Sickle Cell Disease & -Thalassemia

Genes, Disease, and Therapeutics

Fall 2015

November 3
Genetics, newborn screening,
molecular phenotype, case study,
inheritance, prenatal testing, question
and answer

Hemoglobin
Hemoglobin (Hb) protein transports
oxygen in red blood cells
Adult Hb is a tetramer with 2 chains, 2
chains and 4 heme groups
chain encoded on chromosome 16 gene
cluster and chain on chromosome 11
gene cluster

Hemoglobinandthe-GlobinChromosome11GeneCluster

Embryonic Hb: 2/2

5UTR
(50 nt)

(93 nt)

Exon 1

IVS1 (129nt)

Fetal Hb: 2/2

G A

(222 nt)

Exon 2

Adult Hb: 2/2

IVS2 (850 nt)

(129 nt)

3UTR (131 nt)

Exon 3

Adult hemoglobin
Hemoglobin A (22)

The most common type in adults (about 97%)

Hemoglobin A2 (22)

chain synthesis begins late in the third trimester

and in adults, it has a normal level of about 2%

Hemoglobin F (22)

In adults, restricted to a limited population of red

cells called F cells and has a normal level of less
than 1%

Beta-globin LCR
Upstream of the epsilon-globin gene in the
beta-globin gene cluster on chromosome 11
LCR, in addition to individual promoters for
each beta-globin like gene in the beta-globin
gene cluster, provides regulatory control of
gene expression
Switching of beta-globin like genes during
development

Developmental regulation of expression of beta-globin

gene cluster

Two developmental switches in expression

from the cluster
Embryonic to fetal during the first trimester
Fetal to adult around birth

Processes involved
DNA methylation:

Hypermethylation of beta-globin promoter during

embryonic and fetal stages
Hypermethylation of gamma-globin promoter during
adult stage

Developmental regulation of expression of beta-globin

gene cluster

Processes involved (cont)

Histone acetylation and deacetylation

Active globin genes are hyperacytylated

Inactive globin genes are hypoacytylated

Sickle Cell Disease & -Thalassemia

Mutations in either or globin gene cluster
members can lead to a wide variety of abnormal Hb
(structural or numerical chain problems)
SCD is usually a defect in globin chain structure
-Thal is usually a defect in globin chain number
Combinations possible - A sickle allele and one
type of -Thal allele together cause S0-Thal (S0)
which has many features of sickle cell disease

Sickle Cell Disease

SCD alleles are more common in the
following ancestries: African, Mediterranean,
Middle Eastern, Indian, Caribbean, and
parts of Central and South America
One in every 300-500 African-Americans
born/year has SCD (specifically, Hb SS)
One in every 36,000 Hispanic-Americans
born/year has SCD (specifically, Hb SS)

Sickle Cell Disease

SCA allele (S) provides protection against
malaria and thus is more prevalent in
populations where malaria is widespread

RBC of infected heterozygotes (Swt) believed

to express malarial antigens more effectively
than infected homozygote normal (wtwt) so
immune system will clear infection more
rapidly in these RBCs

-Thalassemia
-Thal alleles are more common in the
following ancestries: Mediterranean, Middle
Eastern, Central Asian, Indian, Far Eastern,
and African.
Like the SCD allele, thal alleles (0 and +,
but note not truly alleles per say) provide
protection against malaria and thus are
more prevalent in populations where malaria
is widespread

SCD & -Thalassemia newborn screen

All 50 U.S. states and D.C. screen for SCD
(which includes sickle cell anemia), many
also screen for types of -Thalassemia
SCD screening may include screening for
SCA, sickle cell trait, SC Disease, S0Thalassemia, S+-Thalassemia, SE Disease,
and CC Disease.

SCD & -Thalassemia newborn screen

Currently, most screening done by isoelectric
focusing (IEF) from dried blood spot
If IEF test positive, usually a second test is
performed

If second test is HPLC or DNA testing, this usually

allows for a diagnosis to be made (esp. if DNA test,
HPLC result may not be reliable as we will discuss later)
If the second test is positive, often a final test on a fresh
blood sample is completed around 6 weeks of age

SCD & -Thalassemia newborn screen

Newborn screening may produce a false
negative result if baby has had a transfusion
Prematurity may also confound newborn
screening results

A normal premature infant may only show HbF on

IEF (no HbA made yet)
Premature infant with SCA may also only show HbF
on IEF because of prematurity (the -globin gene is
not yet expressed in large quantities so dont see
that HbS yet)

Texas newborn screening for SCD

Texas currently screens for Sickle Cell
Anemia (HbSS), SC Disease, S+Thalassemia, and S0- Thalassemia
About 1 in 2500 newborns found to have
SCD each year via newborn screening in
Texas

Possible IEF results and interpretation

Result 1: Newborn shows HbF and HbS,
but no HbA
What does this suggest?

Result 2: Newborn shows HbF, HbS, and

HbC, but no HbA
What does this suggest?

Possible IEF results and interpretation

Result 3: Newborn shows HbF, HbS, and HbA
What does this suggest?

Result 4: Newborn shows HbS alone (i.e. no

HbF, HbA, etc.)
What does this suggest?

Result 5: Newborn shows HbF alone (i.e. no

HbA, HbS, etc.)
What does this suggest?

SCD - Genetics
Terminology can be confusing

Most common form of normal adult

hemoglobin is Hb A. When someone has
normal adult hemoglobin, they are sometimes
referred to as being Hb AA. Genotype is wtwt
(the alleles are also wildtype, but they are
not usually noted)

SCD - Genetics
SCD comprises:

Sickle Cell Anemia (involving Hb S with the disease

phenotype sometimes referred to as Hb SS. Genotype SS)
Sickle Cell Trait is Hb AS with genotype wtS. This is the carrier
heterozygote (the carrier state for SCA)

SC Disease (involving both Hb S & Hb C with the disease

phenotype sometimes referred to as Hb SC. Genotype SC)
S0-Thal (involving Hb S with the disease phenotype
sometimes referred to as Hb S0. Genotype S0, but note 0
(and +) not really alleles as discussed previously)
Other conditions usually involving a sickle allele

Hemoglobinandthe-GlobinChromosome11GeneCluster

SCD - Genetics
Embryonic Hb: 2/2

Fetal Hb: 2/2

Adult Hb: 2/2

globin gene is HBB

Gene is 1.6kb with 3 exons
5UTR
(50 nt)

(93 nt)

Exon 1

IVS1 (129nt)

(222 nt)

IVS2 (850 nt)

(129 nt)

3UTR (131 nt)

Exon 3

Upstream of globin gene cluster on

chromosome 11 is locus control region (LCR)
Exon 2

Controls developmental timing of expression of

the genes in the beta globin gene cluster

SCD - Genetics
Numerous types of mutations can lead to numerous
types of structural aberrations in the chain
SCA (Hb SS) (60-70% of SCD in U.S.)
Both alleles S. S results from a point mutation in globin
gene. GAG (glutamic acid) -> GTG (valine) in 6th codon
A to T is in nucleotide 20.

SC Disease (Hb SC)

One allele is S and other is C. C results from a point mutation
in globin gene. Causes glutamic acid to lysine in 6 th codon
G to A in nucleotide 19.

Another confusing aspect of Hb literature

The SCA mutation (and SC disease

mutation) is in the 6th codon of the gene.
Its actually the 7th codon, but because the
mature globin protein does not include
the initiating Met, they say the 6th codon.

SCD - Genetics
S0-Thalassemia (Hb S0)
One allele is S and other allele is 0. As noted earlier, 0 is not
truly a specific mutation (i.e. particular allele). It refers to the
molecular phenotype of the thalassemia. 0 denotes no chain
made from that allele. Numerous different mutations can cause
this.

S+-Thalassemia (Hb S+)

One allele is S and other allele is +. + is not truly a specific
mutation (i.e. particular allele). It refers to the molecular
phenotype of the thalassemia. + denotes some, but less than
wildtype levels, of chain made from that allele. Numerous
different mutations can cause this.

SCD - Genetics
Of most common 4 SCD, SCA and S0Thalassemia are more severe phenotypes
versus SC Disease or S+-Thalassemia

SCD - Genetics
Numerous other SCD mutations possible.
Examples:
SD Disease (Hb SD)
One allele is S and other is D. D is Glu->Gln at codon 121.
Sometimes referred to as D-Punjab reflecting higher
incidence in Indian population

SO Disease (Hb SO)

One allele is S and other is o (not to be confused with form
of thalassemia which is zero). o is Glu->Lys at codon 121.
Sometimes referred to as O-Arab reflecting higher incidence
in Middle Eastern population

SCD - Genetics
Numerous other SCD mutations possible.
Examples (cont.):
SE Disease (Hb SE)
One allele is S and other is E. E is Glu->Lys at codon
26. E found in Sri Lanka, Eastern India, Southeast Asia,
and Southwest China

CC Disease (Hb CC)

Both alleles C. C results from a point mutation in
globin gene. Causes glutamic acid to lysine in 6 th
codon. CC disease is homozygosity for this mutation

Case study
17 year-old male with severe clinical
course

frequent pain crises of increasing intensity

2 episodes of acute chest syndrome requiring
hospitalization and multiple blood transfusions

Had been diagnosed with SC Disease at

age 6 with a confirmation diagnosis of SC
Disease at age 11

Case study
SC Disease is normally associated with a
less severe clinical picture compared to,
for example, SCA
SC Disease

Painful crises usually appear later in life (often

after age 20)
Less frequent episodes of crises

Case study
Methodology used to make the initial
diagnosis in patient (at age 6) was
unknown
Methodology used to make diagnosis at
age 11 was IEF and an older version of
HPLC instrument

Case study
DNA sequencing also performed and indicated
presence of O-Arab -globin mutation (versus C
Harlem which was in the differential here)
Discussion with patient also revealed a relevant
piece of family history
Patients brother was recently diagnosed with S/OArab Disease after an earlier misdiagnosis with SC
Disease (methodology in misdiagnosis unknown)

Patient actually has S/O-Arab Disease (also

known as SO Disease), not SC Disease

Case study
S/O-Arab Disease

More clinically severe than SC Disease

S/O-Arab Disease shows earlier onset, more frequent,

and more severe clinical characteristics
hemolytic anemia, jaundice, vaso-occlusive
complications such as pain crises and stroke,
pneumonia, acute chest syndrome, and sepsis

Patient had frequent pain crises of increasing

intensity and 2 episodes of acute chest syndrome
requiring hospitalization and multiple blood
transfusions all by age 17

Case study
Revised diagnosis led to revised treatment
protocol for patient
Patient was started on hydroxyurea (1000
mg/day) in accordance with NIH consensus
documents

As we will discuss in next lecture, hydroxyurea has

been shown to reduce episodes of pain crises and
acute chest syndrome
At follow-up, patient reported improved health and
anemia had improved somewhat

Case study
Easy to misinterpret the IEF result and older
version of HPLC did not allow one to
distinguish between HbC and HbO-Arab
So understandable that he was
misdiagnosed
However, the emergence of such a severe
clinical picture should have prompted
physicians to question diagnosis perhaps
earlier

Case study
This case also emphasizes importance of using
more than one methodology to determine
diagnosis in these conditions, especially if there
is known possibility of ambiguity
Investigators noted that if a well-known (albeit
not often used now) hemoglobin electrophoretic
technique with citrate agar had been used
initially, could have distinguished between HbC,
HbO-Arab, and HbC-Harlem

SE Disease case study

IEF newborn screen of male infant
suggested SE Disease
At age 14 months, presented with fever
and strong pain in thighs
Investigators sought to confirm diagnosis
(unclear how diagnosis was initially made
after the positive newborn screen)

 HPLC also suggested SE Disease

HPLC results of boys parents

SE Disease case study

Investigators noted that often times it is
difficult to distinguish various hemoglobins
depending on the technique used

They noted that HbE comigrates with HbO-Arab

(among others) in IEF
HbE comigrates with HbC and HbO-Arab (among
others) in cellulose acetate hemoglobin
electrophoresis at alkaline pH
HbE and HbA2 have similar retention times in
HPLC

SE Disease case study

How to resolve these issues?
DNA analysis is useful
They used the primer specific PCR-based
system called Amplification Refractory
Mutation System (ARMS)

ARMS
Two different tubes used for PCR analysis

Tube 1: Patient sample, a common primer and a

mutation-specific primer

Often looking for a point mutation. The mutation-specific

primers 3 end is complementary to the mutant nucleotide
HbS and HbE both due to point mutations, so technique is
applicable here

Tube 2: Patient sample, same common primer as

was used in tube 1 and a normal primer

normal primer = primer with the 3 end complementary

to the normal nucleotide

ARMS
PCR reaction run and result run on a gel
If patient is homozygous normal, will only
see a PCR product on the gel from tube 2
If patient is homozygous mutant, will only
see a PCR product on the gel from tube 1
If patient is heterozygous with one normal
and one mutant allele, will see PCR
products from both tubes 1 and 2

This is not from the case study, but is conventional ARMS

ARMS in the case study

They used a single tube ARMS variation
Can detect mutant and normal in single tube
Still ARMS in that 3 end of primer is the key
use different primers for mutant versus
normal and the 3 end nucleotide reflects the
difference between two
Add another primer set so can differentiate
mutant versus normal also by size on
electrophoresis

SE Disease case study

Boys SE Disease diagnosis could be confirmed

Investigators noted that DNA analysis is useful in diagnosis of

hemoglobinopathies where ambiguity may arise when using
other techniques (IEF, HPLC, etc)

Investigators reviewed a number of SE Disease case

studies and noted:

Although HbS and HbE have higher incidence in somewhat

distinct populations, population migrations and racial
intermarriages over the last century have led to increasing
numbers of individuals who are compound heterozygotes for
Hb S and Hb E
SE Disease not a benign disease

-Thalassemia - Genetics
Terminology is clinically based
-Thalassemia major, intermedia and minor

Numerous types of mutations can lead to numerous

types of numerical aberrations in the chain
-Thalassemia major possible genotypes (again, note
that the thalassemia allele represents one of many
different mutations that may cause either the absence
or reduction of chain production from that allele)
00, 0+, ++ (note ++ may also cause intermedia, it
depends on the severity of the particular mutation)

-Thalassemia - Genetics
-Thalassemia intermedia possible
genotypes
++, 0wt (for example, if have also have
increased amount of globin to be
discussed later)

-Thalassemia minor possible genotypes

0wt (especially if also have compensatory
Thal), +wt

-Thalassemia - Genetics
Prior are all considered simple Thalassemia
Complex -Thalassemia globin gene
and one or more other members of
chromosome 11 globin gene cluster
involved (may also include LCR)

Hb Lepore and Hb Miyada

Meiotic recombination from mispairing
between the similar and globin genes
creates two fusion strands, Lepore and
Miyada (aka a type of anti-Lepore)

Hb Lepore and Hb Miyada

Sometimes classified as Hb structural variant with Thalassemia phenotype

Not only does it produce abnormal beta globin chain, it also has
reduced beta globin chain synthesis

Fusion chain in each (Lepore and Miyada) has reduced

expression (not completely absent) so usually +
Homozygous mutant or compound heterozygote with a
severe -Thalassemia allele leads to -Thalassemia major
Compound heterozygote with s : HbS/HbLepore, for
example, is considered more of a structural variant of
hemoglobin disorder

-Thalassemia - Genetics
The location and type of mutation in the globin gene sometimes allows you to predict
phenotype
Examples that lead to -Thalassemia major
Homozygosity (or compound het with another 0
allele) for IVS1 +1 G->T
Mutation found in Asian Indian and Chinese population
Mutation is in splice donor site and results in no normal
-globin mRNA formed is a 0 allele

Reminder on normal splicing

Consensus splice site sequences for
major class of introns (U2 introns)

MAG/gtragt for EXON/intron (5ss) junction

cag/G for intron/EXON (3ss) junction
Where M = A or C and R = A or G

gt in red is the core splice donor and ag

in blue is the core splice acceptor

-Thalassemia - Genetics
Examples that lead to -Thalassemia
major (cont)
Homozygosity (or compound het with another
0 allele) for IVS1 +1 G->A
Mutation found in Jordanian, Egyptian, Syrian, and
Palestinian populations
Mutation is in splice donor site and results in no
normal -globin mRNA formed is a 0 allele

-Thalassemia - Genetics
Examples that lead to -Thalassemia
major (cont)
Homozygosity (or compound het with another
0 allele) for MET1ARG
Mutation found in Chinese population
Initiator codon mutation, ATG to AGG is a 0
allele

-Thalassemia - Genetics
Examples that may lead to -Thalassemia intermedia
(depending on the other allele)
IVS1 +5 G->C
Mutation found in Asian Indian population
Mutation is in splice donor region of -globin intron 1 and results
in reduced normal -globin mRNA production is a + allele
Note that the consensus sequence on the earlier slide is variable
depending on the gene and intron we are talking about
IVS1 of normal -globin begins with GTTGGT - the nucleotides that differ
from the consensus sequence are in green

-Thalassemia - Genetics
Examples that may lead to -Thalassemia
intermedia (depending on the other allele)
(cont)
IVS1 +5 G->T
Mutation found in Mediterranean and Northern
European populations
Mutation is in splice donor region of -globin intron
1 and results in reduced normal -globin mRNA
production is a + allele

-Thalassemia - Genetics
Examples that may lead to -Thalassemia
intermedia (depending on the other allele)
(cont)
IVS1 +110 G->A
Mutation found in Mediterranean populations
Mutation is in -globin intron 1 and creates a new
splice acceptor site. This results in reduced
normal -globin mRNA production is a + allele

-Thalassemia - Genetics
IVS1 +110 G->A (cont.)
Mutation is in -globin intron 1 and creates a new
splice acceptor site. This results in reduced
normal -globin mRNA production is a + allele

SCD Molecular Pathology SCA

Under deoxygenated state, the codon 6 valine
binds in a hydrophobic pocket of another chain
and rigid strands of hemoglobin polymers form
Attempts of these RBC to flow through narrow
vessels causes them to adopt a sickle shape and
become stuck in the vessel (vascular occlusion)
Ischemia (and eventual necrosis) followed by
inflammation and neuropeptide release cause
damage (hemolysis) and pain

SCD Molecular Pathology SC Disease

Oxygenated Hb C tends to crystallize, causing RBC
to be somewhat rigid
Hb C also induces RBC to dehydrate
K-Cl co-transport thought to be involved

In SC Disease, Hb C enhances pathogenic properties

of the Hb S that is also present
Dehydrated RBC (from presence of Hb C) has an
increased hemoglobin concentration which is a favorable
environment for Hb S polymerization to occur
Leads to a significant disorder, albeit not as severe as SCA

SCD Molecular Pathology SC Disease

-Thalassemia Molecular Pathology

Normal hemoglobin has an equal balance
of and chains
In -Thalassemia, reduced or no chains
creates an excess of free chains
These free chains are insoluble and
precipitate in RBC precursors which are
destroyed in bone marrow so have
ineffective erythropoiesis

SCD and -Thalassemia - Inheritance

Inheritance is autosomal recessive
Parents of an affected individual are usually
heterozygotes (carriers) and not severely
affected

Carriers of SCA allele Have Sickle Cell Trait.

Usually asymptomatic, but may show signs of
disease under extreme exertion, at high altitude or if
severely dehydrated
Carriers of -Thal allele Usually have -Thal minor
with mild anemia, however(next slide)

Carrier of -Thal allele

What do we mean by this?

0wt or +wt: The person has at least one wild

type -globin allele

Usually -Thal minor, but in some cases

may be -Thal intermedia
If person also has triplicated or quadrupled
alpha globin gene rearrangement

SCD and -Thalassemia - Inheritance

Carrier parents have a 25% chance of having
an affected child, 50% chance of having a
carrier and 25% chance of having normal
non-carrier
All children of an affected individual will be at
least carriers
Sibling of an affected person - if the sibling is
known via clinical diagnosis to be unaffected,
what is the chance is a carrier?

SCD and -Thalassemia Prenatal testing

Chorionic villus sampling (CVS) at 10-12

weeks gestation
Amniocentesis at 15-18 weeks gestation

SCD and -Thalassemia Prenatal testing

CVS or amnio

Risk of both is loss of pregnancy

Gene testing from these samples may include
targeted mutation analysis (RFLP often used
for SCA) and sequencing analysis (often used
for -Thalassemia since numerous mutations
possible)
Desirable to have parents mutations identified
prior to prenatal testing