haemoglobinopathies and thalassemias

week 2 blood sciences

regulation of RBC production

• hypoxic inducible factor (HIF)

• blast forming unit (BFU-E)

• colony forming unit (BFU-E)

• transferrin receptor (TfR)

haemoglobin genes

• 2 α- globin genes located on chromosome 16

• 2 β-globin genes located on short arm on chromosome 11

• globin genes are developmentally regulated

Hb production affected by

• globin chain synthesis

• Fe levels

• porphyrin levels

classification of genetic Hb disorders

haemoglobinopathies: inherited single-gene disorders causing defects in globin chain structure

often manifests as haemolytic anaemia

thalassaemias: autosomal recessive blood disorders causing defects in α or β globin chain synthesis

often manifests as hypochromic (MCH) microcytic anaemia

normal Hb

• normal genotype is AA

• represents receiving one normal Hb A from each parent

lab tests of Hb

• wet prep and sickle solubility

• Hb electrophoresis

• immunoassays

• high performance liquid chromatography

• DNA analysis

wet prep

• EDTA whole blood is dropped onto a slide with sodium metabisulfite

• after incubation, 30-60 sample is examined microscopically

sickle solubility

• saponin and sodium dithionite is added to whole blood

• then mixed with phosphate buffer

• sickle Hb becomes opaque against line test card

Hb electrophoresis

used to identify and quantify both normal and variant Hbs

immunoassays

• ELISA of Hb

• flow cytometry

HPLC

• high performance liquid chromatography is used to identify and quantify more complicated Hbs

Hb S

• single point mutation

• glutamic acid replaced by valine at sixth position of β globin chain

• those of African descent exhibit the highest frequency of the homozygous genotype

DNA analysis

• reference laboratories

• confirmation of serious Hb variants

sickle cell trait

• genotype (AS): heterozygous

• carrier

• produce both normal and abnormal Hb

• generally asymptomatic

• 40% Hb S and 60% HbA

• protection against malaria

HPLC (high performance liquid chromatography)

• technique to detect different Hb variants

• if two Hbs have similar charge, difficult to detect on gel electrophoresis

• e.g: S and G variants are similar so HPLC is preferred

sickle cell anaemia

• genotype SS (homozygous)

• produce abnormal Hb

• anaemia as a result of haemolysis

• sickling occurs under conditions of deoxygenation such as exercise, infection and acidosis

• RBC lifespan (20 days)

sickling

• normally no interaction between Hb molecules

• valine substitution causes abnormal β chain interaction

• valine at position 6 on one β chain fits into a pocket between phenylalanine in position 85 and leucine in position 88 of adjacent β chain

determinants of HbS polymerisation

• HbSS red cells

  • MCHC 32g/dl

  • deoxyHbS solubility 16g/dl

• factors affecting polymerisation

  • intracellular Hb, Hb composition, 02 saturation

symptoms

• anaemia:

  • fatigue

  • shortness of breath

  • fast heartbeat/palpitations

• sickle cell crisis (vaso-occlusive episode)

  • inflammation

  • severe pain

• other

  • jaunfice

  • avascular necrosis

  • leg ulcers

clinical presentation

• painful vaso-occlusive crisis

• blood contains reticulocytes, erythroblasts (codocytes (target cells) and drepanocytes (sickle cells) )

• spleen damage from infarctions (autosplenectomy) causes Howell-Jolly bodies

sickle solubility test

• only sickling Hbs produce a positive result

• all other Hbs are negative

Hb E

• single point mutation

• glutamic acid replaced by lysine at 26th position of the β globin chain

• alternative mRNA splicing site at codons 25-27

• production of anomalous β RNA may cause a deficiency of normal β chains, which may cause β thalassaemia

• can be homo or hetero

Hb E disease

• genotype EE (homozygous)

• benign condition despite causing haematological abnormalities in the FBC

• approximately 98% Hb E

• mild microcytic anaemia and MCV 65 fl

• blood often contains codocytes

Hb E trait

• genotype AE (heterozygous)

• asymptomatic

• may cause low mean corpuscular volume and presence of codocytes

• if mutation occurs in the protein coding region, it would have a serious impact but if in splice slice, not very serious

Hb C

• single point mutation

• glutamic acid replaced by lysine at sixth position of β globin chain

Hb C disease

• genotype CC (homozygous)

• Hb C less soluble than Hb A and forms characteristic crystals

• Hb does not polymerise like HbSS but does crystallise with reduction in flexibility of RBC in its survival

• RBC lifespan 32 days

• splenomegaly due to destruction of RBCs containing crystalline structures

clinical presentation of Hb C disease

• blood film with microcytosis, codocytes and nucleated erythrocytes

• 90-95% Hb C

Hb C trait

• genotype AC (heterozygous)

• benign disorder

• may have slight low MCV with occasional codocyte

• 40% HB C and 60% Hb A

haemoglobin SC disease

• genotype SC (doubly heterozygous)

• conc of Hb S and Hb C is 5050

• less sickling than with sickle cell anaemia

• generally benign but with increased avascular necrosis of the femoral head

• low MCV and high MCHC

• occasional codocyte and crystals

other haemoglobinopathies

• Hb D punjab

  • single point mutation, glutamic acid replaced by glutamine at the 121st position of the β globin chain

  • asymptomatic

• Hb SD

  • SD (doubly heterozygous)

  • the presence of Hb D enhances sickling of Hb S due to co-polymerisation, severe sickling disorder

haemoglobin SO disease

• genotype: SO (double heterozygous)

• sickling anaemia: similar in severity to sickle cell anaemia

• Hb 7-8 g/dL with prominent reticulocytes

• clinically mimic sickle cell anaemia and requires blood transfusions

• double heterozygous HbO/β+ thalassemia is also associated with a relatively severe microcytic anaemia with Hb as low as 6 g/dl

Hb G Philadelphia

• genotype: AG or GG

• α- chain point mutation

• asparagine replaced by lysine at 68th position of α globin chain

• mild microcytosis associated to possible gene deletions, greater than 40% Hb G may be present

• migrated with Hb S on electrophoresis, so may be mistaken if further testing such as HPLC is not performed

Hb O Arab

• genotype: AO and OO

• single point mutation

• glutamic acid replaced by lysine at 121st position of the β globin chain

• AO asymptomatic

• OO causes mild microcytic anaemia

Hb Lepore

• genotype: hetero or homozygous

• non homologous crossover of 2 gene loci during meiosis

• 2 α chains, 2 δ β fusion globins that arise from unequal crossover between δ and β globin chains

• fusion of residues 1-87 of the δ chain with residues 116-146 of the β chain

• synthesised at a decreased rate causing thalassaemic picture

• heterozygotes have 12% Hb Lepore, low MCV and high RBC count

• homozygotes have anaemia similar to thalassaemia intermedia

α- thalassaemias

• 2 α globin genes are called HBA1, HBA2

• gene deletions are most common cause of α thalassaemia, however gene mutations may be cause rarely

• disorder more severe if occurring from a gene mutation

• each person should inherit one HBA1 and HBA2 from each parent

α thalassaemia silent carrier

• genotype /α α/ α

• asymptomatic as 3 functional globin genes permit normal Hb production, can be reduced MCV

α thalassaemia trait

• genotype α/α-/- (alpha-thal-1) or -/α-/α (alpha-thal-2)

• 2 functional genes allow almost normal erythropoesis

• mild microcytic hypochromic anaemia may be present that is often treated as an iron deficiency anaemia

α thalassaemia trait continued

• alpha-thal-1 caused by cis deletion of HBA1 and HBA2 on the same chromosome

• alpha-thal-2 caused by trans deletion of HBA1 and HBA2 on different but homologous chromosomes

• alpha-thal-1 is common in the African population

• alpha-thal-2 common in Asian population

Hb H disease

• genotype -/- -/α

• due to lack of α chains, 2 unstable Hb forms exist in the blood, Hb Barts and Hb H

• Hb Barts = tetramer of γ- chains whilst Hb H is tetramer of β-chains

• both have a higher affinity for oxygen than Hb A resulting in little delivery of 02 to tissues

symptoms

• spenomegaly and anaemia

• erythrocytes are microcytic and hypochromic, with codocytes and Heinz bodies present on blood film

• Hb 9.5 g/dl

• haemolytic crises commonly accompany infections

Hb Barts

• genotype -/- - /-

• due to complete lack of α globin chains the foetus is unable to survive outside the uterus and commonly may not survive gestation

• most are stillborn with hydrops fetalis whilst those born alive die shortly after

• they are oedematous with very little circulating Hb of which all if Hb Barts

inheritance of alpha thalassaemias

• each person inherits two alpha globin genes from each parent

• if both are missing at least one alpha globin alleles there children are at risk of having Hb Bart Syndrome, HbH disease or alpha thalassemia

β thalassaemias

• β globin chain (HBB) is located on the short arm of chromosome 11 in region containing the δ globin gene

• most mutations are single nucleotide substitutions, deletions, or insertions of oligonucleotides leading to frameshift

• rarely, β thalassaemias result from gross gene deletion

β thalassaemia genotype

• β indicates the normal production of β chain

• β+ indicates a reduced production of β chain

• β 0 indicates a complete lack of production of β chain

• β thalassaemias are classified on phenotype (severity of symptoms) rather than genotype

β thalassaemia minor

• genotype: β/β+ or β/β0

• one β globin chain allele is mutated

• microcytic anaemia will be present with a low MCV

β thalassaemia intermedia

• genotype: β/β0 or β+/β0 or β+/β+

• 2 globin genes are mutated to produce intermediate symptoms, however β/β0 may also

• fairly severe anaemia which may require transfusions during periods of bodily stress

• frequent blood transfusions may lead to Fe overload (chelation therapy may be required)

β thalassaemia major

• both β-globin alleles have mutations

• presents within the first two years

• causes severe microcytic hypochromic anaemia

• can also cause splenomegaly and bone deformities if left untreated

• frequent transfusions required, along with iron
  chelation.

• splenectomy may be necessary if
  splenomegaly present

• A bone marrow transplant is a possible long term cure