Hemoglobinopathies

Barbara Karczeski, MS
DNA Diagnostic Lab
Board Review Lecture Series 2007

Agenda

Brief overview of hemoglobin

The globin genes
The Thalassemias
Structural hemoglobinopathies
Testing

Hemoglobin

Delivers oxygen to the cells

Tetramer (4 subunits 2 A and 2
B) plus Heme groups
A = Alpha like genes and pseudogenes (Chromosome 16); , ,
B = Beta like genes and pseudo
genes (Chromosome 11); ,,,

The Globin Genes

Hemoglobin structure

heme

Heme

Globin

Hemoglobi Name
n Type
Adult
A

Component
s
22

A2

22

Fetal

22

Embryonic

Portland

22

Gower 1

22

Gower 2

22

Barts

Abnormal

Hemoglobins in normal adults

A

A G

B A

HbA

HbF

HbA2

98%

~1%

<3.5%

Hemoglobinopathy
definition

An inherited mutation of the

globin genes leading to a
quantitative or qualitative
abnormality of globin synthesis

Thalassemia - Defined

A family of genetic anemias

characterized by a reduced rate of
production of 1 or more globin
subunits of hemoglobin (Hb)
Symptoms are caused by the
deleterious effects of the normally
produced subunits that are now in
excess

Structural variant - Defined

Abnormal globin protein that is

produced at a normal rate, with
varying consequences
Oxygen affinity, stability and
function

Normal

Normal
Thalassemia

Normal

Structural
Variant

- and -thalassemia
Alpha
Thalassemia
Deletions of
alpha- globin
gene(s)
Symptoms can
begin in fetal life
Complicated
inheritance 4
alpha genes

Beta Thalassemia
Nonsense, splice and
frameshift mutations
in beta-globin gene
Symptoms begin in
infancy/childhood
Simple AR
inheritance;
genotype-phenotype
correlation

Structural Variants

Missense mutations in any globin

gene that cause amino acid
substitutions
Symptoms begin in childhood
Simple AR inheritance, though the
sheer number of possible
combinations makes it a more
complex issue.

Some terminology

Thal minor and Thal trait carrier

state; generally asymptomatic
Thal major affected, full blown
disease
Thal intermedia somewhere in
between stay tuned

Beta Thalassemia

-thalassemia

One of the most common single

gene disorders in the world
Occurs worldwide, but more
common in SE Asia, Mediterranean,
India, Central/Northern regions of
Africa, Middle East, Caribbean
Carrier frequencies 1/100 to 1/2
Worldwide frequency: 1-2%

Pathophysiology

Excess alpha chains precipitate and

form inclusion bodies that associate
with the RBC cell membrane
Cause membrane damage and
shortened cell survival
Large scale destruction of precursor
cells in bone marrow
Decreased B production causes
increased production and an elevated
A2 (22)

Normal

Carrier

Affected

Factors that affect severity

Specific B-globin mutations

inherited
Co-inheritance of alpha
thalassemia
Fetal hemoglobin production

Clinical Findings: Carrier

State

Generally asymptomatic
May have mild anemia (weakness,
pallor, mild unexplained fever)
Low MCV (measurement of cell size),
elevated A2
Can see some bone marrow changes
Anemia worsens during pregnancy

Identification of the
Carrier State

Important for risk assessment and

prenatal diagnosis

Also important to avoid treating

with iron

Affected Individuals Presenting Symptoms

Failure to thrive
Anemia
Pallor
Feeding difficulties
Diarrhea
Recurrent infections
Abdominal enlargement

Clinical Symptoms
Untreated Child

Appear prior to age

10
Variable anemia
Hepatosplenomegaly
Recurrent infections
Spontaneous bone
fractures
Extra-medullary
hematopoesis
Gallstones
Leg ulcers

B-thalassemia
Intermedia?

Milder form of B-thal

Transfusion requiring (to aid quality
of life); not transfusion dependent
More likely to have B+ mutations
than B0 mutations
A clinical classification more than a
disease

Treatment

Transfusions of packed RBC as needed (up

to every 3-4 weeks)
Iron chelation therapy (desferroxamine)
Splenectomy
Vitamin Supplementation
Prevention of Infection
Psychosocial issues of chronic disease
Bone marrow transplant functions as
cure

Treatment - Risks

Blood born infections (HIV,

Hepatitis, etc)
Transfusion reactions (errors)
Failure of compliance with
chelation regimen

Clinical Symptoms - Treated

Usually due to iron

overload
Liver disease
Endocrine dysfunction
(diabetes, growth
retardation, failure of
sexual maturation,
Cardiac complications
(infections, right
sided failure)
No direct effect on
behavior or
intelligence

Genetic / Molecular Aspects

Mutations cause decreases in

chain production, not normal
production of abnormal B-globin
Nonsense, small insertions; splice
mutations
Some deletions

Types of B-globin mutations

B0 No B-globin chains are produced

Nonsense - Nt169C>T
Frame shifts Nt176delCTTT
Destruction of splice site Nt142+1G>A

B+ - some beta chains produced

Decreased splice efficiency
Nt142+6T>C Cryptic splice site
Nt365+654C>T

Types of B-globin mutations

B+mild subset of B+ with higher rates

of B-globin production
Poly A mutation Nt494+110T>C
Promoter mutations Nt -88C>T
Bsilent - ??, some promoter, deep
intronic, 5 or 3 UTR mutations
Nt-101C>T, 5UTR+10delT

Alpha Thalassemia

Alpha thalassemia
AA / AA

Normal

AA / A -

Mild microcytosis (Silent Carrier)

AA / - A-/AA-/--

Mild microcytosis (Trait or Carrier)

(cis vs trans)

--/--

Hemoglobin H diseaseclinically variable

Hydrops Fetalis (Alpha Thal Major)

Since alpha chains are

synthesized in fetal life,
symptoms of Alpha-Thalassemia
Major begin in fetal life

Hydrops Fetalis:
Ultrasound

Hydrops Fetalis: Path photo

Death occurs in utero or shortly

after delivery
About 17% of these fetuses also
have developmental abnormalities
Maternal complications: anemia,
pre-eclampsia, hemorrhage,
sepsis, retained hydropic placenta

Pathophysiology

Excess of Beta chains leads to Hb

Barts (gamma 4) and Hb H (beta 4)
These Hbs have high O2 affinity and
are unstable useless!
Lead to early RBC death
Hb H precipitates out and damages
membranes

Hb H Disease

Presence, in a person more than 6

years old, of significant amount (1-2%)
of Hb H
Sx variable; but can include acute or
chronic microcytic anemia,
hepatosplenomegaly, mild bone
changes, gallstones, leg ulcers, venous
thrombosis, transient aplasia
May have Hydrops Fetalis

Management

Hb H Hydrops Fetalis
Blood transfusions, iron chelation
may have sequelae of hypoxia (congenital
anomalies; developmental delay)

Hb H Disease

Folate supplementation
Splenectomy
Transfusions as needed

Molecular / Genetic
Aspects

Most common molecular cause is

one of several deletions of one or
both alpha genes of a chromosome
Some point mutations described
(Hb Constant Spring)
Hb variants of alpha also described

Alpha Deletions

Alpha Deletions

Deletions named according to

their size
3.7kb; 4.2kb; 5.2kb; 20.5kb

Deletions named according to

their origin
Med, Fil, Thai, SE Asian

Double gene deletions more

common in SE Asia
These are the populations at higher risk
for severe alpha-thalassemia

Single gene deletions more common

in African, Indian, Caribbean
populations
Not at usually at risk for severe disease

Non-Deletional Alpha
Thal

Usually represented as /T

May be more severe than deletional

form there is an abnormal product
made; can cause more cellular
damage
Most common is Hb Constant
Spring

Other forms of AlphaThalassemia

ATR-16: contiguous gene deletion

syndrome, diverse clinical features
ATR-X: mutations in an X-linked
gene having some regulatory
effect on alpha gene expression.
Sx include hypotonia, seizures,
short stature, skeletal and GU
abnormalities, cardiac defects

Structural
Hemoglobin Variants

More than 750 variants described

by the late 90s
Theoretically, could have multiple
missense mutations for every
amino acid
Alpha chain variants are
individually rare or of little clinical
consequence

Named for how they migrate on

electrophoreses: Hb E
Named for place in which they
were discovered: Hb Rio Grande
Both: Hb D-Iran; Hb D-Ibidan;
Hb D-Los Angeles

Some rare, only described in a

single family
Some very common and occurring
at high frequency in certain
populations
Hb E in SE Asia
Hb S and Hb C in Africa

Hb C

Found mainly in West Africa

Lower oxygen affinity than Hb A
Crystallizes, reduced
deformability of RBC; hemolysis
Milder than Hb S

Hb E

The most common Hb variant in the

world
Nearly exclusively in SE Asia and India
Has Beta thal properties (An amino acid
substitution AND activates a cryptic
splice site)
Homozyotes extremely mild
Compound het with Beta thal can be
very severe

Sickle Cell Anemia

What is Sickle Cell Anemia?

Disorder affecting hemoglobin (HBB gene)

Inherited as autosomal recessive pattern
Characterized by low number of red blood
cells
Life span of red blood cell
-Normal cells 120 days
-Sickle cells 10-15 days
Affects millions through the world:
Sub-saharan Africa, Spanish speaking region,
Mediterranean countries
Most common inherited disorder in USA
- 70000 to 80000 American
1 in 500 African- American
1 in 1000 to 1400 Hispanic-American

Symptoms of SCD

Hand-foot syndrome
Fatigue, paleness, and
shortness of breath.
Pain that occurs
unpredictably in any
body organ or joint.
Eye problems.
Yellowing of skin and
eyes.
Delayed growth and
puberty in children and
often a slight build in
adults.

Complications:
Infections.
Stroke
Acute chest
syndrome.

Pathophysiology
1.
2.

Hemolysis
Occlusion of blood vessels
a. bone (painful crisis)
b. lung (acute chest syndrome)
c. brain
d. heart
e. spleen (Acute splenic
sequestration)
f. hands (dactylitis in children)

Sickle Cell Anemia blood

film
Sickle
Cells

Erythroblasts
HowellJolly Body

Normal vs. Sickle Red

Cells

Normal cell

Normal cell release oxygen and

maintain it biconcave shape

Deformation of the cell due to

the polymerization of
hemoglobin in sickle red cells
when they release oxygen
Sickle cell

Sickle Cell
Anemia
EM of red
cell
showing
tactoids

Fibers of Sickle Hemoglobin

Fibers of
Sickle
Hemoglobin
cross
section

Nature of the problem

Substitution of valine residue for glutamic acid

at position 6 in the beta-subunit of
hemoglobin.

HB A

HB S

Adenine

Thymine

Testing
Hemoglobin electrophoresis:
Hemoglobin electrophoresis is a test
that measures the different types of
hemoglobin in the blood.
The presence of significant levels of
abnormal hemoglobins may indicate:

Hemoglobin C disease
Rare hemoglobinopathy
Sickle cell anemia

Modulators of SCD

Fetal Hemoglobin

High level of fetal hemoglobin protects the

infants during the first year and declines
between one and two year of age.
Patient with hereditary persistence of fetal
hemoglobin (HPFH) have few symptoms.
In HPFH, Hb F usually comprise 20% of
the hemoglobin in the erythrocytes.
Fetal Hb disrupts the polymerization of
dexoxy-Hb-S

Modulators of SCD (Cont.)

Alpha-thalassemia

The rate of SCA is lower in people who

also have two-gene deletion
The mechanism by which alphathalassemia ameliorates red cell
destruction is unknown

Sickle Cell Anemia treatment

Opiates and hydration for painful

crises
Transfusion for serious
manifestations (eg stroke)
Bone Marrow Transplantation: can
cure Sickle Cell Disease
The optimal time of transplantation
is during childhood

Treatment

Hydroxyurea:
Used mainly to enhance fetal
hemoglobin production
-Blocks DNA synthesis and cell division
by inhibiting ribonucleotide reductase
- Induce fetal Hb production
-Reduces the number of dense cells
and irreversibly sickled cells in the
circulation

Experimental Therapies

Erythropoietin: stimulate red cell

production and increase Hb F level
Clotrimazole: reduces the number of
dense cells and the number of
irreversibly sickled cells in patients with
sickle cell disease
Nitric oxide: An inhaled gas that can
reduce the polymerization tendency of
sickle hemoglobin
Gene therapy: replacing the defective
gene with a normal one

Sickle Cell Trait

Heterozygous state for Hb S (Hb AS)

No serious clinical consequences
Sudden death during intensive
training
Hematuria, isosthenuria (renal
papillary necrosis)

Hereditary Persistence of
Fetal Hemoglobin

Significant Hb F production into

adulthood
Molecular heterogeneity
Several deletions of all or part of the
delta and beta globin genes
Point mutations in delta globin
Mutations in genes outside the beta
gene cluster

Frequency and
Distribution of
Hemoglobinopathies

Ethnic Group
Mediterranean
African American
Non-Hispanic Caribbean
West African
Hispanic Caribbean
Mexican/Central Amer.
Asian
SE Asian
India/Pakistan
Middle Eastern

Beta Thal
1/20-30
1/75
1/50-75
1/50
1/75
1/30-50
1/50
1/30
1/30-50
1/50

Alpha Thal
1/30-50 t
1/30 t
1/30 t
1/30 t
Variable
Variable
1/20 c
>1/20 c
Variable
Variable

Ethnic Group
Mediterranean
African American
Non-Hispanic Caribbean
West African
Hispanic Caribbean
Mexican/Central Amer.
Asian
SE Asian
India/Pakistan
Middle Eastern

HbS
1/30-50
1/12
1/12
1/6
1/30
1/30-200
Rare
Rare
1/50-100
1/50-100

HbC
Rare
1/50
1/30
1/20-30
Rare
Rare
Rare
Rare
Rare
Rare

Ethnic Group
Mediterranean
African American
Non-Hispanic Caribbean
West African
Hispanic Caribbean
Mexican/Central Amer.
Asian
SE Asian
India/Pakistan
Middle Eastern

Other Hb
D,G Lepore
O, D
O, D
O, D
Variable
J, E
E
E
D, O, E
D, O, E, J

Hemoglobinopathies
and Malaria

Why is thalassemia so
common?

The thalassemias and other

hemoglobinopathies are so
common that, unless you want to
entertain a ridiculously high new
mutation rate, there must be
some selection for the carrier
state
The Malaria Hypothesis

The Malaria Hypothesis

When mapped out, the global distribution

of malaria and populations with high
carrier frequencies for
hemoglobinopathies are essentially the
same
Being a carrier protects you from Malaria
so you have an evolutionary advantage
over a non-carrier.
How does this work?
Hasnt been pinned down yet, but
theories abound

Hb S reduces oxygen tension in cells

retarding growth of the parasite
A thal confers susceptibility to a milder
form of malaria; once contracted, the
patient has immunity to the more
severe form and increased survival
B-thal - ???; possibly a role in decreasing
cell adhesions in the deadly cerebral
form of malaria

Screening and
Testing

Screening

CBC to look for MCV (mean

corpuscular volume) and MCH
(mean corpuscular hemoglobin)
Will be reduced in both thalassemias
(microcytic anemia)

Hb Electrophresis to look for A2: Will

be elevated in B-thal, normal on Athal. May also be elevated in HbS

Screening

Hb Electrophresis may also identify

abnormal hemoglobins
Structural Hb Variants
Some Hb Barts or Hb H
Wont find unstable variants except in
exceedingly small quantities may be
missed

Iron studies to rule out iron deficiency

Mutation Identification

Not usually a diagnostic tool. You

can narrow down the diagnosis
well with non-molecular blood
testing, smears, etc.
Necessary for prenatal diagnosis
Helpful in estimating severity

Just when you thought you

understood Hemoglobinopathies

Other causes of a + carrier screen (low MCV

and high A2)
Multiple hemoglobinopathies can interact and
alter the results of screening; produce lots of
undescribed phenotypes
Non-classic: B: mutations in LCR, upstream
promoter; A: point mutations, novel/rare
deletions
Other Deletions (thalassemia, Hb Lepore)
Dominant B-thal (Irish family)
Thal plus structural hemoglobinopathies (HbS,
HbC, HbE and any one of the >300 other
abnormal B-globins)