Haemoglobinopathies

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Haemoglobinopathies

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

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