Prenatal Diagnosis of Complex Haemoglobinopathies

1. Prenatal Diagnosis of Complex Haemoglobinopathies Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital Sydney , Australia

2. Outline • Introduction • Complex haemoglobinopathies • Different ethnic groups • Gene-gene interactions • Diagnosis of complex haemoglobinopaties • Prenatal diagnosis • Case studies • Summary

3. HAEMOGLOBINOPATHIES= inherited disorders of globin divided into: Thalassaemia Syndromes (quantitative disorders) Variant Haemoglobins (qualitative disorders)

4. Structure of haemoglobin molecule

5. Haemoglobin compositions • Functioning haemoglobin (Hb) molecules are tetramers made up of two pairs of globin chains • The different types of Hb are characterised by their globin chains (γβαδ) • Hb A (α2β2) ->97% (adult) • Hb A2 (α2δ2)->2.5% (adult) • Hb F (α2γ2)-> <1% (adult) http://globin.bx.psu.edu/hbvar/menu.html

6. Inheritance patterns of thalassaemia http://www.thalassaemia.org.cy • Autosomal recessive (2 parents carriers = 1 in 4 risk ) • Compound states e.g. + & o thalassaemia = HbH disease • Combinations of  and  thalassaemias

7. RPA Experience with Ethnic Mix Mediterranean (Greek, Italian) Chinese / SE Asian Middle Eastern Indian subcontinent Unusual groups (Maldives, Eastern Europe)

8. Haemoglobinopathies & Ethnic variety •  thalassaemia: SEA, Chinese, Mediterranean, African •  thalassaemia: European, Middle Eastern, Indian and SEA populations • HbS: Mediterranean, Middle Eastern and Black African • HbE: SEA

9. α thalassamia ±Low MCV ±Low MCH Blood film ±HbH inclusions HbA2 normal Variant haemoglobins Normal MCV Hb EPG-> variant peak β thalassamia Low MCV Low MCH Blood film HbA2 Diagnosis of haemoglobinopathies:haematological features

10. Why Prenatal Diagnosis ?

11. Prenatal Diagnosis • Indications Couple at high risk of reproducing an affected fetus with • Hb Barts Hydrops Fetalis (if both are αα/-- carriers) • β Thalassaemia Major (if both are β βT/ carriers) • HbH disease (if one is αα/-- and the partner is -α/αα or ααT/αα ) • Various Hb variant in combination with β thalassaemia (ββT/ββE ) • Sources • Chorion villus sample ~11/52 • Amniocentesis ~ 15/52 • Genetic counselling in relation to prenatal diagnosis

12. Summary of mutation categoriesin database α1 globin gene mutations β globin gene mutations α2 globin gene mutations http://globin.bx.psu.edu/hbvar/menu.html

13. Flow chart of DNA testing Thalassaemia α thalassaemia β thalassaemia Common ethnic specific mutation screen (Deletions) Common ethnic specific mutation screen (Point mutations) 5-plex PCR Sequencing β globin gene Sequencing the two genes βMLPA α MLPA

14. Techniques used in DNA testing ARMS (Amplification Refractory Mutation System) for common ethnic specific mutations RFLP analysis for very common variants (HbS, HbE) Gap PCR for common α globin gene deletions Sequencing Direct sequencing using Fluorescent dye terminator Sequencing analysis using SeqScape software MLPA: Multiplex Ligation-dependent Probe Amplification to detect gene doses including deletion and duplication

15. Protocol for reporting Base on: HGVS nomenclature http://www.hgvs.org/mutnomen/ RefSeq sequences from NCBI/Ensembl GenBank (U01317 for the beta-like globin genes, or Z84721 for the alpha-like globin genes) Public Database reports http://www.hgmd.cf.ac.uk/ac/index.php- Cardiff- Human Gene Mutation Database http://www.ncbi.nih.gov/sites/entrez?db= OMIM http://globin.cse.psu.edu/globin/ Globin Gene Server Published sources http://www.ncbi.nlm.nih.gov/pubmed/

16. Protocol for classification of variants as deleterious or neutral Nearly 95% mutations have been described and 5% remains as novel mutation Frequency of a variant in normal population Co-segregation of a sequence variant with the disease in family Assess degree of conservation among different species (with or without Grantham calculation) Assess type of amino acid substitution Protein modeling Splicing studies if relevant Automated splice site analysis http://splice.cmh.edu Functional analysis

17. Diagnosis of complex haemoglobinopathies: interaction of HbE and αthalassaemia

18. Main Diagnostic Challenges in Haemoglobinopathies • MCV and HbA2 – what is normal range? • 2.Can one exclude underlying a thalassaemia? • 3.Importance of detecting HbS carriers HbA2 – interlab variation for NORMAL range + additional caveats creeping in the interpretation of normal ranges. Normal HbA2b thalassaemia. Risk of co-existing a thalassaemia with HbE, HbS and b thalassaemia – i.e. risk for underlying HbH disease within certain populations e.g. SE Asian, Mediterranean and Middle East Traditionally taught that screening for thalassaemia done with MCV / MCH. However, this will miss HbS (Black Africans, Mediterranean, Middle Eastern) and maybe other Hb Variants.

19. Influences on the HbA2 Recommendation: RCPA QA should look at what we are measuring and how we report this result

20. Silent βthalassaemia – Case 1 Southern Chinese couple with a 14 month old child recently diagnosed to have  thalassaemia major. Parents then tested for  thalassaemia. Child: b thalassaemia major Father: b thalassaemia minor Mother: Hb 139 g/L MCV 81.3 fl HbA2 3.0%(1.5-3.4) HbF 0.7% Ferritin 61 μg/l Father is heterozygous for the IVS2, 654 mutation Child is also heterozygous for this mutation Explanation for  thalassaemia major : ?

21. RESULT: Mother hasβ thalassaemia mutation involving poly A tail (AATAAA → AATAGA) Haematologic clues in this case: (1)HbA2 upper limit 3.0% (N = 1.5 - 3.4). (2) 1 parent + brother living in Adelaide tested and shown to have equivocal HbA2 levels and normal MCVs

22. Silent βthalassaemia – Case 2 Normal HbA2 = 1.5 - 3.4% Blue = b thalassaemia; stippled = d thalassaemia

23. Silent α Thalassaemia & Risk Populations - important but: • ethnicity is rarely indicated in request, • risk may reflect ethnicity of earlier generations Bain, 2006.

24. Case 3 Greek couple - pregnant female, with  thalassaemia trait just before Christmas!! Partner MCV 66.2 fl (NR 80-100); HbA2 1.8% (NR 1.5-3.5); HbF 1.3% (NR <1); no HbH inclusions (2 labs) Interpretation ** Normal HbA2 thalassaemia ** ( )o thalassaemia ?? The Haematology Laboratory Holds the Key

25. Family study (parents travelling overseas – mother said to have b thalassaemia) Wife’s b thalassaemia mutation IVS1,110 Fortunately, fetus did not have IVS1,110 Husband did not have 11 b thalassaemia mutations. Before sequencing DNA, Haematology laboratory asked to review results

26. HbH inclusion bodies now found !!!!! DNA testing husband – 0 thalassaemia Mediterranean type (/--) Haematology his mother – HbH inclusions present (also a brother)

27. Summary • In an ethnically-diverse community there are an increasing number of unusual haemoglobinopathies causing significant problems for the health care system. • Prenatal diagnosis of complex haemoglobinopathies becomes more difficult in term of the amount of time undertaken. • Therefore having a knowledge on ethnic origin, good haematology results including Hb EPG, iron studies and possible information on family studies would have a great impact on identification of the molecular defects responsible for the complex haemoglobinopathies. .

28. Acknowledgements Professor RJA Trent Head of Dept of Molecular & Clinical Genetics, RPA Hospital & Central Clinical School, University of Sydney