Haemoglobinopathies

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  • Each globin chain is linked to a haem molecule, an iron containing protoporphyrin ring
  • Hb F is the major foetal haemoglobin and is gradually replaced by Hb A from birth . Most Hb is Hb A by about 6 – 9 months
  • Heterozygote - Rarely of clinical significance but important in A/N screening Homozygote - Often of major clinical significance
  • Majority occur in the beta chain. The position of the substitution governs the effect it will have on the molecule
  • Trait - Rarely has any clinical effects, care should be taken when undergoing anaesthesia and in pregnancy SCD -
  • Trait - Rarely has any clinical effects, care should be taken when undergoing anaesthesia and in pregnancy SCD -

Transcript

  • 1. The Haemoglobinopathies: An Introduction Tony Roscioli Clinical Geneticist, Royal Prince Alfred Hospital & NHMRC Research Post-graduate Fellow, Centre for Vascular Research, UNSW
  • 2. Haemoglobin disorders as a model
    • An Autosomal recessive disease of global importance
      • >5% of the world population are Haemglobinopathy carriers
      • >3/1000 liveborn babies are affected
    • Population carrier screening (basic haematology) and prenatal diagnosis available for 20 years
    • These services have spread globally:
      • extensive experience in community implications of genetic screening
  • 3. Autosomal Recessive Inheritance • When both parents are heterozygotes – 1/4 offspring affected, 2/4 will be carriers, 1/4 unaffected in each pregnancy Unaffected Thalassaemia: An Important Diagnosis because of Recurrence Aa Aa Aa Aa aa AA
  • 4. Haemoglobin Structure
    • 2 dissimilar pairs of globin chains each linked to a single haem molecule forming a tetramer
    • Normal adult Hbs consists of 2  chains + another pair
  • 5. Adult Haemoglobins
    • Hb A (  2  2 )
      • Major adult haemoglobin
      • 95% of total
    • Hb A2 (  2  2 )
      • ~ 2.5% of adult Hb
      •  chain differs from  at ~ 10% of residues
      • function unclear
    • Hb F (  2  2 )
      • about 1%
  • 6. Fetal Haemoglobins
    • HbF  2  2 late fetus and neonate
      • Replaced by HbA at 3-6 months
      •  chain differs from  at 25% residues
      • Higher affinity for O2 than HbA: increases fetal oxygenation
      • 2 HbFs in humans:  chains differing by 1 AA
    • Hb Gower : Embryonic
      • Gower 1:  2  2 (  similar to  , 20% difference)
      • Gower 2:  2  2 (  similar to  , 40% difference)
    •  ,  ,  ,  chains can all form tetramers,
    •  does not form tetramers
  • 7. Developmental Pattern of Human Haemoglobins Based on Voet & Voet (1995)
  • 8. What are Haemoglobinopathies ?
    • Thalassaemia: Quantitative defects:
        • Reduced synthesis of a normal globin chain
      •  + or  + thalassaemia
        • Absent synthesis of a normal globin chain
      •  0 thalassaemia or  0 thalassaemia
        • Unequal  and  globin chain production
    • Haemoglobinopathy: Qualitative defects
      • Normal synthesis of an abnormal globin chain, due to amino acid substitution
        • eg Sickle Cell Disease
    • Heterozygote: Trait
    • Homozygote: Disease
  • 9. Why do Haemoglobinopathies occur?
    • Vast majority are inherited
    • Spontaneous mutations related to inherent gene structure
      • Ancestral gene deletions
      • Occasional spontaneous mutation
  • 10. Haemoglobin Gene Clusters      2  2  1  G  A     40 20 60 Kbp Chromosome 16 Chromosome 11  1   
  • 11. MECHANISM OF GENE DUPLICATION/DELETION
  • 12. Deletions in the  Haemoglobin Gene Cluster HPFH = hereditary persistent fetal haemoglobin  0 thalassaemia Hb Lepore G  A   thalassaemia G  A  HPFH G  HPFH Hb Kenya kbp  G  A     40 20 60
  • 13. Why do Haemoglobinopathies Persist?
    • ?Selecitve advantage in heterozygotes
    • ?protection against malaria?
    • Similar explanation for G6PD deficiency
  • 14.  
  • 15.  
  • 16.  
  • 17. Thalassaemia Phenotype
      • Altered  and  globin chain ratios
        • Anaemia
      • Intramedullary and extramedullary (spleen) haemolysis of abnormal blood cells
        • hypersplenism
        • anaemia
    • Homozygotes usually diagnosed in infancy
        • Pale, unwell
        • Splenomegaly and hepatomegaly common
        • Severe microcytic,hypochromic anaemia
    • Heterozygotes often chance finding
  • 18. Treatment
    • Homozygote patients are transfusion dependent
    • Problems of transfusion
      • Iron overload – chelation is required
        • cardiomyopathy/diabetes/cirrhosis/pituitatry
      • Risk of allo antibody production
      • Risk of transfusion related infection
        • Hep B / C / HIV
  • 19. Diagnosis of Haemoglobinopathies
    • Family history
      • Ethnic origin
    • Clinical signs/symptoms
    • Laboratory investigations
      • Full blood count and film
      • Ferritin
      • HbEPG
      • Sickle screen
      • HbH preparation
    • Molecular Genetic and Family studies
  • 20. Diagnosis of  Thalassaemia
    • microcytic, hypochromic RBC
    • Anaemia
    • Ferritin normal
    • blood film
    • HbEPG:  HbA 2 (  2  2 )
    • (  /  biosynthesis ratio >1)
    • DNA analysis (point mutations)
    • NB Family Studies
  • 21. Effect of β - thalassaemia on adult red cells
    • MCH = 30pg
    • Hb A = 97%
    • Hb A 2 = 3%
    • Normal
    MCH = 20pg Hb A = 96% Hb A 2 >3.5% β thal Trait MCH = 15-30pg Hb F >80% β thal Major/Intermedia         
  • 22. Exon 1 Exon 2 Exon 3 IVS1 IVS2 Splicing Mutations Nonsense Mutations Poly A site Deletions (rare) Initiator Codon Transcription Insertions Frameshift Deletions (-1, -2, -4)  -Globin
  • 23. Diagnosis of  Thalassaemia
    • (microcytic, hypochromic RBC)
    • (anaemia)
    • blood film
    • HbH inclusions ****
    • HbEPG:  HbA2 and HbF in  thal trait
    • (  / b biosynthesis ratio <1)
    • DNA analysis
    • NB Family Studies
  • 24. Effect of  - thalassaemia on fetal red cells MCH = 35pg Hb F = 92% Hb A = 8% Normal MCH = 20pg Hb F = 89% Hb  4 = 3% Hb A = 8%   thal Trait MCH = 20pg Hb  4 = 80% Hb  2  2 = 10%   thal Major Hb Barts Hydrops Fetalis         
  • 25.  Thalassaemia
    • 2 genes inherited from each parent – more heterogenous picture
      • Usually caused by gene deletions
      • Clinical picture varies with number of deletions
    • Can result in:
      • 1. One gene deletion: silent carrier
      • 2. Two gene deletions:  thal trait
     o : both genes on one chromosome deleted  + : one gene on each chromosome deleted or or ( )
  • 26. Forms of  thalassaemia
    •    Thalassaemia caused by intragenic deletions
     2  1   1 4.2 Kb 3.7 Kb  1   1   1  2  1   1  0 -Thalassaemia caused by large intragenic deletions  2  1   1    Thalassaemia caused by point mutations: Hb Constant Spring
  • 27.  Thalassaemia Genotype No. Genes Functional Phenotype α α / α α - α / α α - α / - α -- / α α - α / -- -- / -- 4 3 2* 2* 1 0 Normal Normal MCV, MCH MCV, MCH HbH disease Hb Barts hydrops fetalis
  • 28. Distribution of  thalassaemia
    • Similar geographical range to β -thalassaemia but particularly associated with SE Asians and widespread in Africa
    • Follows malaria distribution
    •    3.7Kb del: Africa, Mediterranean, Bangladesh,
    • India, Pakistan, Melanesia
    •    4.2Kb del: South East Asia, Melanesia
    •    Mediterranean: Cyprus, Greece, Turkey,
    • Sth Italy
    • Asian: China, Thailand, Cambodia,
    • Philippines, Vietnam
  • 29. Molecular Testing
    • Complex but the reference laboratory for NSW is in our health area!
    • Clinical Genetics and Molecular Genetics infrastructure
      • Ron Trent
      • Robert Ogle
      • Tony Roscioli
      • Bronwyn Culling
      • Gayathri Parasivam
  • 30. Substitution Haemoglobinopathy
    • Amino acid change in globin chain
    • May be insignificant with no effect
    • May cause functional changes eg
      • Altered oxygen affinity
      • Loss of molecular stability
      • Polymerisation
    • Eg HbS, HbC, HbE
  • 31. Diagnosis of Sickle Cell Disease
    • Low Hb, increased WBC
    • Marked anisocytosis, poikilocytosis, polychromasia, target cells and sickle cells
    • NB Must do HbEPG as sickle cell trait can be missed on blood film alone
  • 32. Sickle Cell Disease
    • Single amino acid substitution on  chain
      • Valine for Glutamic acid at position 6
    • Sickle cell trait – 40% HbS - AS
    • SCD - homozygote – 90% HbS - SS
      • Change in Hb solubility in low O 2 tension
      • Structural changes  sickling
      • Loss of flexibility  destruction
      • increased blood viscosity
  • 33. Sickle Cell Disease
    • Anaemia
    • Splenic atrophy
    • Increased risk of infection
    • Crises
      • Vaso-occlusive/Painful
      • Haemolytic
      • Aplastic
  • 34. Thalassaemia in NSW
    • 1998: 123 people with transfusion-dependent thalassaemia
      • 106 (86%) homozygous  thalassaemia
      • 17 (14%) other thalassaemia/haemoglobinopathies
    • 1989-1998: 23 new cases
    • Increased detection rates for homozygous  thalassaemia
      • Major risk groups Asian and Middle eastern
  • 35. 100% 23 TOTAL 13.1% 3 India 26.2% 6 Meidterranean/ Africa 26.2% 6 South East Asia 34.5% 8 Middle East Percentage No of Diagnoses Ancestry
  • 36.
    • “ It is important to consider that an individual could be a genetic carrier for thalassaemia or HbE whenever a blood count shows a low MCV or MCH, particularly if an individual or their ancestors have originated from an at-risk group. The blood picture described is frequently mistaken for iron deficiency. It is also important to note that both iron deficiency due to other causes and thalassaemia can co-exist in the same individual. Therefore a low MCV or low MCH which appears to be refractory to iron treatment should be considered to represent thalassaemia minor until proven otherwise.”
    • NSW Health Department Guidelines, 1998
  • 37. Definitions: Genetic Testing and Genetic Screening
    • Genetic testing : providing a genetic test to someone who is thought to be at increased risk eg. Affected relative, member of at risk group
    • Genetic screening : offering a test to all members of a population to identify those at increased risk, for genetic testing
  • 38. Core Ethical Principles of Genetic Counselling*
    • The autonomy of individual and couple
    • Their right to full information
    • Strict confidentiality
    • *Fletcher, Berg and Trannoy 1985, endorsed by WHO, 1985
  • 39.  
  • 40. NSW Health Department Guidelines and take home messages
    • Haemoglobinopathy screening (FBC, Film, HbEPG, Iron studies):
      • Anyone with a blood count showing a low MCH or MCV
      • People whose origins are from Southern Europe, Africa, Middle east and Asia, India
      • All pregnant women when they are having routine antenatal blood examinations
      • Too difficult to target specific groups: surnames and populations changing!
    • Cascade counselling/testing!
    • Clinical Genetic Service RPAH/Thalassaemia Service RPAH
      • 9515 5080 / 9515 6111