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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