You are on page 1of 68

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 βglobin 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