis of other parameters of cardiac function has also been related to myocardial T2*. Diastolic function is significantly affected by T2* in the abnormal range of < 20 ms, and there is a progressive increase in the ratio of early to late peak filling rate (Westwood MA. Cardiovascular magnetic resonance in myocardial siderosis. MD Thesis. Imperial College, London. 2004). However, the early detection of cardiomyopathy using diastolic function is still relatively poor when compared with the more direct measure of myocardial T2*. There is also a strong inverse relation between right ventricular function and myocardial T2*, indicating that myocardial iron is responsible for bi-ventricular dysfunction (Anderson LJ. Assessment of cardiac iron overload in thalassaemia. MD Thesis. Imperial College, London. 2002)
validation of the T2* technique : some have questioned whether the myocardial T2* technique has been adequately validated. In brief, a technique can be validated in relation to established clinical parameters known to reflect a disease process^{ref1,
ref2,
ref3,
ref4}. As described above, there are important relationships between T2* in the range below normal and the remodeling of heart failure, which is the classic response of the heart in cardiomyopathy. There is no doubt, therefore, that since nothing is known to reduce T2* to abnormal levels other than iron loading, iron affects T2*, and T2* is related, in turn, to cardiomyopathy. Therefore, myocardial T2* is validated clinically against cardiac function. What has not yet been achieved is calibration of myocardial T2* against absolute myocardial iron levels. In discussion of whether absolute calibration is vital, the question is whether it is more important to know the relationship between myocardial T2* and cardiac function or between myocardial T2* and directly measured myocardial iron levels. The former is vital, while the latter has additional scientific merit. To date, the myocardial T2* technique can be considered validated but not yet calibrated.
relation of cardiac iron to liver iron and serum ferritin : no significant correlation could be found between liver and heart T2* in a large cohort (r = 0.15, P = 0.11).5 This lack of correlation has been confirmed by Wood et al^ref. Similarly, no significant correlation was found between heart T2* and serum ferritin level at the time of the scan (r = 0.10, P = 0.32). To confirm that this finding was not due to spurious individual ferritin readings, the mean ferritin for 12 months prior to the scan was also compared to heart T2*, and once again there was no significant correlation (r = 0.09, P = 0.35). Wood found a weak correlation between myocardial T2* and ferritin^ref. This strongly suggests that assessment of myocardial iron from the liver iron is unreliable, and that from ferritin is not ideal.
relation of cardiac function to liver iron and serum ferritin : in a cohort study of 205 patients, there was no significant difference in left ventricular ejection fraction in patients with liver iron above 15 mg/g (left ventricular ejection fraction [LVEF] 66.1 ± 6.3%) or below 15 mg/g (LVEF 66.2 ± 9.0%, P = 0.96) or with serum ferritin levels above 2500 mg/L (LVEF 65.1 ± 10.1) or below 2500 mg/L (LVEF 66.6 ± 8.2, P = 0.22).9 No significant correlation could be demonstrated between liver iron and LVEF (r = 0.13, P = 0.07) or serum ferritin and LVEF (r = 0.08, P = 0.27) in the cohort of 205 patients. These findings strongly suggest that assessment of the risk of heart failure from either the liver iron or serum ferritin alone is unreliable
reversibility of cardiac iron in heart failure : a study was performed of 7 thalassemia patients presenting with heart failure who were converted to intravenous DFO treatment, with follow-up over 12 months^ref. One patient died. The survivors showed improved LV function and improved myocardial T2*. This rare prospective study in this condition clearly demonstrates that aggressive iron chelation with DFO is effective in patients with heart failure, and that myocardial T2* improves in concert with parameters of LV function. Of great interest, however, was the clearance rate of iron from the heart, which was substantially slower than from the liver (5.0 ± 3.3% vs 39 ± 23% per month, P = 0.02). Excess myocardial iron persisted in all patients at 1 year, despite liver iron normalization in 4 patients. This suggests that monitoring the duration of treatment by liver iron alone would be misleading in guiding the risk from myocardial iron loading, as the liver would be clear of iron loading very much earlier (by tens of months) than the heart.
cross-sectional study of cardiac iron between deferiprone and desferrioxamine : a cross-sectional study was performed comparing myocardial T2* and LV function between 2 cohorts of thalassemia patients: one treated with standard DFO therapy, and one treated with DFP^ref. The median myocardial T2* was significantly higher in the DFP group, and their ejection fraction was also significantly better, suggesting less cardiac iron and better ventricular function than in patients treated with DFO. The liver T2* was higher in the DFO group, suggesting more rapid removal of iron from this organ than with DFP. These data require prospective confirmation in randomized controlled trials (these are now underway and will be reported in late 2004 or 2005), but the current data suggest that oral DFP may have preferential access to the heart, in which case it has a potentially unique and valuable role in the treatment of iron overload.

allogeneic HSCT in pediatric age is curative and, in the largest reported series, has been carried out in over 800 patients. The proportion of matched sibling donors is a limiting factor and the procedure is associated with an overall mortality of around 20%, the success depending largely on the clinical well being of the patient prior to transplantation^ref. Gaucher-like cells after HSCT^ref. A prompt graft-versus-thalassemia effect upon withdrawal of cyclosporine A in a child who received allogeneic peripheral blood stem cell transplantation^ref
gene therapy : hemoglobin disorders were among the first diseases for which the gene therapy was envisioned. However, the complexities of the globin gene expression and regulation made this a formidable task. Making gene therapy a therapeutic option in ß-thalassemia requires:

efficient, safe and high-level gene transfer into target hematopoietic stem cells (HSCs)
regulated erythroid lineage-specific expression
therapeutic levels of ß-globin gene expression

₂

in vivo

high level of globin gene expression requires distal locus control elements: Expression of human ß-globin gene driven by an internal ß-globin promoter within a retroviral vector is very low^ref. A high-level regulated globin gene expression requires presence of a series of distal control DNAse hypersensitive sites (HS) in the 21 kb of DNA upstream from the ß-globin locus, the LCR, that plays a role in maintaining the ß-globin gene locus in an open configuration to allow for high level expression of the globin genes^{ref1,
ref2,
ref3}
the LCR is too large to be incorporated into oncoretroviral vectors; therefore, small LCR fragments, consisting of 200–300 bp fragments from the HS, containing only the core sequences have been used. However, inclusion of the core LCR elements into Moloney leukemia virus (MLV)-based oncoretroviral vectors leads to low levels of expression that is prone to position effects^{ref1,
ref2,
ref3}. Inclusion of larger LCR elements in transgenic mice results in position-independent, copy number-dependent, high-level transgene expression^ref. However, due to the size constraints of MLV vectors, individual HS site cores or a few combinations of these have been tested in MLV vectors with variable, position-dependent expression^{ref1,
ref2,
ref3}
oncoretroviral long terminal repeats (LTRs) transcriptionally interfere with LCR elements within MLV vectors, resulting in unstable proviral transmission and/or poor transgene expression^ref1,
ref2. Transcriptional interference from the LTR^ref can be overcome by self-inactivating (SIN) vectors. In SIN vectors, the LTR promoter/enhancers are deleted upon integration of the provirus. However, SIN MLV vectors have a loss in titers resulting from the LTR deletion. Replacement of the viral LTR with enhancers from other erythroid genes (the GATA-1^ref, or HS-40^ref1,
ref2, the distal control element from the -globin locus) has met with reasonable success
ß-globin is an intron-dependent gene that destabilizes oncoretroviral vectors, if placed in antisense orientation: Retroviral vectors are limited to incorporating cDNA, since during production of the viral RNA genome, vector plasmids containing introns would be spliced. This becomes a serious handicap in expressing intron-dependent genes, such as ß-globin. The ß-globin cDNA is very poorly expressed, as ß-globin introns contain elements that enhance transcription, allow proper 3' end processing and the nuclear export of transcripts^ref1,
ref2. The ß-globin gene is, therefore, inserted in reverse orientation relative to the open reading frame of the viral LTR transcript. This results in vector instability, as several splice sites and polyadenylation signals are introduced into the open reading frame of the vector transcript
measures taken to overcome instability of globin-containing retroviral vectors:

deletion of a 372-bp fragment of the intron-2 of the ß-globin gene^ref1,
ref2, which contains most of the ‘instability elements,’ helps stabilize the viral genomic RNA
Leboulch et al have mutated numerous deleterious sequences in an MLV vector containing the core LCR elements HS2, 3 and 4 driving expression of ß-globin, showed stable transmission in the majority of mice, but low and variable expression^ref
improve expression from the ß-globin cDNA, by including viral elements that would replace the functions of the globin introns and show that the woodchuck hepatitis virus post-transcriptional regulatory elements improves expression by 5- to 10-fold (Jiang G, Perelman N, Xu D et al. The woodchuck hepatitis virus post-transcriptional regulatory element greatly increases titers from retroviral vectors and improves expression of the beta globin cDNA. Blood. 1999;94:176a), although levels of ß-globin expression were still subtherapeutic. In summary, efforts to impart stability to the ß-globin mRNA in MLV vectors by modifying various ‘instability elements’ and adding RNA export element vastly improved upon previously reported results, but were still suboptimal to be of therapeutic benefit.

^ref

can enter a fully intact nuclear membrane and integrate into non-dividing HSC chromatin without prestimulation with cytokines
are able to package full-length, unspliced RNA due to the presence of a strong RNA export element, the rev response element (RRE). In this regard, May et al have shown that ß-globin gene cassettes that were unstable in MLV vectors were stably transmitted in LV vectors^ref
can hold a much larger cargo (~9–10 kb) than oncoretroviral MLV vectors, allowing for the larger LCR elements to be incorporated into these vectors. LV vectors, therefore, were able to overcome the hurdles encountered with MLV vectors.

^ref

^ref1,
ref2

^ref

Erythroid-specific expression from SIN lentiviral vectors

^ref1,
ref2

^ref

Genetic correction of ß-thalassemia intermedia phenotype with lentiviral vectors

^th3/+

⁰

^ref

^th3/+

^ref

^ref1,
ref2

	transgene	rise in Hb/vector copy	summary of findings
May et al^ref	ß-globin	3.8 gm	correction of anemia in thalassemia intermedia mice
Imren et al^ref	ß⁸⁷-globin	1.5 gm	correction of anemia in thalassemia intermedia mice
Rivella et al^ref	ß-globin	2.3 gm	rescue of lethality in a thalassemia major mouse model
Persons et al^ref	g-globin	1.1 gm	correction of anemia in thalassemia intermedia mice
Hanawa et al^ref	g-globin	2.0 gm	correction of anemia in thalassemia intermedia mice reduced position effects
Imren et al^ref	ß⁸⁷-globin	—	high level expression of an antisickling ß⁸⁷ globin in human erythroid cells derived from cord blood progenitors in immune deficient mice integration of vector near potential oncogenes
Puthenveetil et al^ref34	ß-globin	—	correction of human ß-thalassemia major phenotype in vitro and in immune deficient mice reduced position effects

Gene therapy for thalassemia major

^ref

^{ref1,
ref2,
ref3}

^{ref1,
ref2,
ref3,
ref4}

^ref

⁺

^ref

⁺

₂

^null

^ref

Safety of randomly integrating vectors for gene therapy of ß thalassemia

^ref

^ref1,
ref2

^ref

⁺

regulated, erythroid lineage-specific expression^ref1,
ref2 restricts their expression to differentiated erythroid cells rather than stem or other hematopoietic cells
a self-inactivating (SIN) lentiviral design^{ref1,
ref2,
ref3,
ref4} renders the integrated provirus devoid of viral transcriptional elements^ref1,
ref2
insertion of the cHS4 insulator such that it flanks the integrated vector would additionally reduce position effect variegation (PEV) and have enhancer blocking effects in SIN-LV vectors^ref.

^ref1,
ref2

^{ref1,
ref2,
ref3,
ref4}

^ref1,
ref2

^ref

ex vivo

in vivo

augmentation of fetal hemoglobin (HbF) : the ß thalassemia syndromes are caused by > 175 molecular mutations affecting the ß globin gene complex, which result in decreased synthetic ratios of non-a to a globin chains, precipitation of excess unbalanced globin chains, and programmed cell death of erythroblasts early in their development^ref. It is well-established that affected patients do not become anemic until the fetal (g) globin genes are developmentally silenced, and that patients with persistent high levels of fetal globin typically have less severe anemia, have milder clinical syndromes, and are often transfusion-independent^ref. The ß thalassemias are thus one of a few clinical conditions in which a gene that is transiently expressed during fetal life can functionally replace a mutant gene normally expressed later in development^ref. Reactivation of fetal g globin expression is appealing as a therapeutic approach to the ß thalassemias, as the fetal globin genes are universally present and appropriately contextually integrated in the ß-globin locus in hematopoietic stem cells in virtually all humans. Several mechanisms for optimal correction of ß thalassemia at the molecular and cellular levels are established. 3 classes of potential therapeutic agents have been investigated in ß thalassemia syndromes:

chemotherapeutic agents

hydroxyurea improves the clinical course of patients with S/ß thalassemia and homozygous sickle cell disease^ref, although its precise mode of action is not clear. Hydroxyurea has not proved to be of similar benefit in patients with ß thalassemia due to molecular heterogeneity of the latter disease. Xmn1 and IVSII-1 (homo- and/or heterozygosis) are relevant markers in most responding patients^ref.

short-chain fatty acid derivatives (SCFADs) (of which some are histone deacetylase [HDAC] inhibitors )

butyrate compounds : a few well-documented, albeit anecdotal, cases of ß thalassemia have responded well (Steinberg MH, Forget BG, Higgs DR, Nagel RL. Disorders of Hemoglobin. Cambridge: Cambridge University Press; 2001)

everolimus ^ref
the recombinant growth factor erythropoietin (EPO)

therapeutic agent	action	thalassemia syndromes	increase in total Hb (g/dL) [range]	responses	authors
5-azacytidine	hypomethylation +/– cytotoxicity	thalassemia intermedia	2.5 [1.5–4]	1/1	Ley 1982^ref
				1/1	Dunbar 1989^ref
				3/3	Lowrey CH, Nienhuis AW. Brief report: treatment with azacitidine of patients with end-state ß-thalassemia. N Eng J Med. 1993;329:945
hydroxyurea (HU)	cytotoxicity and erythroid regeneration => high-F BFU-E	HbE/ß-thalassemia	2.0 [1–3.3]	3/3	Hajjar 1994^ref
			2.7 [2.2–3.2]	2/2 (ß globin)	Zeng 1995^ref
			0.6 [0–1.7]	11/13	Fucharoen 1996^ref
		thalassemia intermedia	1.0	3/8	Loukopoulos 1998^ref
		HbE/ß-thalassemia	0.9	7/19	Singer ST, Kuypers FA, Coates TD, et al. The effect of single and combination drug therapy on previously transfused E/beta-thalassemia patients: Implications on decision-making for therapy. Blood. 2002;100:119a–120a.
sodium phenylbutyrate (SPB)	? HDACi effect	thalassemia intermedia and major	2.1 [1.2–2.8]	4/8 untransfused	Collins AF, Pearson HA, Giardina P, McDonagh KT, Brusilow SW, Dover GJ. Oral sodium phenylbutyrate therapy in homozygous beta thalassemia: a clinical trial. Blood. 1995;85:39–43
arginine butyrate (AB) and AB + Erythropoietin (EPO)	activates g globin gene promoter, (stage selector protein(s) (SSP) binding), (? HDACi effect)	thalassemia intermedia and major	2.7 [0.6–5]	7/10 (AB +/– EPO)	Perrine 1993^ref Perrine SP, Boosalis MS, Emery DW, Castaneda SA, Bohacek RA. A pharmacophore model for screening Hb-F-inducing agents [abstract]. Blood. 2003;102:122a.
isobutyramide	activates g globin gene promoter	thalassemia intermedia	increase in HbF, reduced transfusion requirements	6/10	Cappellini, 2000^ref
isobutyramide	activates g globin gene promoter	thalassemia major		2/4	Reich 2000^ref
EPO	stimulates erythroid proliferation, promotes erythroid cell survival	thalassemia intermedia	2 [1–3]	5–7/10	Rachmilewitz 1998^ref
		thalassemia major	2.5	4/4	Bourantas 1997^ref
			2	8/10	Nisli 1996^ref
			reduced transfusion requirements	3/26	Nisli 1997^ref
				5/10	Chaidos 2004^ref
darbepoietin	stimulates erythropoiesis	thalassemia intermedia	1.6 [0.7–3.8]	4/6	Singer ST, Sweeters N, Vichinsky E, Wagner AJ, Rachmilewitz EA. A dose-finding and safety study of darbepoetin alfa (erythropoiesis stimulating protein) for the treatment of anemia in patients with thalassemia intermedia [abstract]. Blood. 2003;102:268a
HU + EPO	as above	thalassemia intermedia	1.7 [0.5–4]		Loukopoulos 1998^ref
HU + EPO	as above	HbE/ß-thalassemia	1.2	1/5	Singer ST, Kuypers FA, Coates TD, et al. The effect of single and combination drug therapy on previously transfused E/beta-thalassemia patients: Implications on decision-making for therapy. Blood. 2002;100:119a–120a
HU + SPB	as above	ß Lepore	3 [2–4]	2/2	Olivieri 1997^ref

^{ref1,
ref2,
ref3,
ref4,
ref5,
ref6}

^ref1,
ref2

^{ref1,
ref2,
ref3,
ref4,
ref5}

^ref1,
ref2

^ref

^ref1,
ref2

^{ref1,
ref2,
ref3,
ref4,
ref5,
ref6}

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ref2,
ref3,
ref4}

^{ref1,
ref2,
ref3}

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ref2

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ref2,
ref3}

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ref2

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ref2

be orally active at tolerable doses
not inhibit erythroid cell proliferation
not be mutagenic.

costs of new drug development, including the challenges of pharmaceutical companies weighing the potential market and academic investigators securing financing for drug development
perceptions regarding the ultimate efficacy (and competitiveness) of this therapeutic approach compared to other approaches.

orphan drug development requires smaller pivotal clinical trials than drug candidates for larger populations
chronic blood transfusions have significant risks that chelation cannot abrogate
thalassemia patients will require long-term treatment.

thalassemia intermedia, low HbF (< 30%) and low EPO (< 100 mU/mL), total Hb > 5 g/dL

EPO
fetal globin gene activator

thalassemia intermedia/major, high HbF (> 60%) and

high EPO (> 160 mU/mL)
total Hb > 4–5 g/dL • Fetal globin gene activator or
hydroxyurea

thalassemia major, total Hb > 4–5 g/dL

EPO
fetal globin gene activator
hypomethylating agent/HDACi (?)

^ref

synthesis of an ultra-pure compound, with identification of impurities, and validation of all methods of detection
mutagenicity testing, by three methods
formulation of the compound into a medicinal preparation
stability testing to determine shelf life and breakdown products
formal toxicology testing with dose-ranging and 28-day dosing in 2 species
cardiac toxicity screening, metabolism and distribution, and other system-specific safety pharmacology tests

splenectomy
EPO

Complications

hepatitis C virus : worldwide, no patients get more red cell products than those with thalassemia major. The life-long need for transfusion renders patients vulnerable to transfusion-transmitted viral infections. HCV has emerged as the paramount risk. HCV was identified in 1989 and serologic screening of the blood supply was initiated in 1990. Data from the Registry of the Thalassemia Clinical Research Network (TCRN) demonstrate that this screening has been successful in preventing new HCV infection via blood transfusion in younger patients with thalassemia major in North America. Only 5% of patients under age 15 (transfused since screening was available) are positive for HCV antibody or RNA, whereas the prevalence of HCV exposure in those over age 25 years is 70%^ref These results are encouraging. However, there are still hundreds of adults in North America and thousands worldwide with chronic HCV who are at risk for significant complications of liver fibrosis, cirrhosis and HCC. Additionally, in developing countries, the prevalence of HCV is as high as 63.8% in patients with thalassemia, including young patients, as shown in a recent report from the Pasteur Institute of Iran^ref. The major issues influencing the rate of HCV exposure and chronic infection in patients with thalassemia (and other patients requiring blood transfusions) are appropriate choice of donors and viral testing of donated units. Voluntary donation has been demonstrated to be a critical component in maintaining a safe blood supply. At the time of a 2000 Pan American Health Organization press release, only 5 countries in the Americas exclusively accepted volunteer donors and 16 nations tested 100% of donated units for human immunodeficiency virus (HIV), hepatitis B virus (HBV) and HCV. According to the American Association of Blood Banks, the chance of becoming infected with HCV from a blood transfusion in the United States is currently 1 per 1 million units transfused. However, the risk is much higher in other countries, so many patients with thalassemia worldwide continue to become infected with HCV. Until universal testing is a reality, treatment must be a focus for these patients. HCV infection and iron overload are risk factors for cirrhosis and HCC. Patients with thalassemia major often suffer from both of these complications. The only large survey, from 52 Italian centers, regarding HCC was reported in 2004 by Borgna-Pignatti et al.^ref 22 patients (estimated denominator of 5000) were identified with HCC. The mean age was 43 years. Only one patient had no evidence of exposure to either HCV or HBV, and 17 of 19 HCV exposed patients were HCV-RNA positive. The most common presenting symptoms were ascites, fatigue, edema, and jaundice. The survival of patients with HCC is negatively associated with size of tumor, and the authors recommended screening of HCV-RNA⁺ patients with ultrasound and serum alpha-fetoprotein (AFP) every 6 months^ref. Although there are no prospective, randomized studies of screening for HCC in patients with chronic, active HCV infection, many hepatologists perform biannual screening with serum AFP and liver ultrasound^ref. Other centers, including ours, perform annual screening. The apparent rate of spontaneous HCV clearance among North American patients with thalassemia major in the TCRN Registry was 33%,1 which exceeds the rate of 10%–20% reported in the literature but still leaves many patients chronically infected. Combination therapy with interferon and ribavirin is now considered standard treatment for patients with chronic HCV without hemolytic anemia based on a randomized, controlled clinical trial of 1121 previously untreated adults^ref. However, the product information for ribavirin still states, "patients with hemoglobinopathies should not be treated with [Rebetol] capsules or oral solution." Because hemolysis is a common side effect of ribavirin therapy, the concern in patients with transfusion-dependent thalassemia and other hemolytic anemias is that the increased iron burden and damage to the liver will outweigh the benefits of clearing the virus. This potential harm would be magnified, theoretically, in patients who fail to respond to treatment including ribavirin since they would have both increased hepatic iron and continuing infection with HCV. Interferon alone in patients with thalassemia has been reported to have a successful clearance rate of only 15–40%^ref1,
ref2. Several reports have demonstrated the safe and successful treatment of chronic HCV infection with combination therapy in patients with thalassemia major^{ref1,
ref2,
ref3}. These studies and case series demonstrate variable but substantial, sustained biochemical and virological response rates of 45.5%7 to 72.2%^ref at 6 months following completion of therapy. The increase in transfusion requirements is highly variable but may be as great as 152% over baseline^ref. The paucity of large prospective trials and the cautionary notes about use of ribavirin in the face of hemolytic anemia pose challenges for clinicians who must decide on therapy for adults with thalassemia who have chronic, active HCV infection. Until adequate data become available, we have taken an individualized approach. Whenever possible, we enroll such patients in clinical trials designed to answer the outstanding questions. We optimize chelation regimen and assess cardiac status prior to therapy. We consider treatment with PEG-interferon/ribavirin with careful monitoring of viral load, ferritin, liver enzymes, transfusion requirements, liver biopsy prior to and after 1 year of therapy and alteration of chelation regimen according to increases in transfusion requirements. Should the viral load not substantially decrease after 6 months of therapy, it is unlikely that further treatment will be effective and we would discontinue therapy.
thrombosis : in a recent review, Eldor and Rachmilewitz summarize data from many series and present compelling clinical evidence for increased risk of thrombosis in patients with not only ß-thalassemia intermedia, but also ß-thalassemia major, -thalassemia syndromes and hemoglobin E/ß-thalassemia (E/ß-thal)^ref. The difficulty in ascertaining clinical risk and determining preventive and treatment care is that many of these patients are not fully characterized in that their transfusion regimens are not known. Additionally, although in one series the incidence of deep venous thrombosis and pulmonary embolism was inversely correlated with hemoglobin levels, the details are lacking^ref. A retrospective review by Cappellini et al of 83 patients with thalassemia intermedia and 65 patients with thalassemia major demonstrated a prevalence of venous thromboembolic events (VTE) of 29% and 2%, respectively^ref. 23 of the 24 patients with thalassemia intermedia and VTE had been splenectomized. The author recommends short-term antithrombotic therapy both perioperatively and when patients are exposed to thrombotic risk factors^ref. We recommend this approach and additionally recommend low-dose daily aspirin for all of our splenectomized patients with thalassemia and also for our splenectomized patients with thalassemia major who have platelet counts in excess of 600,000/µL. Evidence of hypercoagulable pathology in patients with thalassemia syndromes : data included are from multicenter studies, retrospective autopsy reviews and observational studies. Not all studies looked for evidence of all findings, nor did all report on the presence or absence of the spleen in patients^ref

diagnosis	no. of patients	thromboembolic diagnosis	%	comments
ß-TM	138	stroke	0.02	not regularly transfused
		stroke	20	not regularly transfused
ß-TM	735	stroke	0.02	not regularly transfused
ß-TI	41	asymptomatic cerebral ischemia	37	by MRI, Incidence correlated positively w/age and negatively w/hgb
ß-TM	685	DVT or PE	3.95
ß-TI	52	DVT or PE	9.61
ß-TM	1146	DVT or PE	1.1
ß-TM	421	DVT or PE	3.3	median age 28 y, 15% congenital or acquired risk factors
ß-TI	74	DVT or PE	16.2	median age 28 y, 15% congenital or acquired risk factors
ß-TI	83	DVT, PE or portal vein thrombosis	29	11% of total with recurrent VTE, 99% splenectomized
ß-TM	33	pulm HTN	85	age range 2–24 y, suspected microthrombi
ß-TM+/-E	16	pulm HTN	94	age range 5–14 y, suspected microthrombi
ß-TM	35	pulm HTN	75
ß-TM/E	43	pulm arteriolar organized thrombi	19	autopsy findings, 17 splenectomized
ß-TM	2	pulm arteriolar platelet microthrombi	2	autopsy findings

^{ref1,
ref2,
ref3}

^ref

₂

^ref

hypogonadotrophic hypogonadism is common in young adults with thalassemia major and is thought to contribute to low fertility in this population. In addition, endocrinologic and cardiac complications of iron overload may cause pregnancy complications in women with thalassemia. Nevertheless, case reports and small published series demonstrate that successful pregnancy and delivery of healthy babies is possible in women with thalassemia major^{ref1,
ref2,
ref3} (Cunningham MJ, Macklin EA, Muraca G, Neufeld EJ. Successful pregnancy in thalassemia major women in the Thalassemia Clinical Research Network. Pediatr Res. 2004;55:294A). Spontaneous pregnancy without hormonal assistance has been reported^ref1,
ref2 (Cunningham MJ, Macklin EA, Muraca G, Neufeld EJ. Successful pregnancy in thalassemia major women in the Thalassemia Clinical Research Network. Pediatr Res. 2004;55:294A). Recent literature on hypogonadal hypogonadism suggests that early intervention with hormonal therapy and aggressive iron chelation therapy to prevent permanent damage may help to preserve innate fertility^ref. The TCRN Registry recently reported data on successful delivery of healthy babies in 8 women with thalassemia major (Cunningham MJ, Macklin EA, Muraca G, Neufeld EJ. Successful pregnancy in thalassemia major women in the Thalassemia Clinical Research Network. Pediatr Res. 2004;55:294A). The ages of the birthing mothers, pre- and post-delivery ferritin and liver iron content, and need for hormonal replacement therapy were reviewed. Age-matched women with thalassemia major who were not birth-mothers were the controls. The Registry includes 109 women over age 18 years. The review reported 9 term deliveries in 8 women 23–32 years of age. All of the women discontinued DFO during the pregnancy but the date of discontinuation relative to conception was not known. 4 of 9 pregnancies (44%) were assisted by ovulation induction therapy. Significant preconception to postdelivery increases were found for ferritin (n = 7 women, mean increase 590 ng/mL, 95% confidence interaval [CI] 311–1113 ng/mL, P < .03) but not for liver iron (n = 5 women, mean increase 2.2 mg/g, 95% CI –3.8–8.1 mg/g, P = .59). Ferritin values of non-birthing women were 60% greater than the pre-conception ferritins of the pregnant women (2251 vs 1392 ng/mL after controlling for age); however, the difference was not significant (P = .35) (Cunningham MJ, Macklin EA, Muraca G, Neufeld EJ. Successful pregnancy in thalassemia major women in the Thalassemia Clinical Research Network. Pediatr Res. 2004;55:294A). As more women with thalassemia have successful pregnancies and deliveries, careful review of iron accumulation, cardiac stress and potential deterioration, appropriate increase in transfusion support and use of DFO or other chelators during pregnancy is imperative. Successful pre-implantation genetic diagnosis has recently been reported for carriers of thalassemia^ref. Using in vitro fertilization technology, single cells from an early embryo can be assayed by polymerase chain reaction (PCR) for known mutations, including those of globin genes. This method of biopsy of blastomeres and subsequent transfer in utero of blastomeres unaffected by ß-thalassemia major may be used instead of prenatal testing in utero. This technology has also recently been utilized to determine ß-thalassemia and HLA genotype for planned HLA-matched cord blood transplantation for an affected sibling^ref. Selection of nonthalassemia-affected, HLA-matched siblings as a source of cells for subsequent hematopoietic stem cell transplantation for affected older siblings is gaining favor, but this expensive, "cutting edge" technology should not be considered standard therapy at present.

Prognosis

(g-d-b)⁰-thalassemia : only heterozygous, from deletion of LCR on chromosome 11p
d-thalassemia : that involving suppression of the d chains of hemoglobin; it is usually symptom-free.
d-b⁺-thalassemia :

Lepore hemoglobin : Lepore chromosome arises from unequal crossing-over => both d and b chains of hemoglobin is decreased

heterozygous : it resembles thalassemia minor

double heterozygosis for Hb Lepore/b-thalassemia

homozygous (Papua New Guinea) : it resembles thalassemia major

Sicilian db-thalassemia : 13.4-kb deletion involving the d-globin and b-globin genes. Patients have elevated HbF (6.0 to 19.0% in one series) and microcytosis^ref.

the XmnI mutation in the g-globin promoter is known to increase the production of g-globin and result in HbF levels ranging from 2.3 to 3.8% in heterozygotes. The combined effect of these mutations is not completely understood. An analysis of 19 persons with Sicilian db-thalassemia in an area with a high prevalence of the disorder in regions surrounding the Arachthos River in the Epirus region of Greece^ref found that 10 persons who did not have the XmnI mutation had levels of HbF between 6.8 and 12.5%, 7 who were heterozygous for XmnI had hemoglobin F levels ranging from 7.3 to 9.8%, and 2 who were homozygous for XmnI had HbF levels of 8.1 and 9.7%. These values are still lower than those we observed in this patient.

double heterozygosis of db and b-thalassemia : intermediate b-thalassemia (Hb 6-9 g/dl)
increased fetal hemoglobin (HbF)

Aetiology

hereditary

Shwachman-Diamond syndrome (SDS)
Fanconi anemia (FA) or syndrome
dyskeratosis congenita (DC)
Diamond-Blackfan anemia (DBA)
hereditary persistence of HbF (HPFH) : continued production of fetal hemoglobin beyond the point when it is normally replaced by HbA.

the first type involves large deletions in both the d-globin and b-globin genes that result in increased binding of a-globin to g-globin^ref
the second type involves nondeletional mutations in the promoter regions of g-globin genes that result in increased transcription of -globin

one mutation, a CT transversion at base pair –158 in the G promoter that creates a site cleavable by the XmnI restriction enzyme^ref, results in increased levels of hemoglobin F at times of erythrocyte stress, such as pregnancy^ref
other mutations involving the action of transcription regulators that are at a distance from the g-globin genes are possible and still undergoing characterization.
Sicilian db-thalassemia

Symptoms & signs :

heterozygotes are asymptomatic (HbF 15-20%, reduced HbA and HbA₂)
homozygotes (extremely rare ) have hypochromic microcytic erythrocytes and may have a mild type of thalassemia, but usually no anemia

acquired

intentional pharmacologic manipulation (hydroxyurea^ref1,
ref2)
juvenile myelomonocytic leukemia (JMML)

Laboratory examinations

identification of beta-thalassaemia mutations

amplification refractory mutation system (ARMS)
gap-PCR

fetal hemoglobin is assessed by 2 methods:

Betke quantitative biochemical hemoglobin F alkaline denaturation method (Betke K, Marti HR & Schlicht I (1959). Estimation of small percentage of foetal hemoglobin. Nature, 184: 1877-1878)
Kleihauer qualitative cytological method (Kleihauer E, Braun H & Betke K (1957). Demonstration von fetalem Hämoglobin in den Erythrocyten eines Blutausstrichs. Klinische Wochenschrift, 35: 637-638) based on the fact that hemoglobin F does not elute from red cells which are washed with acid solution.

Laboratory examinations :

leptocytosis , target cells
cresyl blue or methyl violet staining to detect RBC inclusion bodies due to HGB precipitation
hemoglobin electrophoresis (iron deficiency upregulates HbF and HbA₂)
Simmel test
ferritinemia = 50-300 ng/ml
increased erythrocyte ferritin concentration (decreased in iron deficiency)
screen for globin gene mutations in partners of known thalassaemia carriers, regardless of MCV and endemicity, to identify pregnancies at risk of Hb H disease or Hb E/b thalassaemia^ref

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