The Haemoglobinopathies:

An Introduction

Haemoglobin disorders as a model

An Autosomal recessive disease of global
importance
>5% of the world population are Haemglobinopathy
carriers
>3/1000 liveborn babies are affected

Population carrier screening (basic haematology)

and prenatal diagnosis available for 20 years
These services have spread globally:
extensive experience in community implications of
genetic screening

Autosomal Recessive Inheritance

When both parents are heterozygotes
1/4 offspring affected, 2/4 will be carriers, 1/4
unaffected in each pregnancy

Aa

AA

Aa

Aa

Aa

aa

Unaffected

Thalassaemia: An Important Diagnosis because of Recurrence

Haemoglobin Structure
2 dissimilar pairs of
globin chains each
linked to a single
haem molecule
forming a tetramer
Normal adult Hbs
consists of 2 chains
+ another pair

Adult Haemoglobins
Hb A (22)
Major adult haemoglobin
95% of total

Hb A2 (22)
~ 2.5% of adult Hb
chain differs from at ~ 10% of residues
function unclear

Hb F (2 2)
about 1%

Fetal Haemoglobins

HbF 22 late fetus and neonate

Replaced by HbA at 3-6 months

chain differs from at 25% residues

Higher affinity for O2 than HbA: increases fetal oxygenation
2 HbFs in humans: chains differing by 1 AA

Hb Gower : Embryonic
Gower 1: 22 ( similar to , 20% difference)
Gower 2: 2 2 ( similar to , 40% difference)

, , , chains can all form tetramers,

does not form tetramers

Developmental Pattern of Human Haemoglobins

Based on Voet & Voet (1995)

What are Haemoglobinopathies ?

Thalassaemia: Quantitative defects:

Reduced synthesis of a normal globin chain

+ or + thalassaemia
Absent synthesis of a normal globin chain

0 thalassaemia or 0 thalassaemia
Unequal and globin chain production

Haemoglobinopathy: Qualitative defects

Normal synthesis of an abnormal globin chain, due to amino acid

substitution

Heterozygote:
Homozygote:

eg Sickle Cell Disease

Trait
Disease

Why do Haemoglobinopathies occur?

Vast majority are inherited
Spontaneous mutations related to inherent
gene structure
Ancestral gene deletions
Occasional spontaneous mutation

Haemoglobin Gene Clusters

Chromosome 16

2 1 2 1

Chromosome 11

20

40

60 Kbp

MECHANISM OF GENE DUPLICATION/DELETION

Deletions in the Haemoglobin Gene Cluster

kbp

20

40

60
0thalassaemia
Hb Lepore
GA
thalassaemia
GAHPFH
GHPFH
Hb Kenya

HPFH = hereditary persistent fetal haemoglobin

Why do Haemoglobinopathies Persist?

?Selecitve advantage in heterozygotes

?protection against malaria?
Similar explanation for G6PD deficiency

Thalassaemia Phenotype
Altered and globin chain ratios
Anaemia

Intramedullary and extramedullary (spleen)

haemolysis of abnormal blood cells
hypersplenism
anaemia

Homozygotes usually diagnosed in infancy

Pale, unwell
Splenomegaly and hepatomegaly common
Severe microcytic,hypochromic anaemia

Heterozygotes often chance finding

Treatment
Homozygote patients are transfusion
dependent
Problems of transfusion
Iron overload chelation is required
cardiomyopathy/diabetes/cirrhosis/pituitatry

Risk of allo antibody production

Risk of transfusion related infection
Hep B / C / HIV

Diagnosis of Haemoglobinopathies
Family history
Ethnic origin

Clinical signs/symptoms
Laboratory investigations
Full blood count and film
Ferritin
HbEPG
Sickle screen
HbH preparation

Molecular Genetic and Family studies

Diagnosis of Thalassaemia

microcytic, hypochromic RBC

Anaemia
Ferritin normal
blood film

HbEPG: HbA2 ( 2 2)

( / biosynthesis ratio >1)

DNA analysis (point mutations)
NB Family Studies

Effect of -thalassaemia on adult red cells

MCH = 30pg
Hb A = 97%
Hb A2 = 3%

MCH = 20pg
Hb A = 96%
Hb A2 >3.5%

MCH = 15-30pg
Hb F >80%

Normal

thal Trait

thal
Major/Intermedia

-Globin

IVS1
Exon 1

IVS2
Exon 2

Exon 3

Splicing Mutations

Insertions

Nonsense Mutations

Frameshift Deletions (-1, -2, -4)

Deletions (rare)
Transcription

Poly A site
Initiator Codon

Diagnosis of Thalassaemia

(microcytic, hypochromic RBC)

(anaemia)

blood film

HbH inclusions ****

HbEPG: HbA2 and HbF in thal trait

( / b biosynthesis ratio <1)

DNA analysis
NB Family Studies

Effect of -thalassaemia on fetal red cells

MCH = 35pg
Hb F = 92%
Hb A = 8%
Normal

MCH = 20pg
Hb F = 89%
Hb

= 3%

Hb A = 8%

thal Trait

MCH = 20pg

= 80%
Hb = 10%
Hb

thal Major
Hb Barts Hydrops Fetalis

 Thalassaemia
2 genes inherited from each parent more
heterogenous picture
Usually caused by gene deletions
Clinical picture varies with number of deletions

1. One gene deletion: silent carrier

2. Two gene deletions: thal trait
o: both genes on
one chromosome
deleted

+: one gene
on each
chromosome
deleted

or

( )
or

Forms of thalassaemia
Thalassaemia caused by intragenic deletions

1
3.7 Kb

4.2 Kb
1

Thalassaemia caused by point mutations: Hb Constant Spring

1

0-Thalassaemia caused by large intragenic deletions

1

 Thalassaemia
Genotype

No. Genes
Functional

Phenotype

Normal

- /

Normal

-/-

2*

MCV, MCH

-- /

2*

MCV, MCH

- / --

HbH disease

-- / --

Hb Barts hydrops

fetalis

Distribution of thalassaemia
Similar geographical range to -thalassaemia but particularly
associated with SE Asians and widespread in Africa
Follows malaria distribution
3.7Kb del: Africa, Mediterranean, Bangladesh,
India, Pakistan, Melanesia
4.2Kb del: South East Asia, Melanesia
Mediterranean: Cyprus, Greece, Turkey,
Sth Italy
Asian: China, Thailand, Cambodia,
Philippines, Vietnam

Molecular Testing
Complex but the reference laboratory for
NSW is in our health area!
Clinical Genetics and Molecular Genetics
infrastructure
Ron Trent
Robert Ogle
Tony Roscioli
Bronwyn Culling
Gayathri Parasivam

Substitution Haemoglobinopathy
Amino acid change in globin chain
May be insignificant with no effect
May cause functional changes eg
Altered oxygen affinity
Loss of molecular stability
Polymerisation

Eg HbS, HbC, HbE

Diagnosis of Sickle Cell Disease

Low Hb, increased
WBC
Marked anisocytosis,
poikilocytosis,
polychromasia, target
cells and sickle cells
NB Must do HbEPG
as sickle cell trait can
be missed on blood
film alone

Sickle Cell Disease

Single amino acid substitution on chain
Valine for Glutamic acid at position 6

Sickle cell trait 40% HbS - AS

SCD - homozygote 90% HbS - SS
Change in Hb solubility in low O2 tension
Structural changes sickling
Loss of flexibility destruction
increased blood viscosity

Sickle Cell Disease

Anaemia
Splenic atrophy
Increased risk of infection
Crises
Vaso-occlusive/Painful
Haemolytic
Aplastic

Thalassaemia in NSW
1998: 123 people with transfusiondependent thalassaemia
106 (86%) homozygous thalassaemia
17 (14%) other
thalassaemia/haemoglobinopathies

1989-1998: 23 new cases

Increased detection rates for homozygous
thalassaemia
Major risk groups Asian and Middle eastern

Ancestry

No of
Diagnoses

Percentage

Middle East

34.5%

South East Asia 6

26.2%

Meidterranean/ 6
Africa

26.2%

India

13.1%

TOTAL

23

100%

It is important to consider that an individual

could be a genetic carrier for thalassaemia or
HbE whenever a blood count shows a low MCV
or MCH, particularly if an individual or their
ancestors have originated from an at-risk group.
The blood picture described is frequently
mistaken for iron deficiency. It is also important
to note that both iron deficiency due to other
causes and thalassaemia can co-exist in the
same individual. Therefore a low MCV or low
MCH which appears to be refractory to iron
treatment should be considered to represent
thalassaemia minor until proven otherwise.
NSW Health Department Guidelines, 1998

Definitions: Genetic Testing and

Genetic Screening
Genetic testing: providing a genetic test to
someone who is thought to be at
increased risk eg. Affected relative,
member of at risk group
Genetic screening: offering a test to all
members of a population to identify those
at increased risk, for genetic testing

Core Ethical Principles of Genetic

Counselling*
The autonomy of individual and couple
Their right to full information
Strict confidentiality

*Fletcher, Berg and Trannoy 1985, endorsed by WHO, 1985

NSW Health Department Guidelines

and take home messages
Haemoglobinopathy screening (FBC, Film, HbEPG, Iron studies):
Anyone with a blood count showing a low MCH or MCV
People whose origins are from Southern Europe, Africa, Middle
east and Asia, India
All pregnant women when they are having routine antenatal
blood examinations
Too difficult to target specific groups: surnames and
populations changing!
Cascade counselling/testing!
Clinical Genetic Service RPAH/Thalassaemia Service RPAH
9515 5080 / 9515 6111