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

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

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

Structure of haemoglobin molecule

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 5

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 The inheritance pattern is autosomal recessive as both parrent need to be carrier for the child to have thalassaemia major 6

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

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 8

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 Hypochromic (pale – indicating less haemoglobin than normal) Vary in size (anisocytosis) and shape (poikilocytosis) Be nucleated (not normal in a mature RBC) Be distorted to produce “target cells”, which look like a bull’s-eye under the microscope. 9

Why Prenatal Diagnosis ? 10

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 11

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

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 β MLPA α MLPA 13

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

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/

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

Diagnosis of complex haemoglobinopathies: interaction of HbE and α thalassaemia Genotype Clinical finding Haemoglobin (estimated %) αα/αα, ββE Normal Slightly hypochromic red cells A + E 27% -α/αα, ββE hypochromic red cells A + E 20-25% --/αα, ββE A + E 17-20% 17

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 HbA2 b 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.

Influences on the HbA2 Elevated β Thalassaemia Thyrotoxicosis, megaloblastic anaemia Low Coexistent iron deficiency (falsely low) Coexistent δ thalassaemia δβ thalassaemia Very High e.g. 6-7% Deletions of the β globin gene and its promotor “Normal” Some β thalassaemia mutations particularly in those of Greek origin e.g. -101 C >T, IVS and 3’ UTR mutations Recommendation: RCPA QA should look at what we are measuring and how we report this result

Silent β thalassaemia – 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 : ?

Haematologic clues in this case: RESULT: Mother has β 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

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

Silent α Thalassaemia & Risk Populations - important but: ethnicity is rarely indicated in request, risk may reflect ethnicity of earlier generations Location ao thalassaemia a+ thalassaemia Non-del a+ / variant SEA, S/China √ Mediterranean √ Greece, Cyprus, (UK) Middle East √ Israel, UAE Africa X ? Polynesia, Melanesia India / Sri Lanka Bain, 2006.

Case 3 The Haematology Laboratory Holds the Key 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

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

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

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. . since many of these mutations responsible for the disease are ethnic-origin specific 27

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