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 2

3. Thalassaemia Syndromes (quantitative disorders) Variant Haemoglobins
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) 5

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

7. (Maldives, Eastern Europe)
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 8

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
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

10. Why Prenatal Diagnosis ?10

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 11

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

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
β MLPA
α MLPA 13

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
RefSeq sequences from NCBI/Ensembl
GenBank (U01317 for the beta-like globin genes, or Z84721 for the alpha-like globin genes)
Public Database reports
Cardiff- Human Gene Mutation Database
OMIM
Globin Gene Server
Published sources

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
Functional analysis

17. 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 %
--/αα, ββE
A + 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 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.

19. 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 C >T, IVS and 3’ UTR mutations
Recommendation: 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 major
Father: b thalassaemia minor
Mother: Hb 139 g/L MCV 81.3 fl HbA2 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 : ?

21. 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 = ).
(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 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.

24. Case 3 The Haematology Laboratory Holds the Key
Greek couple - pregnant female, with  thalassaemia trait just before Christmas!!
Partner
MCV 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 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. .
since many of these mutations responsible for the disease are ethnic-origin specific 27

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