HEMOGLOBINOPATHY

DR. LAXMI KANTA SAY

DEFINITIONDEFINITION

Hemoglobins with altered amino acid Hemoglobins with altered amino acid sequences that result in deranged sequences that result in deranged function or altered physical or function or altered physical or chemical properties.chemical properties.

CLASSIFICATION OF HEMOGLOBINOPATHIES

1) STRUCTURAL GENE MUTATION1) STRUCTURAL GENE MUTATION Replacement of a single amino acid residue

of HbA1, e.g. Hb-S, Hb-M, Hb-C, Hb-D(Panjab)

2) REGULATOR GENE MUTATION2) REGULATOR GENE MUTATION Rate of synthesis of peptide chains , the Rate of synthesis of peptide chains , the

amino acid sequence remaining unaffectedamino acid sequence remaining unaffected

e.g. e.g. -- thalassemia, thalassemia, ßß –thalassemia, –thalassemia, ß, ß, ß ß,etc,etc..

3) ABNORMAL Hb. WHICH PRODUCE 3) ABNORMAL Hb. WHICH PRODUCE METHAEMOGLOBINEMIAMETHAEMOGLOBINEMIA

e.g. Hb-M(Iwate), Hb-Sabinee.g. Hb-M(Iwate), Hb-Sabine

CLASSIFICATION CONTINUEDCLASSIFICATION CONTINUED

4) ABNORMAL Hb. ASSOCIATED WITH HIGH O2 –4) ABNORMAL Hb. ASSOCIATED WITH HIGH O2 –AFFINITYAFFINITY

e.g. Hb-Chesapeake, Hb-Rainere.g. Hb-Chesapeake, Hb-Rainer

5)ABNORMAL Hb- WHICH INTERFER WITH 5)ABNORMAL Hb- WHICH INTERFER WITH

m-RNA FORMATIONm-RNA FORMATION

e.g. Hb-Constant Spring ,Hb-D(Panjab), Hb-e.g. Hb-Constant Spring ,Hb-D(Panjab), Hb-E,E,

Hb-CHb-C

6)6)HEREDITARYHEREDITARY persistence of fetal persistence of fetal hemoglobinhemoglobin

Historical aspects of Historical aspects of sicklesickle cell cell diseasedisease

Earliest paper describing SCD in the West Earliest paper describing SCD in the West was published in 1846 in the Southern was published in 1846 in the Southern Journal of Medical Pharmacology.Journal of Medical Pharmacology.

11stst case described in literature is a case described in literature is a Caribbean dental student named Walter Caribbean dental student named Walter Clement Noel reported in 1910 by Dr Clement Noel reported in 1910 by Dr James Herrick.James Herrick.

Mason gave the term Sickle cell anemia Mason gave the term Sickle cell anemia in 1922.in 1922.

Substitution of valine or lysine residue Substitution of valine or lysine residue for glutamic acid at position 6 in the for glutamic acid at position 6 in the ß-subunit of hemoglobin. ß-subunit of hemoglobin.

HB A

HB S

SICKLE CELL ANAEMIASICKLE CELL ANAEMIA

DIAGRAMATIC REPRESENTATION OF DIAGRAMATIC REPRESENTATION OF STICKY PATCHSTICKY PATCH

FACTORS AFFECTING RATE OF FACTORS AFFECTING RATE OF SICKLINGSICKLING

Amount of HbS Amount of HbS Rate of HbS polymerization depends upon Rate of HbS polymerization depends upon

Hb concentrationHb concentration Decrease in PDecrease in PHH – increased tendency for – increased tendency for

sicklingsickling Sluggish blood flow in microvascular beds Sluggish blood flow in microvascular beds Decreased body temperature causes Decreased body temperature causes

vasoconstriction,leadsvasoconstriction,leads

to sickling.to sickling. Increased blood viscosity due to Increased blood viscosity due to

dehydrationdehydration

MANIFESTATIONSMANIFESTATIONS

Hemolytic crisisHemolytic crisis

Characterized by rapidly developing anaemia , Characterized by rapidly developing anaemia , leukocytosis, jaundice and fever.leukocytosis, jaundice and fever.

Vicious circle of events – low oxygen tension in Vicious circle of events – low oxygen tension in tissues causes sickling, leads to ruptured red cells, tissues causes sickling, leads to ruptured red cells, which causes a further decrease in oxygen tension which causes a further decrease in oxygen tension and still more sickling and red cell destruction.and still more sickling and red cell destruction.

Serious decrease in red blood cells within a few Serious decrease in red blood cells within a few hourshours

Characteristic finding – reticulocytes in peripheral Characteristic finding – reticulocytes in peripheral smear, fragmented RBCs smear, fragmented RBCs

Acute Splenic Sequestration Crisis Acute Splenic Sequestration Crisis

Defined by decrease in Hb by 2gm/dl from steady Defined by decrease in Hb by 2gm/dl from steady

state Hb concentrationstate Hb concentration Due to intrasplenic trapping of red Due to intrasplenic trapping of red

cells,erythrostasis cells,erythrostasis

leads to tissue leads to tissue hypoxia,thrombosis,infarction,fibrosishypoxia,thrombosis,infarction,fibrosis

Continuous scarring-- shrinkage of spleen, Continuous scarring-- shrinkage of spleen, Autosplenectomy Autosplenectomy

11stst attack between 3months - 5yrs age attack between 3months - 5yrs age Manifests as: sudden weakness, pallor, Manifests as: sudden weakness, pallor,

tachycardia, tachycardia,

tachypnea, abdominal fullnesstachypnea, abdominal fullness Risk of infection by Streptococcus pneomoniaeRisk of infection by Streptococcus pneomoniae

Aplastic Crisis Aplastic Crisis

Decreased red cell survival is compensated by 6-Decreased red cell survival is compensated by 6-8 fold 8 fold

increase in bone marrow output.increase in bone marrow output. Temporary cessation of marrow activity due to Temporary cessation of marrow activity due to

viral orviral or bacterial infections causes hematocrit to fall by bacterial infections causes hematocrit to fall by

10-15% / day.10-15% / day. Organ involvement:Organ involvement: Retina: vessel occlusion – hemorrhage, Retina: vessel occlusion – hemorrhage, - neovascularization,detachments- neovascularization,detachments Priapism: due to involvement of penile venous Priapism: due to involvement of penile venous outflow tracts.outflow tracts. - painful erection & permanent impotence- painful erection & permanent impotence

Pregnancy outcome: increased Pregnancy outcome: increased incidence spontaneous incidence spontaneous abortions,IUGR, premature laborabortions,IUGR, premature labor

postpartum infections.postpartum infections. complications increase in complications increase in

pregnancypregnancy Pregnancy outcome unaffected by Pregnancy outcome unaffected by

sickle cell trait.sickle cell trait.

DIAGNOSISDIAGNOSIS

1.1. Full blood count: Hb level low,between 6-9 g/dlFull blood count: Hb level low,between 6-9 g/dl

2. Peripheral smear: sickle cells,Howell jolly 2. Peripheral smear: sickle cells,Howell jolly bodies,bodies,

sideroblasts. target cellssideroblasts. target cells

3.3. Sickling test:Sickling test:

4.4. Hb electrophoresis: HbS pedominates,No Hb electrophoresis: HbS pedominates,No normalnormal

HbA, HbF present around 2-20% HbA, HbF present around 2-20%

5.5. Hemoglobin HPLC: for identification and Hemoglobin HPLC: for identification and quantification of variant Hb as well as HbA2 , quantification of variant Hb as well as HbA2 , HbFHbF

6.6. DNA analysis: for defining the mutation DNA analysis: for defining the mutation

Sickle Cell Anemia – blood filmSickle Cell Anemia – blood film

Sickle Cells

Erythroblasts

Howell-Jolly Body

RECENT ADVANCES IN TREATMENTRECENT ADVANCES IN TREATMENT1.1. Bone marrow transplantation -Bone marrow transplantation - - - in < 16 yrs, to treat sickle cell anaemia with in < 16 yrs, to treat sickle cell anaemia with repeated complications.repeated complications. - can be curative.- can be curative. - barrier is a HLA – matched sibling as donor- barrier is a HLA – matched sibling as donor - expensive, not widely available- expensive, not widely available2.2. Anti-tumor drug - Anti-tumor drug - 5-deoxyazacytidine in low 5-deoxyazacytidine in low

dose,dose, 3.3. Gene therapy - Gene therapy - under study ,to inactivate sickle under study ,to inactivate sickle

gene,gene, - increase expression of gene for HbF- increase expression of gene for HbF3.3. Cord-blood stem cells - Cord-blood stem cells - 4.4. Arginine butyrate Arginine butyrate - increase fetal Hb production- increase fetal Hb production5.5. Poloxamer 188 Poloxamer 188 - to reduce length of pain crisis - to reduce length of pain crisis 6.6. Nitric oxide - Nitric oxide - to manage acute chest syndrometo manage acute chest syndrome

THALASSEMIA THALASSEMIA

Heterogenous group of inherited disordersHeterogenous group of inherited disorders Genetic lesions leads to decreased synthesis of either Genetic lesions leads to decreased synthesis of either

alpha or alpha or beta - globin chain of HbAbeta - globin chain of HbA BETA THALASSEMIASBETA THALASSEMIAS Diminished synthesis of structurally normal beta- globin Diminished synthesis of structurally normal beta- globin

chainschains unimpaired synthesis of alpha- chainsunimpaired synthesis of alpha- chains MOLECULAR PATHOGENESIS:MOLECULAR PATHOGENESIS: - HbA- tetramer of 2alpha,2beta chains encoded by a pair - HbA- tetramer of 2alpha,2beta chains encoded by a pair

of a-of a- globin genes on chromosome 16 and a single b-globin globin genes on chromosome 16 and a single b-globin

gene ongene on chromosome 11 resp.chromosome 11 resp. - B- B00 – thalassemia- total absence of b- globin chains – thalassemia- total absence of b- globin chains - B- B++ -- thalassemia- reduced b-globin chains -- thalassemia- reduced b-globin chains - most are point mutations - most are point mutations

PATHOGENESIS OF BETA THALASSEMIA PATHOGENESIS OF BETA THALASSEMIA MAJORMAJOR

SCANSCAN

THALASSEMIA MAJOR :THALASSEMIA MAJOR : - Homozygous for beta-thalassemia genes.- Homozygous for beta-thalassemia genes. - Severe transfusion dependent anaemia- Severe transfusion dependent anaemia THALASSEMIA MINOR /TRAIT :THALASSEMIA MINOR /TRAIT : - Heterozygous with 1 beta-thalassemia - Heterozygous with 1 beta-thalassemia

gene gene and 1 normal geneand 1 normal gene - mild hemolytic anaemia,no symptoms- mild hemolytic anaemia,no symptoms BETA-THALASSEMIA INTERMEDIA BETA-THALASSEMIA INTERMEDIA - intermediate form- intermediate form

THALASSEMIA MAJOR (COOLEY’S THALASSEMIA MAJOR (COOLEY’S ANAEMIA)ANAEMIA)

In 1925 Thomas B Cooley gave the first In 1925 Thomas B Cooley gave the first description of severe thalassemia .description of severe thalassemia .

11stst case in India reported by Dr case in India reported by Dr Mukherjee in 1938.Mukherjee in 1938.

Common in Mediterranean Common in Mediterranean countries,parts of Africa,Southeast Asia.countries,parts of Africa,Southeast Asia.

Anaemia manifests 6-9 months after Anaemia manifests 6-9 months after birth as Hb synthesis switches from HbF birth as Hb synthesis switches from HbF to HbA.to HbA.

Hb levels remain low between 3-9 g/dlHb levels remain low between 3-9 g/dl

Clinical features:Clinical features: Child presents in 1Child presents in 1stst yr of life with yr of life with

lethargy,poor lethargy,poor

feeding,recurrent infections,massive bone feeding,recurrent infections,massive bone marrowmarrow

expansion,delayed growth and development.expansion,delayed growth and development. Characteristic facies Characteristic facies –chipmunk ,with –chipmunk ,with

maxillary marrow hyperplasia, frontal maxillary marrow hyperplasia, frontal bossingbossing

Hemolytic anaemia Hemolytic anaemia - - hepatosplenomegaly- - hepatosplenomegaly Cardiac problems Cardiac problems - - secondary to iron - - secondary to iron

overload, cardiomegaly,l eft ventricular overload, cardiomegaly,l eft ventricular overload, heart failure, arrhythmiasoverload, heart failure, arrhythmias

EndocrineEndocrine -- diabetes mellitus, -- diabetes mellitus, hyperthyroidism, hypogonadism, hyperthyroidism, hypogonadism, hyperparathyroidismhyperparathyroidism

Spinal cord compression Spinal cord compression -- epidural -- epidural extramedullary hematopoiesis,vertebral extramedullary hematopoiesis,vertebral fracturesfractures

Hemolytic Facies

THALASSEMIA MINOR:THALASSEMIA MINOR: Resistance against falciparum malariaResistance against falciparum malaria Asymptomatic,mild anaemiaAsymptomatic,mild anaemia Mild erythroid hyperplasia in bone marrowMild erythroid hyperplasia in bone marrow ALPHA – THALASSEMIAS:ALPHA – THALASSEMIAS: Reduced or absent synthesis of alpha-globin Reduced or absent synthesis of alpha-globin

chainschains Normally 4 alpha-globin genes presentNormally 4 alpha-globin genes present 4 types of clinical syndromes:4 types of clinical syndromes: Silent Carrier State Silent Carrier State - - single alpha-globin gene single alpha-globin gene

deleted, deleted, Barely detectable reduction in alpha-globin chain Barely detectable reduction in alpha-globin chain

synthesissynthesis Completely asymptomaticCompletely asymptomatic Alpha-thalassemia trait- Alpha-thalassemia trait- 2 alpha-globin gene 2 alpha-globin gene

deletiondeletion No anaemia,no physical signs,like b-thalassemia minorNo anaemia,no physical signs,like b-thalassemia minor

ALPHA – THALASSEMIASALPHA – THALASSEMIAS

Hemoglobin H Disease- Hemoglobin H Disease- 3 alpha-globin 3 alpha-globin gene deletion(gene deletion(4)4)

- most common among Asians- most common among Asians

- amount 10-20%- amount 10-20%

- extremely high affinity for oxygen,- extremely high affinity for oxygen,

- tissue hypoxia,- tissue hypoxia,

- prone to oxidation,- prone to oxidation,

- precipitates of oxidised HbH form- precipitates of oxidised HbH form

- moderately degree of haemolytic - moderately degree of haemolytic anaemiaanaemia

Hydrops Fetalis- Hydrops Fetalis- all 4 alpha-globin gene all 4 alpha-globin gene deletiondeletion

In fetus excess gamma globin chains form In fetus excess gamma globin chains form tetramers (tetramers (Hb Barts - Hb Barts - 44))

very high affinity for oxygenvery high affinity for oxygen No oxygen delivery to tissuesNo oxygen delivery to tissues Fetal distress by 3Fetal distress by 3rdrd trimester,IUD results trimester,IUD results Fetus – pallor, generalized edema, massive Fetus – pallor, generalized edema, massive

hepatosplenomegalyhepatosplenomegaly Intra-uterine transfusion can save such infantsIntra-uterine transfusion can save such infants

DIAGNOSIS OF THALASSEMIASDIAGNOSIS OF THALASSEMIAS

Thalassemia majorThalassemia major Diagnosed in childhood – severe anaemia Hb ,3-6g/dlDiagnosed in childhood – severe anaemia Hb ,3-6g/dl

characteristic facies with fronto-parietal & occipital characteristic facies with fronto-parietal & occipital bossingbossing

malar prominence,malocclusion of teethmalar prominence,malocclusion of teeth HepatosplenomegalyHepatosplenomegaly Peripheral smear Peripheral smear – anisocytosis , poikilocytosis, – anisocytosis , poikilocytosis,

hypochromia,target cells,fragmented RBCshypochromia,target cells,fragmented RBCs Increased reticulocytes 2-4%Increased reticulocytes 2-4% Hb electrophoresis- Hb electrophoresis- Lack of HbA, elevated HbF, Lack of HbA, elevated HbF,

HbA2HbA2 Iron studies Iron studies – increased serum iron level & ferritin– increased serum iron level & ferritin Skull X-ray Skull X-ray – characteristic hair on end appearance– characteristic hair on end appearance

Beta-Beta- Thalassemia minor/traitThalassemia minor/trait Mild anaemiaMild anaemia Microcytosis,hypochromia,target Microcytosis,hypochromia,target

cellscells Hb electrophoresis – HbA2,HbF Hb electrophoresis – HbA2,HbF

increasedincreased

Alpha- Thalassemia traitAlpha- Thalassemia trait Mild hypochromia,micrcytosis Mild hypochromia,micrcytosis

without anaemiawithout anaemia

MANAGEMENT OF THALASSEMIASMANAGEMENT OF THALASSEMIAS

1.1. Chronic transfusion therapy- maintain Chronic transfusion therapy- maintain hematocrit at 25-30% to suppress hematocrit at 25-30% to suppress erythropoiesis, prevent anaemiaerythropoiesis, prevent anaemia

2.2. Splenectomy- in splenomegaly with Splenectomy- in splenomegaly with hypersplenism signs & child is >5yrs of agehypersplenism signs & child is >5yrs of age

3.3. Chelation therapy- prevention and Chelation therapy- prevention and treatment of iron overloadtreatment of iron overload

4.4. Vaccination against PneumococcusVaccination against Pneumococcus

5.5. Bone marrow transplantationBone marrow transplantation

6.6. Cord blood transfusionCord blood transfusion

7.7. Gene therapy – insertion of normal genes in Gene therapy – insertion of normal genes in the stem cells of recepientsthe stem cells of recepients

METHAEMOGLOBINEMIAMETHAEMOGLOBINEMIA

Hb-M(Iwate)Hb-M(Iwate)

Histidine at position 87 of Histidine at position 87 of - - chain is replaced by chain is replaced by Tyrosine. Tyrosine.

The O2 –affinity is much lower than normal variant.The O2 –affinity is much lower than normal variant.

Heme the Hb of the variant chains is spontaneously Heme the Hb of the variant chains is spontaneously oxydized to the ferric state,thus producing oxydized to the ferric state,thus producing methaemoglobinmethaemoglobin..

Hb-Sabine;Hb-Sabine;

Substitution in Substitution in ß-ß-chain of proline for Leucine at position chain of proline for Leucine at position 91.91.

This makes possible for methaemoglobin. This makes possible for methaemoglobin.

HIGH O2-AFFINITYHIGH O2-AFFINITY

Hb-Chesapeake;Hb-Chesapeake;

Substitution of Substitution of ArginineArginine for for LysineLysine in in 9292 position of position of -chains. Formation of ‘R’ form, -chains. Formation of ‘R’ form, increasing Oincreasing O22 affinity resulting in tissue affinity resulting in tissue hypoxia & compensatory polycythemia.hypoxia & compensatory polycythemia.

Hb-RainierHb-Rainier;;

Substitution of Substitution of TyrosineTyrosine at at 145145 by by HistidineHistidine, , which modifies salt bridges of de-oxy form which modifies salt bridges of de-oxy form and favours stabilization of ‘R’ form resulting and favours stabilization of ‘R’ form resulting in tissue hypoxia and compensatory in tissue hypoxia and compensatory polycythemia polycythemia

ABNORMAL Hb. WHICH INTERFERE ABNORMAL Hb. WHICH INTERFERE WITH m-RNA FORMATIONWITH m-RNA FORMATION

Hb-Constant Spring;Hb-Constant Spring;

It produces an unstable m-RNA.It produces an unstable m-RNA.

Stop codon ‘UAA’ mutated for ‘CAA’ which codes Stop codon ‘UAA’ mutated for ‘CAA’ which codes for Glutamine. Hence excess for Glutamine. Hence excess --chain of 31 chain of 31 residues longer than normal 172 in place of 141.residues longer than normal 172 in place of 141.

-Unstable & degraded easily-Unstable & degraded easily

Hb-C;Hb-C;

Glutamic acid acid at 6 replaced by Lysine of Glutamic acid acid at 6 replaced by Lysine of

ß-chain,ß-chain,

Seen in Negros,Seen in Negros,

Mild hemolytic anaemiaMild hemolytic anaemia

OTHER ABNORMAL Hb.sOTHER ABNORMAL Hb.s