THALASSEMIA

The word thalassemia was derived from two Greek words - Thalassa meaning the sea and haima meaning blood. The word originated from a common condition peculiar to the geographical area in the Mediterranea . It is a quantitative defect in hemoglobin

 Thomas Benton Cooley (1871 - 1945 ) an American Physician was the first to describe the clinical presentation and features of unexplained severe anemia and hence it was coined Cooley's anemia. Countries like Italy, Greece and Cyprus have the highest frequency of Thalassemia cases in the world. There exists a Thalassemic belt that includes the Mediterranean passing through West and Central Asian countries like Turkey, Iran, Afghanistan onto Pakistan & India and passes on to the South East Asian countries like Indonesia, Burma & Thailand

Prevalance And Geographic distribution

It is considered as the most prevelant genetic disorder in the world.The estimated carrier rates in some States are as follows. Punjab (3.8%), Bengal (3.7%), Rajasthan (3.5%), Kerela (0.6%). Very high rates have been found in certain communities such as Sindhis 12.4% and in Lohana Gujratis 13.6 %. The reason is attributed to intra-caste and intra-community marriages. It is an established fact that every year about 10,000 children are born in India with the disorders.Apporoximately 3 % of the world population carry beta thalassemia gene

Role of malaria in geographic distribution

Vezzoso noted that the distribution of cooley anemia in Italy coincided with that of malaria Haldane proposed that the small red cells of the carriers of thalassemia could be more resistant to malaria The selective pressure of malaria has amplified the beta thalassemia genes to high frequency A recent study shows increased susceptibility to P.VIVAX and resistance to P.FALCIPARUM

Globin gene
3 exons & 2 introns Promoter TATA 30bp,CAAT -70 bp, CACCC -110 bp Transcription factors GATA -1,Nuclear factor erythroid 2 , erythroid kruppel like factor Motifs CAP site which indicates start of transcription, AGT initiation code for translation, poly A tail

Genetic mechanisms
Two gene clusters required for Hb synthesis alpha & beta Alpha duplicated; in the telomeric region of chromosome 16(16p13.3) Cluster contains- embryonic alpha like gene zeta ,3 pseudogenes , a gene theta with unknown function 2 adult alpha gene Several regions of the cluster contain tandem arrays of short GC rich sequence(minisatellites) identified as hypervariable regions & Alu family repeats

The zeta-globin gene is expressed exclusively in the primitive erythroblasts of the embryonic yolk sac and is selectively silenced during the transition from primitive to definitive erythropoesis. The two alpha-globin genes are expressed through development; they are expressed at equivalent levels in embryonic cells at a 2.6:1 ratio of alpha 2:alpha 1 in fetal and adult cell.Developmental silencing of the zetaglobin gene reflects both transcriptional and posttranscriptional mechanisms. Transcriptional silencing is mediated by an interaction between the zeta-globin gene promoter and a silencer located in the 3' flanking region. This transcriptional silencing is only partial, and residual levels of zeta-globin mRNA are subject to subsequent degredation. This instability of zeta-globin mRNA relative to that of alpha-globin mRNA reflects differences in their respective 3'UTR segments; the zeta-globin mRNA 3'UTR has a lower affinity for a sequence-specific mRNP stability complex which assembles at this site. The alpha-globin mRNA assembles this complex at a higher efficiency and mutations which interfere with 3'UTR function result in corresponding loss of alpha-globin gene expression. These data outline a developmental pathway for the alpha-globin gene cluster which reflects transcriptional and posttranscriptional controls.

Genetic mech contd.

The alpha 1 & alpha 2 genes differ only in the IVS-2 & IN 3NONCODING REGION EMBRYONIC GENE SHOWS ONLY 58% HOMOLOGY TO THE 2 ALPHA GENES The level of transcription of the two alpha gene differs alpha 2 encodes 2-3 times more alpha globin than alpha 1 However the reported abnormal Hb in hetrozygotes with alpha 2 mutations (23.5%) isonly slightly higher than alpha 1 (19.7%)

Genetic mech contd

This is because other factors such as stabilityof the variant,affinity of the variant for beta chain & no. of active alpha genes may influence the final level of abnormal Hb Expression of alpha gene is regulated by a region 40 kb upstream from the alpha cluster defined as hypersensitive site (HS-40)-contains an erythroid specific DNAase I HS & 350 bp core element with multiple binding sites for transacting factors(nuclear factor erythroid 2,GATA-1 ),several CACC motifs& a YY1 transcription factor bibnding site

Genetic mech. Contd.

The beta globin gene is located on short arm of chromosome 11contains delta gene,embryonic epsilon gene, fetal Ggamma & A gamma genes & the pseudogene Interspersed repititive sequences (microsatellite repeats of (CA)n, (ATTTT)n repeat, Alu I & Kpn I families of repeat DNA sequences

Genetic mech contd

The region also contains many polymorphic base sustitutions which produce restriction fragment length polymorphisms The major regulatory region is the locus control region (LCR) located 5 to 25 kb upstream from the epsilon globin gene . 5 DNA ase HSs ve been described in the region

SWITCH OVER
2 switch overs take place Embryonic to fetal switch( epsilon to gamma & zeta to alpha ) which starts early in pregnancy & is completed at 10 wks of gestation Fetal to adult switch ( gamma to beta ) occurs during perinatal period The switches comes with changesin morphologic & biochemical characteristics of RBCs,shift in the site of erythropoises, changes in membrane antigenic profile & red cell glycolytic activity

Deletion alpha thalassemia

3 homologous subsegments (X,Y,Z ) separated by non homologus elements Reciprocal recombination between Z boxes which are 3.7 kb apart & b/w X boxes which are 4.2 kb apart gives rise to chromosome with only one alpha gene These are called 3.7 kb rtward deletion 4.2 ltward deletion

 Besides, non reciprocal crossover produces chromosomes with 3 alpha globin genes

Non deletion alpha thalassemia

Constant spring mutation Common in south east asia Change the stop codon to an amino acid alowing m RNA translation to continue to next in phase stop codonlocated in polyadenylation signal Result is alpha chin elongated by 31 AA Other mutations described are Hb Herakilon,Hb Agrinio

Non deletion beta thalassemia

Transription mutation 1) promoter mutations : A) in the 5flanking sequence

785-4144; Fax: (203) 785-7232; E-mail: bernard.forget@yale.edu Increased levels of fetal hemoglobin (HbF) can ameliorate the clinical course of inherited disorders of -globin gene expression, such as thalassemia and sickle cell anemia. In a group of disorders called hereditary persistence of fetal hemoglobin (HPFH), expression of the -globin gene of HbF persists at high levels in adult erythroid cells. Molecular studies of the HPFH syndromes have identified several important regulatory elements for the normal pattern of -globin gene expression. Deletion as well as nondeletion types of HPFH have been identified. The nondeletion types of HPFH are characterized by the presence of point mutations, in the promoter region of one or another -globin gene, that are thought to alter interactions between various transcription factors and the promoter. The deletion types of HPFH are thought to deregulate the normal developmental pattern of -globin gene expression due to the juxtaposition of normally distant cis-acting factors into the vicinity of the genes. These findings have provided us with a more sophisticated understanding of the molecular basis for the persistent -gene expression in these syndromes and point to certain strategies for potential future attempts at gene therapy for -globin gene disorders.

Hereditary persistence of fetal hemoglobin

Increased Hb F (2-30%) Deletion defects : extend from 13 kb to 106 kb; remove delta and beta globin genes & spare Ggamma & A gamma Nondeletion defects : usually mutation in promoter region Hb F may segregate unlinked to the beta globin cluster AD,AR& X linked

Pathophysiology of beta thal

Reduced /absent beta chains with excess of alpha chains: net decrease in Hb ,low MCV, low MCH Premature destruction of RBC precursor in marrow : INEFFECTIVE ERYTHRPOISES Hemolysis of the inclusions containing erythrocytes is a minor cause of anemia

v
Excess of alpha chains undergo proteolysis / become associated with erythroid precursors & RBC membranewith deleterious effects on erythroid maturation The inclusion bodies are the precipitated alpha chains. Oxidation of the excess alpha chains result in formation of hemichromes

o
Alpha chain precipitation in RBC membrane causes structural & functional changes in deformability, stability &red cell hydration Protein 4.1 undergoes partial oxidation altering cytoskeletal stability Association of hemichromes with protein band 3 creates a neoantigen which is subject to opsonization with autologous IgG

RBCs loose K+, store Ca++ & are dehydratedresulting in altered deformability Free iron via fenton reaction generates reactive oxygen species which cause lipid & protein peroxidation These alterations in RBCs result in increase apoptosis which occurs primarily in polychromatophillic erythroblast stage causing ineffective erythropoises

 The first response to anemia is increased erythropoietin causing marked erythroid hyperplasia Anemia may cause cardiac enlargement,skeletal deformities,splenomegaly,retarded growth,increased Fe absorption Another consequence of RBC memrane damage is loss of normal asymmetric distribution & increased surface exposure of procoagulants, -ve charged phospholipids

 The anionic phospholipids increase thrombin generation which leads to activation of platelets & endothelial cells The adherence of thalassemic cells to endothelial cells is increased

PATHOPHYSIOLOGY OF ALPHA THALASSEMIA

Excess of beta, gamma or both genes Unlike alpha chains which are highly unstable ,excess gamma chains in fetal life & beta chains in extrauterine life formsHb bart& HbH .These excess non alpha chains damage mostly mature red cells & to a lesser extent erythroid precursors leading mainly to hemolysis & minimally to ineffective erythropoeisis

 RBCs in alpha thalassemia are rigid but unlike beta thalassemia they are hyperhydrated & have red cell membranes that are hyperstable The cause of hyperhydration is likely the effect of beta chain excess on the KCL cotransporter system

 Beta globin tetramers precipitate as the red cell age forming inclusions Membrane bound inclusions bodies perturb flow velocity during transit through the spleen capillaries

GENOTYPE PHENOTYPE CORRELATION

Factors which reduce the globin chain imbalance are coinheritance of alpha thalassemia, presence of silent or mild beta alleles associated with a high residual output of beta globin

 There are four types of alpha thalassemia that range from mild to severe in their effect on the body. Silent Carrier State : This results due to single alpha globin gene defect 3.7 or 4.2 kb deletion. In the newborn period mild increase (1-2%) of Hb bart It is called "silent carrier" . Silent carrier state is "diagnosed" by deduction when an apparently normal individual has a child with hemoglobin H disease or alpha thalassemia trait.4.2 deletion has a more severe phenotype than 3.7 deletion Hemoglobin Constant Spring. This is an unusual form of Silent Carrier state that is caused by a mutation of the alpha globin. It is called Constant Spring after the region of Jamaica in which it was discovered. As in silent carrier state, an individual with this condition usually experiences no related health problems. Alpha Thalassemia Trait or Mild Alpha Thalassemia. In this condition, the lack of alpha protein is somewhat greater. Patients with this condition have smaller red blood cells and a mild anemia, although many patients do

not experience symptoms. Higher levels of Hb barts (5-6%) . However, physicians often mistake mild alpha thalassemia for iron deficiency anemia and prescribe iron supplements that have no effect on the anemia. Hemoglobin H Disease. There is only one functional alpha gene.In this condition, the lack of alpha protein is great enough to cause severe anemia and serious health problems such as an enlarged spleen, bone deformities and fatigue. It is named for the abnormal hemoglobin H (created by the remaining beta globin) that destroys red blood cells. Hb H is unstable Hb barts is elevated (25%) Hemoglobin H-Constant Spring. This condition is more severe than hemoglobin H disease. Individuals with this condition tend to have a

 Homozygous Constant Spring. This condition is a variation of hemoglobin H-Constant Spring that occurs when two Constant Spring carriers pass their genes on to their child (as opposed to hemoglobin H Constant Spring, in which one parent is a Constant Spring Carrier and the other a carrier of alpha thalassemia trait). This condition is generally less severe than hemoglobin H Constant Spring and more similar to hemoglobin H disease. Hydrops Fetalis or Alpha Thalassemia Major. In this condition, there are no alpha genes in the individual's DNA, which causes the gamma globins produced by the fetus to form an abnormal hemoglobin called hemoglobin Barts. Most individuals with this condition die before or shortly after birth. In some extremely rare cases where the condition is discovered before birth, in utero blood transfusions have allowed the birth of children with hydrops fetalis who then require lifelong blood

Unusual forms of alpha thalassemia

Alpha thalassemia/myelodysplasia syndromes : unusual form of alpha thal in pt of myelodysplasia characterized by +ence of classic HbH inclusion bodies in RBCs ,detectable HbH(1-57%)& a severe microcytic hypochromic anemia with anisopoikilocytosis The alpha to beta globin m RNA ratio is reduced Possibly alpha genes are downregulated by a mutation in trans acting genes that control alpha gene expressionas structural analysis of alpha gene has revealed no abnormalities

Alpha thalassemia & mental retardation syndromes

1) alpha thal mental retardation-16 syndrome : mild mental retardation,facial & skeletal abnormalities. They ve extended deletions resulting from rearrangements of short arm of chromosome 16 2)alpha thal mental retardation X :

Alpha thalassemia associated with structural variants

1)HbQ/alpha0 thal: similar to HbH disease .No synthesis of HbA 2)HbG-Philadelphia/alpha0 thal : like alpha thal minor 3)HbS trait produces a trimodal distribution

Beta thal
There are three types of beta thalassemia Thalassemia Minor or Thalassemia Trait. In this condition, the lack of beta protein is not great enough to cause problems in the normal functioning of the hemoglobin. A person with this condition simply carries the genetic trait for thalassemia and will usually experience no health problems other than a possible mild anemia. As in mild alpha thalassemia, physicians often mistake the small red blood cells of the person with beta thalassemia minor as a sign of iron-deficiency anemia and incorrectly prescribe iron supplements

Thalassemia Intermedia.This term is used to designate a form of anemia that does not require transfusions irrespective of genotype, In this condition the lack of beta protein in the hemoglobin is great enough to cause a moderately severe anemia and significant health problems, including bone deformities and enlargement of the spleen. However, there is a wide range in the clinical severity of this condition, and the borderline between thalassemia intermedia and the most severe form, thalassemia major, can be confusing. The deciding factor seems to be the amount of blood transfusions required by the patient. The more dependent the patient is on blood transfusions, the more likely he or she is to be classified as thalassemia major. Generally speaking, patients with thalassemia intermedia need blood transfusions to improve their quality of life, but not in order to survive.

 Thalassemia Major or Cooley's Anemia. This is the most severe form of beta thalassemia in which the complete lack of beta protein in the hemoglobin causes a life-threatening anemia that requires regular blood transfusions and extensive ongoing medical care. These extensive, lifelong blood transfusions lead to iron-overload which must be treated with chelation therapy to prevent early death from organ failure CLINICAL FEATURES: 1)anemia : mean age of presentation 6 months 2)bone deformities :metatarsals are the first to expand 3)osteoporosis 4)cholelithiasis 5)thrombotic complications 6)pseudoxanthoma elasticum