Dr.S.Chakravarty MD

Specific Learning Objectives At the end of today’s lecture you should be able to – – Enumerate the various hemoglobinopathies with their associated genetic defects. – Describe the molecular basis and the basis of laboratory diagnosis of Sickle cell anaemia, alpha and beta thalassemia in detail.

β β α α

βδγαα Chromosome 16Chromosome 11 25% ααβδγ 48% 1.5%0.5% 1.5%0.5%

α α α αα α βγδ βδγ HbAHbFHbA 2 98%~1% <3.5%

Steps in globin chain synthesis : 1.Transcription 2.Modification of mRNA precursor by splicing 3.Translation by ribosomes & further modifications (i.e. glycosylation)

An inherited mutation of the globin genes leading to a qualitative or quantitative abnormality of globin synthesis. Hereditary disorders that can result in moderate to severe anemia

Structural hemoglobinopathies Thallasemias Thallasemic Hb variants Hereditory persistance of fetal Hemoglobin Acquired hemoglobinopathies 10

1. Structural Hemoglobinopathies a)Altered Hb polymerization b)Altered affinity c)Unstable Hb Variants. 11

A number of factors may precipitate a sickle cell crisis, these include: Hypoxia Acidosis Dehydration Infections Severe exercise Increase physical / physiological demand (Pregnancy, physical exercise) Hb S is less soluble in acidosis and dehydration

Lippincott’s Illustrated Biochemistry Hydrophobic valine causes stickiness

1.Hemolysis  Anaemia 2.Occlusion of blood vessels by sickled red cells- SEVERE PAIN ( DUE TO TISSUE ANOXIA)

Complete Hemogram  Anemia Sickling test :- Blood smear prepared after adding reducing agent sodium dithionite  See under microscope (NOT SPECIFIC ) Solubility test :- Hemolysate is prepared in the presence of reducing agent.Opalescence is suggestive of sickle cells. (NOT SPECIFIC ) Hb electrophoresis- CONFIRMATORY Southern Blot -CONFIRMATORY

@ pH 8.6 Glutamic acid carboxy group is –ve charged Lack of this charge of HbS makes it less negatively charged and decreases the electrophoretic mobility towards positive pole. However at acidic pH (citrate buffer)HbS moves faster than Hb A Lippincott’s Illustrated Biochemistry

Repeated Blood transfusions- MAINSTAY IRON OVERLOAD is a problem CHELATION therapy with Desferrioxamine

CHRONIC HEINZ BODY ANAEMIAS -Unstable Hb variants have a tendency to denature. They tend to form molecular aggregates called Heinz Bodies within cells  hemolysis – Hb Köln (Hb β 98val  met) – Hb Poole – γ chain unstable variant

INCREASED O 2 AFFINITY – Hb Chesapeake – erythrocytosis (ODC shifts to left ) DECREASED O 2 AFFINITY – Hb Kansas – Cyanosis Hb M- – Most HbM are produced by substitution of Tyr for proximal/Distal His in the haem pocket of the alpha or ß-chains  This results in facilitated oxidation of the hemoglobin to yield excess methemoglobin which leads to cyanosis. – Hb M Hyde Park, β92His→Tyr; Hb M Boston, α58His→Tyr; Hb M Saskatoon, β63His→Tyr; Hb M Milwaukee-1, β67Val→Glu.

1925: Described by Dr. Thomas Cooley and Dr. Pearl Lee of Detroit 1920’s: Osmotic fragility test 1932: Dr. George Whipple of Rochester coined the name “thalassa anemia” from Greek story about Xenophon’s army returning from Persia 1930’s: Familial pattern recognized 1950’s: Alkali denaturation test for Hb F, Hb ELP 1956: Coulter model A 1960’s: RBC indices 1980’s: Histogram, DNA analysis, PCR

Hereditary disorders that can result in moderate to severe anemia Basic defect is reduced production of globin chains.

Found most frequently in the Mediterranean, Africa, Western and Southeast Asia, India and Burma Distribution parallels that of Plasmodium falciparum Hb D

Symbolism Alpha Thalassemia Greek letter used to designate globin chain: 

Symbolism Alpha Thalassemia / : Indicates division between genes inherited from both parents:  /  Each chromosome 16 carries 2 genes. Therefore the total complement of  genes in an individual is 4

Symbolism Alpha Thalassemia - : Indicates a gene deletion: -  / 

Classification & Terminology Alpha Thalassemia Normal  /  Silent carrier-  /  Minor-  /-  --/  Hb H disease--/-  Barts hydrops fetalis --/--

Symbolism Other Thalassemia Greek letter used to designate globin chain: 

Symbolism Other Thalassemia + : Indicates diminished, but some production of globin chain by gene:  +

Symbolism Other Thalassemia 0 :Indicates no production of globin chain by gene:  0

Classification & Terminology Beta Thalassemia Normal  /  Minor  /  0  /  + Intermedia  0 /  + Major  0 /  0  + /  +

 Some mutations lie within promoter regions and typically lead to reduced globin gene transcription.  In some cases a single-nucleotide change in one of the exons leads to the formation of a termination, or "stop" codon, which interrupts translation of β- globin messenger RNA (mRNA) and completely prevents the synthesis of β- globin. Such alleles are designated β 0.  Mutations that lead to aberrant mRNA processing are the most common cause of β-thalassemia. Most of these affect introns, but some have been located within exons. If the mutation alters the normal splice junctions, splicing does not occur, and all of the mRNA formed is abnormal. Unspliced mRNA is degraded within the nucleus, and no β-globin is made

Abnormal associations of otherwise normal subunits. With severe α-thalassemia, the β-globin subunits begin to associate into groups of four (tetramers) due to the paucity of potential α -chain partners. These tetramers of b-globin subunits are functionally inactive and do not transport oxygen. No comparable tetramers of alpha globin subunits form with severe beta-thalassemia. Alpha subunits are rapidly degraded in the absence of a partner from the beta-globin gene cluster (gamma, delta, beta globin subunits).

β-Thalassemias Thalassemia majorHomozygous or compound heterozygous (β 0 /β 0, β 0 /β +, or β + /β + ) Severe, requires blood transfusions regularly Defects in transcription, processing, or translation of mRNA, resulting in absent (β 0 ) or decreased (β + ) synthesis of β- globin β-thalassemia traitβ/β + or β/β 0 Asymptomatic, with mild microcytic anemia, or microcytosis without anemia α-Thalassemias Hydrops fetalis--/--Fatal in utero Gene deletions spanning one or both α-globin loci HbH disease--/-αModerately severe anemia α-thalassemia trait--/αα(Asian) or -α/--α(African) Similar to β- thalassemia trait Silent carrier-α/ααAsymptomatic, normal red cells

Excessive RBC BREAKDOWN

Anaemia Bone changes (hair on end) Ethnicity: Mediterranean, Africa, Southeast Asia Hypo-Micro, Poikilocytosis NRBC’s, reticulocytosis, basophilic stippling Siderocytes (with repeated transfusions)

Thalassemia Blood Smears

X-ray of scull in Thalassemia: “Hair-on-end”

MRI showing marked widening of the diploic space containing alternating bands (arrows) of hypointense trabeculae and hyperintense marrow.

Beta thalassemia major Male 18 years HEPATOSPLENOMEGALY OVERALL DECREASED GROWTH

Hb Lepore:  fusion seen in some types of  thalassemia Hb Constant Spring  chain with 31 additional amino acids --/  cs  Hereditary persistence of fetal hemoglobin (HPFH) This is usually caused by mutations in the β-globin gene. Beneficial to patients with sickle cell or thalassemia.

Hb H  4 tetramer and γ 4 tetramers Associated with --/-  thalassemia

Hb Barts & hydrops fetalis Barts is a  4 tetramer Associated with --/-- Lethal High concentrations are capable of sickling

Peripheral smear – Severe cases present with Microcytosis Hypochromia Poikilocytosis Hb Electrophoresis DNA studies (PCR + S.blot)

Findings in severe cases can mimic those seen in other microcytic/hypochromic anemias. Results of the reticulocyte count are variable(although increased retic count is found) Nucleated RBCs (NRBCs) may be present (contrast with iron deficiency anemia)

Time of presentation Related to degree of severity Usually in first few years of life Untreated severe  thalassemia --/--: Prenatal or perinatal death --/-  & --/  cs  : Normal life span with chronic hemolytic anemia

Untreated  thalassemia Major: Death in first or second decade of life Intermedia: Usually normal life span Minor: Normal life span

Repeated Blood transfusions - MAINSTAY

Repeated blood transfusions leading to Iron excess and Hemosiderosis. Pigment stones in Gall Bladder and CBD are more common in such patients.

Hemoglobinopathies Thalassemia genetics Hb synthesis Hb A, A2, F Hb ELP Hb Constant-Spring Hb Bart’s Hb H Hb Lepore Hb E Hb S Hb C Hb SC disease HPFH Hb POINT MUTATION POSITION AMINO ACID SUBSTITUTION Hb Sβ6β6Glu- Val Hb Cβ6β6Glu-LysHomozygotes – CC Mild anaemia Heteozygous – AC no disease Double heterozygous SC –Moderate disease Hb Eβ26Glu-LysHeterozygous – Asymptomatic Homozygous – Mild disease Hb D (Punjab or Los Angeles) β121Glu-GlnHbSD – severe disease Hb O (Arab) β121Glu-LysHomozygous –mild anaemia Hb G (Philadelphia) α68Glu-Lys HbG Philadelphia is an α variant, often associated with deletions of the nonaffected a genes. With no deletions, there is approximately 20% HbG, with one deletion about 30% G is present, and with two about 40% is present. Hb Lepore δ(1-87) β( )Lepore is the product of the indicated crossover during meiosis.(NOT A MUTATION)

Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A 2 /C S F A + Normal

Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A 2 /C S F A + Normal Hb SS

Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A 2 /C S F A + Normal Hb SS Hb AS

Secondary Laboratory Investigation Cellulose Acetate Hb Electrophoresis - A 2 /C S F A + Normal Hb SS Hb AS Hb SC Hb CC

Can you identify which person has severe sickle cell anemia and who is heterozygous for the condition ?

In general on alkaline electrophoresis in order of increasing mobility are hemoglobins A2, E=O=C, G=D=S=Lepore, F, A, K, J, Bart's, N, I, and H. In general on acid electrophoresis in order of increasing mobility are hemoglobins F,A=D=G=E=O=Lepore, S, and C

Solubility testing-Dithionite tube test Alkali denaturation test for quantification of fetal hemoglobin Unstable hemoglobin testing for Heinz bodies

Principle Other hemoglobins are more susceptible than hgb F to denaturation at alkaline pH Denaturation stopped by addition of ammonium sulphate Denatured hemoglobin precipitates Remaining hemoglobin (F) can be measured spectrophotometrically

Test partners of heterozygous or affected individuals Antenatal diagnosis from DNA obtained by chorionic villus sampling, or by amniocentesis

67 An Asian child has severe anemia with prominence of the forehead (frontal bossing) and cheeks. The red cell hemoglobin concentration is dramatically decreased, and it contains only beta-globin chains with virtual deficiency of alpha-globin chains. Which of the following mechanisms is the most likely explanation? A. A transcription factor regulating the alpha-globin gene is mutated B. A regulatory sequence element has been mutated adjacent to an alpha-globin gene C. A transcription factor regulating the beta-globin gene is mutated D. A transcription factor regulating the alpha and beta -globin genes is deficient E. A deletion has occurred surrounding an alpha-globin gene MCQ 1

HbC disease is caused by a single amino acid substitution (lysine instead of Glutamic acid) at position 6 in the bete-globin chain of the hemoglobin molecule. Patients homozygous for HbC have a mild chronic hemolytic anemia. HbS disease generally causes a more severe condition compared to HbC disease because HbS disease: A.Impairs oxygen binding to the heme moiety B. Impairs proper folding of the alpha-helix in the beta-globin chain C. Allows hydrophobic interaction among hemoglobin molecules D. Impairs beta-globin interaction with 2,3-bisphosphoglycerate E. Stabilizes iron moiety at ferric state (fe3+). MCQ 2

69 An infant to a greek immigrant appears healthy at birth but develops transfusion dependent hemolytic anemia by the age of 6 months. his erythrocytes contain insoluble aggregates of hemoglobin subunits. The child developed normally in utero because at that time he produced high quantities of: A.Alpha globin B. Beta globin C. Gamma globin D. Delta globin E. Epsilon globin MCQ 3

which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell disease is correct? A.acidosis influences the shape of hemoglobin B. Acidosis decreases the solubility of Hb S C. Acidosis favours the conversion of hemoglobin from the taut to the relaxed conformation D. Acidosis shifts the oxygen-dissociation curve to the left E. Acidosis decreases the ability of 2,3-BPG to bind to Hemoglobin. MCQ 4