5 Haemoglobin compositions Functioning haemoglobin (Hb) molecules are tetramers made up of two pairs of globin chainsThe 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)5
6 Inheritance patterns of thalassaemia Autosomal recessive (2 parents carriers = 1 in 4 risk )Compound states e.g. + & o thalassaemia = HbH diseaseCombinations of and thalassaemiasThe inheritance pattern is autosomal recessive as both parrent need to be carrier for the child to have thalassaemia major6
8 Haemoglobinopathies & Ethnic variety thalassaemia: SEA, Chinese, Mediterranean, African thalassaemia: European, Middle Eastern, Indian and SEA populationsHbS: Mediterranean, Middle Eastern and Black AfricanHbE: SEA8
9 Diagnosis of haemoglobinopathies: haematological features α thalassamia±Low MCV±Low MCHBlood film±HbH inclusionsHbA2 normalVariant haemoglobinsNormal MCVHb EPG-> variant peakβ thalassamiaLow MCVLow MCHBlood filmHbA2Hypochromic (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
11 Prenatal Diagnosis Indications Couple at high risk of reproducing an affected fetus withHb 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 )SourcesChorion villus sample ~11/52Amniocentesis ~ 15/52Genetic counselling in relation to prenatal diagnosis11
12 Summary of mutation categories in database α1 globin gene mutationsβ globin gene mutationsα2 globin gene mutations
13 Flow chart of DNA testing Thalassaemiaα thalassaemiaβ thalassaemiaCommon ethnic specific mutation screen(Deletions)Common ethnic specific mutation screen(Point mutations)5-plex PCRSequencing β globin geneSequencing the two genesβ MLPAα MLPA13
14 Techniques used in DNA testing ARMS (Amplification Refractory Mutation System) for common ethnic specific mutationsRFLP analysis for very common variants (HbS, HbE)Gap PCR for common α globin gene deletionsSequencingDirect sequencing using Fluorescent dye terminatorSequencing analysis using SeqScape softwareMLPA: Multiplex Ligation-dependent Probe Amplification to detect gene doses including deletion and duplication
15 Protocol for reporting Base on:HGVS nomenclatureRefSeq sequences from NCBI/EnsemblGenBank (U01317 for the beta-like globin genes, or Z84721 for the alpha-like globin genes)Public Database reportsCardiff- Human Gene Mutation DatabaseOMIMGlobin Gene ServerPublished sources
16 Protocol for classification of variants as deleterious or neutral Nearly 95% mutations have been described and 5% remains as novel mutationFrequency of a variant in normal populationCo-segregation of a sequence variant with the disease in familyAssess degree of conservation among different species (with or without Grantham calculation)Assess type of amino acid substitutionProtein modelingSplicing studies if relevantAutomated splice site analysisFunctional analysis
17 Diagnosis of complex haemoglobinopathies: interaction of HbE and α thalassaemia GenotypeClinical findingHaemoglobin(estimated %)αα/αα, ββENormalSlightly hypochromic red cellsA + E 27%-α/αα, ββEhypochromic red cellsA + E %--/αα, ββEA + E %17
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 carriersHbA2 – interlab variation for NORMAL range + additional caveatscreeping 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 populationse.g. SE Asian, Mediterranean and Middle EastTraditionally taught that screening for thalassaemia done withMCV / MCH. However, this will miss HbS (Black Africans,Mediterranean, Middle Eastern) and maybe other Hb Variants.
19 Influences on the HbA2 Elevated β Thalassaemia Thyrotoxicosis, megaloblastic anaemiaLowCoexistent iron deficiency (falsely low)Coexistent δ thalassaemiaδβ thalassaemiaVery Highe.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 C >T, IVS and 3’ UTR mutationsRecommendation: RCPA QA should look at what we are measuring and how we report this result
20 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 majorFather: b thalassaemia minorMother: Hb 139 g/L MCV 81.3 fl HbA2 3.0% ( )HbF 0.7% Ferritin 61 μg/lFather is heterozygous for the IVS2, 654 mutationChild is also heterozygous for this mutationExplanation for thalassaemia major : ?
21 Haematologic clues in this case: RESULT: Mother has βthalassaemia mutationinvolving poly A tail(AATAAA → AATAGA)Haematologic clues in this case:(1) HbA2 upper limit 3.0% (N = ).(2) 1 parent + brother living in Adelaide tested and shown to have equivocal HbA2 levels and normal MCVs
22 Silent β thalassaemia – Case 2 Normal HbA2 = %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 generationsLocationao thalassaemiaa+ thalassaemiaNon-del a+ / variantSEA, S/China√Mediterranean√ Greece, Cyprus, (UK)Middle East√ Israel, UAEAfricaX?Polynesia, MelanesiaIndia / Sri LankaBain, 2006.
24 Case 3 The Haematology Laboratory Holds the Key Greek couple - pregnant female, with thalassaemia trait just before Christmas!!PartnerMCV 66.2 fl (NR );HbA2 1.8% (NR );HbF 1.3% (NR <1);no HbH inclusions (2 labs)Interpretation** Normal HbA2 thalassaemia** ( )o thalassaemia ??The HaematologyLaboratoryHolds the Key
25 Family study (parents travelling overseas – mother said to have b thalassaemia) Wife’s b thalassaemia mutation IVS1,110Fortunately, fetus did not have IVS1,110Husband did not have 11b thalassaemia mutations.Before sequencing DNA,Haematology laboratoryasked 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 SummaryIn 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 specific27
28 Acknowledgements Professor RJA Trent Head of Dept of Molecular & Clinical Genetics, RPA Hospital & Central Clinical School, University of Sydney