The Haemoglobinopathies Model

Of Molecular Diseases
Diseases of the Blood

Red blood cells

White blood cells
Coagulation diseases

Red blood cells diseases

Anaemias
 Anaemias

Haemoglobinopathies … Hb (protein)
∴ Genetic diseases

Haemoglobinopathies were the first genetic

diseases to be characterized at the molecular
levels.
Genes in Chromosomes
23 chromosome pairs (46 chromosomes)
22 pair autosomes
1 pair sex chromosome

Genetic disorders

• Chromosome abnormalities (entire or large

segment)
• Single gene abnormality
• Multifactorial ( in multiple genes)
• Mitochondrial ( non-chromosomal DNA)
Single gene abnormality has 4 patterns of
passing disorders:

• autosomal dominant
• autosomal recessive
haemoglobinopathies (i.e. heterozygous or
homozygous)
• x-linked dominant
• x-linked recessive
Genes of Globin Chains

α- and β- chains (subunits) of Hb are encoded by

two gene families.
Members of each family are structurally closely
related and their expression is regulated as
different forms (members) are required through
development : embryonic, fetal , and adult.
.FIGURE 1
In α-globin locus : ζ gene in embryo, α1 and α2
in both fetus and adult.
In β-globin locus : ε gene in embryo, fetal Gγ and
Aγ , and δ and β in adult.
ψβ, ψζ and ψα are pseudogenes.
Each gene is a separate transcriptional unit with:
5' upstream regulatory sequences and coding
sequences (3 exons) split by two introns
followed by transcriptional termination signals.
Figure 2
At the end of the primary RNA there is a
sequence AAUAA site for endonuclease that
removes 15-30 nucleotides leaving site for poly
A to be added.
There are two consensus sequences: on 5' end
of intron and 3' end of intron.
They are important for splicing out introns
involving complemintary sn RNA.
α-globin is clustered within 25 kb
.FIGURE 3
Mutations ( extent)

A. Point mutations .
B. Deletion mutations .

Mutations ( effect )

1. Transcriptional mutants.
2. RNA processing mutants.
3. RNA translation mutants.
4. Post-translational instability mutants.
Point mutations :
:Silent mutations.1
In third base of codon no change
.e.g. UCC UCA both give serine
: Mutations in promoter .2
Lead to inefficient transcription into mRNA
e.g.β .- thal
Cap site mutations : e.g. .3β- thal
:Read- through mutations .4
Change in termination codon longer Protein
e.g.α.- thal
:Missense mutations .5
.One amino acid change e.g. HbS
.Intron or exon e.g. B-thal
Mutations in splicing signals in forming mRNA .6
consensus mutation ) : defect in mRNA )
synthesis e.g.β. - thal
Mutations in polyadenylation of mRNA .7
precursor e.g.β .- thal
 Mutation in initiation codon .8
:Non sense mutations .9
Formation of termination codon truncated
protein
e.g.β.- thal
:Frameshift mutations .10
Insertion or deletion of a single nucleotide
. within codon region
Reading frame is altered and subsequent
. codons are read
e.g.β- thal
Hb Wayne
.Table 1
 β-Thalassaemia
80 – 90 million are carriers of β-thalassaemia gene (5%).
~170 different point mutations or small insertion/deletion
mutations, and ~17 gene deletions (25 bp – 67 kb) in
β-globin was discovered. Result in gene of :
β°- no β-globin, β+- severe deficiency, β++- mild .

Phenotypes are :
β-thalassaemia major,
β-thalassaemia intermedia,
β-thalassaemia minor,
Depending on the combination of β°- , β+- , β++-
Which comes from a low level or total absence of β-
globin chain.
• Mutations :-
2. Mutations in promoter:
Affect transcription (inefficient transcription
or mRNA synthesis) resulting in low level
of mRNA (20% normal),
i.e. β+- thal.
Table 2
Figure 4
Table 3 •
Cap site mutations. .2β+ .- thal
Table 2
Figure 4
Table 3 •
.3
a) Mutations at the splice junctions of introns,
where other donor cryptic sites lead to
untranslatable mRNA.β° . - thal
Table 2
Figure 4
Table 4
Figure 5
 b) Changes in consensus sequence on either of
conserved dinucleotides of splice site lead to
normal and cryptic sites.β+.- thal
mutation could affect 3' acceptor site of intron 2
in which cryptic site lead to mRNA with a part
of 3' intron 2, but cryptic 3' site could spliced
. out with normal 5' acceptor
β+ .- thal
Figure 5
Table 2
Figure 4
 c) Mutations within intron creating new 3'
acceptor splice site
Mutations within exon creating new 5, donor
splice site lead to new splice sites competing
. with normal site
β+ - thal.,β++ .- thal
Table 5
 Mutations in polyadenylation of mRNA .4
. precursor

β++ .- thal
Table 2
Figure 4
Table 6
Mutations in initiation codon. .5
β°-thal
Table 6
 Nonsense mutations giving rise to translation .6
termination codon ( leading to premature
termination of peptide synthesis ).β° . - thal
e. g. codon 17, AAG (lys) becomes UAG
.(a stop codon)
Table 2
Figure 4
Table 6
Frameshift mutations .7

Small deletion or insertion in coding sequence

. causing shift in reading frame
β° . - thal
Table 2
Figure 4
Table 7
Table 8
B.Deletions Mutations
Table 9
Table 10
Table 11
Table 12
 α-Thalassaemia
million carriers (i.e. 250α+ -) , 26 millionα° - carriers
.(in East Asia , 1% in Middle East % 5-15 )
/different deletions , and ~47 point or small insertion 32~
.deletion mutations
Results inα° - noα-globin
α+ - one gene in theα.-globin cluster
: Phenotypes are
α (-thalassaemia major ( Hb Bert hydrops faetalis
.all 4 genes deleted
α-thalassaemia minor one –three genes are deleted
α-thalassaemia carrier one –three genes are deleted
-A.: Mutations
RNA- processing mutants .1
- a) splice site mutationsα+
- b) polyadenylation signal mutationsα+ -α°
RNA- translation mutants .2
- a) initiation codon mutationsα+ - andα+ -α°
- b) termination codon mutationsα+
Table 13
 normal stop codon UAA mutates to one of
sense codons giving amino acid at position 142,
continue to 172 amino acid (instead of 141 amino
acids). Then come a triplet normally untranslated
.to be a termination codon at normal position 173
Table 15
- c) frameshift mutationsα+
- d) nonsense mutationsα+

Post-transcriptional instability mutations .3

- Point mutationsα+
- Small deletionsα+
Table 13
Table 14
 B.Deletions
- deletions involving one .1α -globin geneα+
- deletions involving both .2α -globin geneα°
- deletions of locus control region .3α°
Table 16
 Haemoglobin S
Clinically the most important -20% in some
areas- missense mutation is HbS/S in which a
change of A→U
in either GAA or GAG codon of glutamate ,at
position 6 ofβ-globin chain, into GUA or GUG
.codons of valine
Or A→.T in GAG of glutamate into GTG of valine

.in 10 Afriamericans carries these mutations 1

The homozygous gives sickle cell anaemia
.disease
Figure 6
: Other genotypes
Hb S/Cβ6 GAG→ AAG (glu→(lys
,Where both oxyHb and deoxyHb are affected
.but as crystals (dehydrated RBC). It is milder than HbS/S
Hb S/β° - thal (heterogeneity in phenotype due to type of
Hb S/β+ - thalβ(.-thal

Hb S/δβ -thal mild due to↑( Hb F (10- 12 g/dl

Hb S/ D Punjabβ 121 glu→( gln mild to moderate(5-10g/dl
Hb s/ O arabβ 121 glu→ lys severe, similar to sickle cell
Hb S/ C Harlemβ6 glu→ val ,andβ 73 asp→ asn
severe sickle cell
Hb S/ Omanβ6 glu→ val ,andβ 121 glu→ lys
Combined withα-thal
Clinically (phenotypically) Hb S involve interaction with other
factors . e.g.↑ Hb F ( -158 Gγ C→ . % T ) 5- 10 upto 30
.Other mutations as above