B E T A - T H A L A S S E M I A S Y N D R O M E S , C L I N I C A L A N D L A B O R A T O R Y A P P R O A C H
Emine T ü rk k a n , M .D . / Su Gülsün B erra k, M .D . / Cengiz C an polat, M .D .
S u b -d e p a r tm e n t o f P a e d ia tric H e m a to lo g y - O n c o lo g y , D e p a r t m e n t o f C h ild H e a lth a n d P e d ia tric s , S c h o o l o f M e d ic in e , M a r m a r a U n iv e rs ity , Is ta n b u l, T u rk e y .
ABSTRACT
The Beta ((3) th ala sse m ia syndrom es are a heterogeneous group of genetic disorders. The frequency of thalassem ia is dependent on the ethnic origins of the patient population. Turkey is located in a geographic area of the world where th ala sse m ia syn d ro m e s a re com m on. Th e incidence rate of (3-thalassemia carriers w as stated to be 2 per cent in Tu rkey. Clinical m anifestations are d ive rse and range from asymptomatic hypochromia and m icrocytosis to profound ane m ia leading to death in e arly childhood if untreated. Individuals who are homozygous for the (3-thalassemia genes have se vere , transfusion-dependent anem ia and are said to have (3-thalassemia major. T h a la sse m ia intermedia is a condition in which the degree of hem olysis is milder even though the patient may have a deficiency of both (3 genes. Therefore, th a la sse m ia interm edia is e sse n tia lly a descriptive term that refers to minimal or no need for transfusions. T h e presence of one normal gene in the heterozygotes u sually lead s to enough normal (3-globin chain syn th esis so that the affected individuals are usually asym ptom atic with only a mild anem ia. Th is condition is referred to as (3-thalassemia minor or (3-thalassemia trait. In this report clinical and laboratory findings of 13- th a la sse m ia syn d ro m e s in childhood are reviewed.
Key W ords:
Beta th alasse m ia, Childhood, Anem ia, Hypocromic microcytic anem iaINTRODUCTION
T h e Beta ((3) th ala sse m ia synd rom es are a heterogeneous group of genetic disorders, all characterised by a lack of or d ecreased synthesis of (3-globin chain of Hb A (a 2 (32) (1).
T h a la sse m ia w as not recognized a s a clinical entity until 1925, when Th o m as Cooley described a syndrom e among children of Italian descent c h a ra c te rise d by profound a n e m ia , splenom egaly, and bone deformities (2). Th e first two patients with (3-thalassemia major in Turkey were reported in 1941 (3).
Prevalence and Geographic Distribution
The frequency of th alasse m ia is dependent on the ethnic origins of the patient population (1 ,4 ) . The geographic a re a s in which th alasse m ia is prevalent closely parallel the regions in which P. falcip arum m alaria w a s form erly en d em ic. R e s is ta n c e to lethal m alarial infections by c a rrie rs of th a la sse m ia g e n e s apparently represented a strong selective force that favored their survival in these a re a s of endem ic d ise a se (5-7). About 3% of the world's population (150 million people) carry (3 th alasse m ia g en es. Th e se g enes are particularly prevalent in inhabitants of (A c c e p te d 2 3 May, 20 02 ) M arm ara M e d ic a l J o u rn a l 2 0 0 2 ;1 5 (3 ):1 9 4 -20 0
Correspondence to: Emine Turkkan, M.D, - Sub-department of Pediatric Hemotology-Oncology, Department of Pediatrics. School of Medicine, Marmara University Hospital. Altunizade, 81190 Istanbul, Turkey,
Italy, G re e ce and C yprus (8, 9). Turkey is located In a geo g rap hic a re a of the world w here th alasse m ia syndrom es are common. The first report about the prevalence of (3-thalassemia carriers in Tu rkey w a s published in 1971 in which the incidence rate w a s stated to be 2 per cent (1 0 ).Different stu d ie s noted a frequency variability, ranging between 3.4 (E a st Anatolia) and 11 per cent (W estern Th race and Antalya) in Turkey (11, 12).
The pathophysiology
T h e "globin" part of the hemoglobin predom inating in adults (hem oglobin A) is com posed of four ch ain s, 2 a- and 2 3-chains (13, 14). Th e pathophysiologic m echanism in th a la sse m ia is related to an unbalanced syn th esis of globin ch ain s or 3 chains. Normally, the syn th esis of a and (3 chains is balanced, resulting in normal hemoglobin A (a 2
32
)- An unbalanced syn th esis of either chain can lead to a failure in the matching of these chains and a defect in hemoglobinization within erythrocytes. In hom ozygous 3 -th a la sse m ia , there is an e x c e ss of a ch ain s (1, 15). B e ca u se of their great instability, free a ch a in s aggregate to form insoluble inclusions in bone marrow erythroid precursors, causing premature destruction of maturing eryth ro b lasts within the marrow (ineffective erythropoiesis) a s well as lysis of mature red cells in the spleen (hem olysis)(1). There are two 3 genes. Deficiency of one leads to e sse n tia lly no sig n ifican t h em o lysis (3 th alasse m ia trait/thalassem ia minor); deficiency of both g enes leads to significant hemolytic ane m ia (th a la sse m ia m ajor). T h a la ss e m ia intermedia is a condition in which the degree of hem olysis is milder even though the patient may have a deficiency of both 3 genes. Therefore, th a la sse m ia interm edia is e sse n tia lly a descriptive term that refers to minimal or no need for transfusions (16-19).Genetics of (3-thalassemia
Th e non-a g enes reside on chrom osom e 11. The 3-thalassem ias result from the interaction of a large number of different m olecular defects in the (3-globin g e n e s. U nderlying genetic d efects include total or partial deletions of globin chain g en es, nucleotide substitutions, deletions, or insertions (1, 13, 18), which result in none ((3°) or sm all am ounts (0-20 % ) ((3+) of globin chain
synthesis. 3-Thalassem ia trait refers to mutations in one of two 3-globin genes.
In contrast to the a-thalassem ia syndrom es, the 3 -th alasse m ias are rarely ca u se d by major structural gene deletions. Most of the 3 thalassem ia syndrom es result from one or more nucleotide substitutions or deletions in genes that are otherwise intact. The well-known clinical heterogeneity of the thalassem ia syndrom es is a reflection of the great heterogeneity of mutations affecting the globin genes. Using techniques such a s, restriction end onuclease digestion, gene blotting studies, cloning and sequencing of beta-globin genes, examination of expression of mutant genes in tissue culture cells with the use of plasmid expression vectors, investigators have identified more than 150 different mutations (18). Different studies showed that the molecular b asis of 3 -th alasse m ia in Tu rke y is quite heterogeneous and that more than 40 different mutations are responsible for the great variability in clinical expression of this disorder (20-24). The IV S-l-110 (G-A) mutation is the most common 3 thalassem ia defect in Turkey (20, 22).
Clinical Features of (3 Thalassem ia Syndromes
Individuals who are hom ozygous for the
3
- thalassem ia genes (3+/3+ or 3°/3°) have severe, transfusion-dependent anem ia and are said to have3
- thalassem ia major (Table I). Children with thalassem ia major usually develop signs and symptoms of severe anem ia in the latter part of the first year of life when normal hemoglobin synthesis becom es increasingly dependent on3
- globin. On presentation, affected infants usually have pallor, poor growth and development, and abdom inal enlargem ent (1, 16, 2 5 ). In the absence of transfusion therapy, the hemoglobin concentration slowly falls to 3 to 6 g/dl (26). The associated pathophysiologic changes resulting from the su b se q ue nt anem ia include splenom egaly, which may lead to hypersplenism , osteoporosis, and other skeletal and soft tissue changes associated with an expanded bone marrow, and iron overload resulting from a combination of enhanced gastrointestinal iron absorption and red cell transfusions (27, 28). Prom inence of the cheek bones tends to obscure the base of the nose and to expose the upper teeth. Thickening of the cranial bones produces frontal bossing (28). T h e s e d istu rb an ces ofcraniofacial growth constitute the thalassém ie facies. The liver, heart, p an creas, pituitary, and other endocrine organs se rve a s the major sites of e xce ssive iron deposition, which ultimately leads to dam age and failure of these organs (1, 16, 26).
The definition of th alasse m ia intermedia is used to designate a le ss se vere hemolytic anem ia. Chronic transfusion therapy is not required except in association with intercurrent illness, and survival into adult life is the rule. Th e se patients usually experience normal growth and development (1). H ow ever the phenotype of thalassem ia intermedia can be variable ranging from mildly to se v e re ly affected patients. G e n e tica lly, th a la sse m ia interm edia is hom ozygous (3-thalassem ia with alleviating factors such as inheritance of mild (3-thalassemia mutations ((3+/(3+), Hb F enhancing factors and co-inheritance of a-thalassem ia (29).
Th e p re se n ce of one norm al gene In the heterozygotes ((3+/(3 or (3°/(3) usually leads to enough normal (3-globin chain synthesis so that the affected individuals are usually asymptomatic with only a mild anem ia. Th is condition is referred to as (3- thalassem ia minor or (3- thalassem ia trait (3 0 ,3 1 ). T h e synd rom e is alm ost a lw a y s discovered accidentally during exam ination for unrelated sym ptom s or as a consequence of a study designed to characterize better the nature of symptomatic anem ia in a fam ily member. Failure to respond to therapy for presumptive iron deficiency anem ia often leads to a diagnosis of thalassem ia trait. Th ere is no enlargem ent of the liver or sp leen in (3-thalassem ia trait. Organomegaly suggests a more se vere form of thalassem ia.
The term th alasse m ia minima described the situation in individuals who, although obligate carriers of a th alasse m ia gene, had neither anem ia nor abnormal red cell morphology. Each of these clinical phenotypes e n co m p asses a heterogeneous group of genetic d iso rd ers including inheritance of heterozygous state for some types of mild (3-thalassemia mutations ((3+/(3+). It is undetectable except by inference from family studies (16, 32, 33).
Laboratory Features and Definitive Diagnosis of (3 Thalassem ia Syndrom es
In p T h a la sse m ia major, the anem ia is se vere and first becom es m anifest 6 to 9 months after birth, as hemoglobin syn th esis sw itches from Hb F to Hb A. In untransfused patients, hemoglobin levels range between 3 and 6 gm/dl (1, 16, 26). Th e peripheral blood sm e a r sh o w s se v e re ab n o rm alities; there is m arked a n iso c y to sis (variation in size) with many sm all and virtually colorless (m icrocytic, hypochrom ic) red cells. Abnormal forms, including target cells, stippled red cells, and fragmented red cells, are common. Inclusions representing aggregated a ch ain s are removed by the spleen and hence are not visible in peripheral blood (34). Th e reticulocyte count is elevated , but b e c a u se of ineffective erythropoiesis, it is lower than would be predicted from the se ve rity of an e m ia (2 6 ). V ariab le numbers of poorly hemoglobinized norm oblasts are seen in the peripheral blood. Th e red cells contain either no Hb A at all (P° /p° genotype) or sm all amounts ((3+/p+ genotype) (0 to 20% ) (16). Hb F is m arkedly increased (20-100% ) and indeed constitutes the major hemoglobin of red cells (1, 16). Hb A 2 levels may be normal, low, or high (2 to 7 % ). W B C counts are often mildly increased, and there may be mild granulocytic immaturity, som etim es even with m yelocytes present (26). Platelets are normal. Increased unconjugated bilirubin le v e ls and other biochemical evidence of hem olysis can be found. The urine often contains increased quantities of urobilin or urobilinogen and may be dark brown b e ca u se of the p re se n ce of dipyrroles and m esobilifuscin (26). Th e results of ferrokinetic stu d ie s are in keeping with ineffective erythropoiesis; plasm a iron turnover is increased out of proportion to the increase in erythrocyte iron turnover. Serum iron levels are increased, serum transferrin is often fully saturated, and a non- transferrin-bound iron fraction m ay be present. The osmotic fragility of red co rp uscles is strikingly d e c re a se d . T h e bone m arrow is rem arkab ly h yp e rce llu la r with profound normoblastic hyperplasia. Deficient hemoglobin content of red cell p re cu rso rs, a s w ell a s cytoplasm ic inclusions, is apparent (1, 16, 26). In th a la sse m ia interm edia, parental stu d ies reveal the anem ia and m icrocytosis. However, baseline hemoglobin concentration is higher than thalassem ia major with a range of 6 to 9 g/dl
without tran sfu sio n (2 6 ). P erip h eral blood erythrocytes show ch ang e s com parable to those of th alasse m ia major: significant anisocytosis, hypochromia, target cells, basophilic stippling, and num erous nucleated form s. Bone marrow h yp e rp lasia is prom inent and in clu sio n s of denatured a ch ain s can be demonstrated in late norm oblasts by using supravital stains (34). The hemoglobin electrophoretic pattern is highly variable, a reflection of the heterogeneity of genotypes producing this clinical syndrom e. Other laboratory fe a tu re s are d erivatives of accelerated heme turnover and iron overload. Infants destined to develop thalassem ia minor are hem atologically normal at birth. By 4 months, the hemoglobin concentration, M CV, M CH, Hb A 2 and Hb F levels are all outside normal ranges (3 5 ) .The postnatal decline in Hb F is delayed, and the normal increase in Hb A 2 is accelerated (3 6 ) . Anem ia is mild or absent. The hemoglobin co ncentration rem ain s app roxim ately 2 g/dl below normal standards but parallels the normal upward trend during, childhood. The red cell count is elevated, and the M CV and MCH values are reduced. The degree of reduction in the MCV is directly related to the degree of reduction in (3- globin production (36). Th e M CVs produced by P° mutations are lower than those produced by P+ mutations. Co-inheritance of a p° thalassem ia gene and a th alasse m ia (one or two a-globin gene deletion) norm alizes the red cell indices but not the level of Hb A 2 (37, 38). The M CH C is normal or only slightly d ecreased . In contrast to the minor d eg ree of a n e m ia , morphologic alterations of peripheral blood erythrocytes are prominent. T h e se ch ang e s include microcytosis, hypochromia, aniso cyto sis, poikilocytosis, target cells, and basophilic stippling Nucleated R B C s are not p re se n t. T h e reticulocyte count is frequently elevated. The osmotic resistance of erythrocytes is strikingly increased . Erythrocyte- free protoporphyrin usually is normal, in contrast to elevated levels in iron deficiency (39, 40). The most consistent feature of heterozygous Pj and P+ th alasse m ia is an increase in Hb A 2 (A2 is not elevated in a th alasse m ia trait). A 2 is a variant of adult Hb A and is normally present in quantities up to 2 .5 % or 4 % , depending on the method used. In p th alasse m ia trait, A 2 is elevated to som e degree with a maximum of approximately 10% . More than 9 0 % of p erso n s with p- thalassem ia trait have diagnostic elevations of
Hb A2 of 3.4-7% . Hemoglobin F is usually normal but can be present in quantities up to 5% . Hemoglobin A2 cannot be identified on paper electrop h oresis, and dem onstration or quantitation necessitates cellulose acetate or polyacrylam ide gel electrop h oresis or resin column m ethods. Chronic iron deficiency d ecreases Hb A2 levels so that iron deficiency coexistent with p thalassem ia trait could lead to falsely normal Hb A 2 results (41). Th alassem ia minor is easily confused with mild iron deficiency (T ab le II). S e ve ra l sim ple ob servations distinguish the two conditions. W h ereas m icrocytosis, hypochromia, aniso cyto sis, and poikilocytosis are minimal in iron deficiency at a hemoglobin concentration of 10 to 11 g/dl, they are conspicuous features of the blood sm ear in patients with th a la sse m ia trait having com parable levels of hemoglobin. Basophilic stippling and an increase in the icterus index are also prom inent, w h e re as iron d eficien cy is associated with little or no stippling and with a d e c re a se in serum bilirubin level. S e ve ra l discriminative functions have been developed to facilitate differentiation of the two disorders on the b asis of routine blood counts (42). Patients who have p-thalassem ia trait, in contrast to those who have iron deficiency, typically have an increased number of red cells that are sm aller than normal, leading to a ratio of the MCV/red cell count per milliliter of less than 13. The ratio in iron deficiency is usually more than 13 (Mentzer index) (43). The coefficient of variation of red cell siz e is higher in iron d eficiency than in thalassem ia minor, a derivative readily generated by electronic cell counters (44). Laboratory a sse ssm e n t of iron status can be used to differentiate p th a la sse m ia trait and iron deficiency (Table II). Bone marrow iron is absent in iron deficiency and normal or increased in p- thalassem ia trait. Patients with p thalassem ia trait may have concurrent chronic iron deficiency and anem ia of chronic d ise a se m ay be concurrent with either condition. The anem ia of chronic d isease may itself be microcytic and hypochromic in some c a se s (1).
Silent p thalassem ia is inferred in the ostensibly normal parent of a child with th ala sse m ia intermedia if the other parent has high-Hb A 2 thalassem ia minor. The M CV and MCH values are normal or only minimally decreased and the hemoglobin pattern is normal. The condition can
be established with certainty only by globin chain synthesis studies, which show an a:p-globin chain ratio of approxim ately 1.3 (32).
S c re e n in g fo r |3-thalassem ia
Ideally, thalassem ia carriers should be detected as part of a screening programme, such as premarital screening in Cyprus (9) and Sardinia (4 5 ), or during ea rly p re g nan cy in most Mediterranean countries. Th is allows screening of the partner and if the couple are both carriers then prenatal diagnosis will be possible. In particular, the finding of a non-iron-responsive microcytic anem ia in the pregnant woman should prompt a careful evaluation for thalassem ia trait that, if present, should lead to a sim ilar
evaluation of the father. Th is approach permits the family to consider in utero diagnosis. Fetus of heterozygote parental pairs can be screened for hom ozygous th ala sse m ia in utero employing DNA a n alysis of a chorionic villus biopsy at 8-10 w eeks or amniotic fluid cells from am niocentesis at 16-18 w e e k s (46, 4 7 ). T h e e m p h asis is prevention of an affected birth by giving the parental pair the ch o ice of term inating an affected pregnancy, and this can only be done by early detection of the carrie rs. A previous study dealing with prenatal diagnosis of (3 thalassem ia in Turkey has indicated that prenatal diagnosis is feasible in Turkey when early methods of fetal sampling are combined with the advent of P C R - based techniques (24).
T a b l e I: Clinical and Hematologic Features of the (3 Thalassemia Syndromes
Nomenclature (3 Thalassemia Major/ Homozygous (3 Thalassemia
(3 Thalassemia Intermedia (3 Thalassemia Minor/ Heterozygous (3 Thalassemia Genetics (3° /(3°
P* /P+ P°/p'
Homozygous |3-thal. inheritance of p+ /(3+ mutation,
Coinheritance of cc thal. Hb F enhancing factors
(30 /|3 P+/P
Clinic Severe anemia
Requires blood transfusions regularly
Moderately severe anemia,
does not require regular blood transfusions
Asymptomatic child with mild or absent anemia Hematologic features Normoblastemia
Microcytosis, Hypochromia Anisocytosis Poikilocytosis Basophilic stippling Fragmented red cells
Normoblastemia Microcytosis, Hypochromia Anisocytosis Poikilocytosis Basophilic stippling
Fragmented red cells
Microcytosis, Hypochromia Anisocytosis Poikilocytosis Basophilic stippling Anemia (Hb) (g/dl) 3-6 6-9 9-10
(3°: Mutations that lead to a decreased level of normal (3-chain production (3+: Mutations that lead to absence of normal (3-chain production Thai.: Thalessemia
T a b l e II: Differential Diagnosis of Iron deficiency and Beta Thalassemia Trait
Iron deficiency Beta Thalassemia Trait
MCV Low Very Low
Serum iron Decreased Normal TIBC Increased Normal Trans. Sat. Decreased Normal FEP Increased Normal Serum Ferritin Decreased Normal Hb A2 Normal Increased MCV: Mean Corpuscular Volume, TIBC: Total Iron Binding Capacity, Trans. Sat: Transferrin iron saturation, FEP: Free erythrocyte porphyrin Hb: Hemoglobin
CONCLUSION
Appropriate scre e n in g and su b se q u e n t diagnostic testing will allow the family physician to diagnose most c a s e s of anem ia in children . If there is a question about the diagnosis, the genetics, appropriate counseling, or subsequent evaluation, children su sp e cte d of having 13- thalassem ia should be referred to a pediatric hem atologist. T h e s e would include patients w hose anem ia is more se ve re than would be expected; they m ay have a confounding second
reason for a n e m ia or they m ay have 13- th ala sse m ia interm edia, (3-thalassemia major, which present more com plex diagnostic and therapeutic problems.
Although there are excellent treatment modalities today, there is no definitive cure for (3- th alasse m ia yet with the exception of bone marrow transplantation, which is not available to most patients. Until novel therapies, such as Hb F reactivation and gene therapy becom es a reality, the only approach to the control of haem oglobinopathies is prevention. Avoidance of consanguineous m arriage, which is 2 1 .2 1 % in T u rk e y (4 8 ), will a lso help to significantly d e cre ase the frequency of affected births. The most important challenge for the eradication of the haem og lob ino p athies in Tu rke y is the organization of a national genetic preventive programme for the eradication of (3-thalassemia, like those going on in several high-risk a reas of the M editerranean basin such as Cyprus and Sard inia. T h is could be performed either as premarital screening of reproductive couples (49) or by education of the population at risk and their physicians.
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