Prenatal Diagnosis

of Complex

Haemoglobinopathies

Huong LeSenior Hospital Scientist

Department of Molecular & Clinical GeneticsRoyal Prince Alfred Hospital

Sydney , Australia

Outline

1. Introduction

2. Complex haemoglobinopathies– Different ethnic groups

– Gene-gene interactions

3. Diagnosis of complex haemoglobinopaties– Prenatal diagnosis

– Case studies

4. Summary

HAEMOGLOBINOPATHIES

= inherited disorders of globin

Thalassaemia Syndromes (quantitative disorders)

Variant Haemoglobins(qualitative disorders)

divided into:

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

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

http://www.thalassaemia.org.cy

RPA Experiencewith 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

Diagnosis of haemoglobinopathies:haematological features

• α thalassamia±Low MCV±Low MCHBlood film±HbH inclusionsHbA2 normal

• Variant haemoglobinsNormal MCVHb EPG-> variant peak

• β thalassamiaLow MCVLow MCHBlood filmHbA2

Why Prenatal Diagnosis ?

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

Summary of mutation categoriesin database

http://globin.bx.psu.edu/hbvar/menu.html

β globin gene mutations

α1 globin gene mutations

α2 globin gene mutations

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 PCRSequencing β globin gene

β MLPA

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

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 mutation1. 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 http://splice.cmh.edu

7. 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 Normal

hypochromic red cells

A + E 20-25%

--/αα, ββE Normal

hypochromic red cells

A + E 17-20%

Main Diagnostic Challenges in Haemoglobinopathies

1. MCV and HbA2 – what is normal range?

2. Can one exclude underlying thalassaemia? 3. Importance of detecting HbS carriers

HbA2 – interlab variation for NORMAL range + additional caveatscreeping in the interpretation of normal ranges. Normal HbA2 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.

Influences on the HbA2 Elevated β Thalassaemia

Thyrotoxicosis, megaloblastic anaemia

Low Coexistent iron deficiency (falsely low)Coexistent δ thalassaemiaδβ thalassaemia

Very 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. -101 C >T, IVS and 3’ UTR mutations

Recommendation: RCPA QA should look at what we are measuring and how we report this result

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 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 : ?

Silent β thalassaemia – Case 1

RESULT: Mother hasβthalassaemia mutation involving poly A tail (AATAAA → AATAGA)

(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

Haematologic clues in this case:

Normal HbA2 = 1.5 - 3.4%

Silent β thalassaemia – Case 2

Blue = thalassaemia; stippled = thalassaemia

1) ethnicity is rarely indicated in request, 2) risk may reflect ethnicity of earlier generations

Location o thalassaemia + thalassaemia Non-del + / variant

SEA, S/China √ √ √

Mediterranean √ Greece, Cyprus, (UK)

√ √

Middle East √ Israel, UAE √ √

Africa X √ ?

Polynesia, Melanesia

X √ ?

India / Sri Lanka X √ √

Bain, 2006.

Silent α Thalassaemia & Risk Populations - important but:

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 ??

Case 3

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

Wife’s thalassaemia mutation IVS1,110 Fortunately, fetus did not have IVS1,110

DNA testing husband – 0 thalassaemia Mediterranean type (/--)

Haematology his mother – HbH inclusions present (also a brother)

HbH inclusion bodies now found !!!!!

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.

.

Acknowledgements

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