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

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Presentation on theme: "Prenatal Diagnosis of Complex Haemoglobinopathies Huong Le Senior Hospital Scientist Department of Molecular & Clinical Genetics Royal Prince Alfred Hospital."— Presentation transcript:

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

2 Outline 1.Introduction 2.Complex haemoglobinopathies –Different ethnic groups –Gene-gene interactions 3.Diagnosis of complex haemoglobinopaties –Prenatal diagnosis –Case studies 4.Summary

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

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)

6 Inheritance patterns of thalassaemia 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 Diagnosis of haemoglobinopathies: haematological features 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

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 β T / carriers) HbH disease (if one is αα/-- and the partner is -α/αα or αα T /αα ) Various Hb variant in combination with β thalassaemia (ββ T /ββ E ) Sources Chorion villus sample ~11/52 Amniocentesis ~ 15/52 Genetic counselling in relation to prenatal diagnosis

12 Summary of mutation categories in database β globin gene mutations α1 globin gene mutations α2 globin gene mutations

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

14 Techniques used in DNA testing 1.ARMS (Amplification Refractory Mutation System) for common ethnic specific mutations 2.RFLP analysis for very common variants (HbS, HbE) 3.Gap PCR for common α globin gene deletions 4.Sequencing –Direct sequencing using Fluorescent dye terminator –Sequencing analysis using SeqScape software 5.MLPA: Multiplex Ligation-dependent Probe Amplification to detect gene doses including deletion and duplication

15 Protocol for reporting Base on: –HGVS nomenclature RefSeq sequences from NCBI/Ensembl GenBank (U01317 for the beta-like globin genes, or Z84721 for the alpha- like globin genes)U01317Z84721 –Public Database reports Cardiff- Human Gene Mutation Databasehttp://www.hgmd.cf.ac.uk/ac/index.php- OMIM Globin Gene Serverhttp://globin.cse.psu.edu/globin/ –Published sources

16 Protocol for classification of variants as deleterious or neutral Nearly 95% mutations have been described and 5% remains as novel mutation 1.Frequency of a variant in normal population 2.Co-segregation of a sequence variant with the disease in family 3.Assess degree of conservation among different species (with or without Grantham calculation) 4.Assess type of amino acid substitution 5.Protein modeling 6.Splicing studies if relevant Automated splice site analysis 7.Functional analysis

17 Diagnosis of complex haemoglobinopathies: interaction of HbE and α thalassaemia GenotypeClinical findingHaemoglobin (estimated %) αα/αα, ββ E Normal Slightly hypochromic red cells A + E 27% -α/αα, ββ E Normal hypochromic red cells A + E 20-25% --/αα, ββ E Normal hypochromic red cells A + E 17-20%

18 Main Diagnostic Challenges in Haemoglobinopathies 1.MCV and HbA 2 – what is normal range? 2. Can one exclude underlying thalassaemia? 3. Importance of detecting HbS carriers HbA 2 – interlab variation for NORMAL range + additional caveats creeping in the interpretation of normal ranges. Normal HbA 2 thalassaemia. Risk of co-existing thalassaemia with HbE, HbS and 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 HbA 2 Elevatedβ Thalassaemia Thyrotoxicosis, megaloblastic anaemia LowCoexistent iron deficiency (falsely low) Coexistent δ thalassaemia δβ thalassaemia Very High e.g. 6-7% Deletions of the β globin gene and its promotor NormalSome β thalassaemia mutations particularly in those of Greek origin e.g C >T, IVS and 3 UTR mutations Recommendation: RCPA QA should look at what we are measuring and how we report this result

20 Southern Chinese couple with a 14 month old child recently diagnosed to have thalassaemia major. Parents then tested for thalassaemia. Child: thalassaemia major Father: thalassaemia minor Mother: Hb 139 g/L MCV 81.3 fl HbA 2 3.0% ( ) 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 : ? Silent β thalassaemia – Case 1

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

22 Normal HbA 2 = % Silent β thalassaemia – Case 2 Blue = thalassaemia; stippled = thalassaemia

23 1)ethnicity is rarely indicated in request, 2)risk may reflect ethnicity of earlier generations Location o thalassaemia + thalassaemiaNon-del + / variant SEA, S/China Mediterranean Greece, Cyprus, (UK) Middle East Israel, UAE AfricaX? Polynesia, Melanesia X? India / Sri LankaX Bain, Silent α Thalassaemia & Risk Populations - important but:

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

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

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

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


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