Investigating
haemoglobinopathies
7% of the world’s population are carriers
of haemoglobin disorders
Carrier frequencies of thalassaemia alleles (%)
β-Thalassaemia
α0-Thalassaemia
α+-Thalassaemia
Americas
0–3
0–5
0–40
Eastern
Mediterranean
2–18
0–2
1–60
Europe
0–19
1–2
0–12
Southeast Asia
0–11
1–30
3–40
Sub-Saharan
Africa
0–12
0
10–50
Western Pacific
0–13
0
2–60
Region
Weatherall D, et al. Inherited Disorders of Hemoglobin. In: Disease Control Priorities in Developing Countries.
2nd ed. New York: Oxford University Press; 2006: 663-80. Available from: www.dcp2.org/pubs/DCP.
Types of haemoglobinopathies
 Thalassaemias – result from an imbalance
in a and b globin gene production, most
commonly due to
–
Point mutations in the b gene
–
Deletion of one or more a genes
 Haemoglobin variants - result from point
mutation in the a or b genes leading to
amino acid substitution, producing a
different haemoglobin, sometimes with
different properties

Heterozygous:
– Thalassaemia trait/minor
• Mild/no microcytic
anaemia

Homozygous:
– Thalassaemia major
• Marked anaemia (usually
transfusion dependence)
• Iron overload
• Transfusion complications
Clinical impact of
thalassaemia major


Transfusion dependence
Iron overload
– Cardiac complications
– Endocrine (diabetes, hypothyroidism,
hypogonadism, hypoparathyroidism)


Transfusion complications (eg hepatitis C)
Osteoporosis – bone disease
Copyright ©1997 BMJ Publishing Group Ltd.
Mutations in thalassaemia

b thalassaemia
– 200 point mutations in the b globin gene
– Deletions are rare

a thalassaemia
– Deletion of one a globin gene on an allele
• Common, many ethnic groups
– Deletion of both a globin genes on an allele
• Less common, some ethnic groups
– Occasional point mutations
b globin under-production leads to excess of other haemoglobins
a globin under-production leads to excess of other haemoglobins
Microcytosis

Thalassaemia trait
– Anisocytosis, poikilocytosis
– Target cells

Iron deficiency
– Hypochromic red cells
– Pencil cells


Anaemia of chronic disease (eg rheumatoid
arthritis) – upregulation of hepcidin, functional
iron deficiency (poor release of iron from
enterocytes, hepatocytes)
Very rare: sideroblastic anaemia
Iron deficiency
Thalassaemia trait
Cellulose acetate and citrate agar
gel electrophoresis
A2 C
S FA
Barts H
CS AF
Thalassaemia trait - phenotype

b thalassaemia trait
– Variable microcytosis, mild anaemia

a thalassaemia
– Single gene deletion: none/microcytosis
– Two gene deletion: mild anaemia/variable
microcytosis
– Three gene deletion (Hb H disease)
• Variable
– Four gene deletion
• Hb Barts hydrops fetalis

Compound heterozygotes
– Sickling disorders (HbSS, HbSC, HbS-b-thal
Interpreting the haemoglobin EPG



An elevated HbA2 is diagnostic of b thalassaemia trait
Hb H inclusions are diagnostic of a thalassaemia trait
Elevated Hb F
–
–
–
–

May be seen in b thalassaemia trait
Other disorders of erythropoiesis
Pregnancy
Hereditary persistence of fetal haemoglobin
Abnormal bands
– Haemoglobin E (with or without a thalassaemia), Lepore
• Matters because of compound heterozygosity with b thalassaemia
trait
– Hb S, C
• Matter because can contribute to sickling
– Other D, O, etc, etc
• Often don’t matter
• Some produce unstable haemoglobins
• Can’t be easily characterised on standard HbEPG
Lab Testing


FBC
Characterisation of abnormal haemoglobins
– Haemoglobin electrophoresis
– HPLC
– Supravital staining for H inclusions

Iron studies
–
–
–
–

Ferritin
Transferrin/TIBC
Transferrin saturation
Serum iron
(inflammatory markers)
Common problems in Hb
EPG interpretation

H inclusions are rare
– a thalassaemia cannot be excluded
The findings of b thalassaemia may
be masked by iron deficiency
(reduction in Hb A2)
 Rare problem of normal Hb A2 b
thalassaemia trait
 Unexplained elevated Hb F

Solutions to difficult Hb
EPG results
Family studies
 Repeat testing when iron replete
 DNA testing

– a globin gene PCR testing
– b globin gene sequencing
When does it matter?


Pre-pregnancy
Early pregnancy
– DNA testing is rapid if the mutations are
known
– DNA testing is slow and may not yield a
result if the mutation is not known
– CVS possible at 11 weeks
– Second trimester amniocentesis
– Genetic counselling takes time and causes
anxiety

The role for screening?
Important patterns

Microcytosis in early pregnancy
– Partner should have FBC, iron studies and Hb EPG (to
exclude both thalassaemia trait and a sickling disorder).
– Don’t wait for the woman’s results

Microcytosis (in a male or female)
– Test the partner
– Test other family members – it may help someone else

Microcytosis in both partners
– Is there a risk of Hb H disease or Hb Barts hydrops fetalis
– “masked” b thalassaemia major

Hb S or Hb E trait
– Think of compound heterozygosity
– Test the partner and other family members