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 pseudo-
genes (Chromosome 16); , ,
B = Beta like genes and pseudo
genes (Chromosome 11); ,,,
The Globin Genes
Hemoglobin structure

A B

B A

heme
Heme
Globin
Hemoglobi Name Component
n Type s
Adult A 22
A2 22
Fetal F 22
Embryonic Portland 22
Gower 1 22
Gower 2 22
Abnormal H 4
Barts 4
Hemoglobins in normal adults

A B A G A D

B A G A D 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 Beta Thalassemia

Thalassemia Nonsense, splice and

Deletions of frameshift mutations

alpha- globin in beta-globin gene
gene(s) Symptoms begin in

Symptoms can infancy/childhood

Simple AR
begin in fetal life
Complicated inheritance;
genotype-phenotype
inheritance 4
correlation
alpha genes
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 / - - Mild microcytosis (Trait or Carrier)

A-/A- (cis vs trans)
A-/-- Hemoglobin H disease-
clinically 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 Alpha-
Thalassemia
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 Complications:

Fatigue, paleness, and Infections.
shortness of breath.
Stroke
Pain that occurs
unpredictably in any Acute chest
body organ or joint. syndrome.
Eye problems.
Yellowing of skin and
eyes.
Delayed growth and
puberty in children and
often a slight build in
adults.
Pathophysiology
1. Hemolysis
2. 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
Howell-
Jolly 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 alpha-

thalassemia 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 Beta Thal Alpha Thal
Mediterranean 1/20-30 1/30-50 t
African American 1/75 1/30 t
Non-Hispanic Caribbean 1/50-75 1/30 t
West African 1/50 1/30 t
Hispanic Caribbean 1/75 Variable
Mexican/Central Amer. 1/30-50 Variable
Asian 1/50 1/20 c
SE Asian 1/30 >1/20 c
India/Pakistan 1/30-50 Variable
Middle Eastern 1/50 Variable
Ethnic Group HbS HbC
Mediterranean 1/30-50 Rare
African American 1/12 1/50
Non-Hispanic Caribbean 1/12 1/30
West African 1/6 1/20-30
Hispanic Caribbean 1/30 Rare
Mexican/Central Amer. 1/30-200 Rare
Asian Rare Rare
SE Asian Rare Rare
India/Pakistan 1/50-100 Rare
Middle Eastern 1/50-100 Rare
Ethnic Group Other Hb
Mediterranean D,G Lepore
African American O, D
Non-Hispanic Caribbean O, D
West African O, D
Hispanic Caribbean Variable
Mexican/Central Amer. J, E
Asian E
SE Asian E
India/Pakistan D, O, E
Middle Eastern 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 A-
thal. 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)