Post on 27-Oct-2014
transcript
SAJAD AHMAD
Introduction The inherited diseases of hemoglobin
are the most common single-gene disorders
About 7 % population of the world are carriers
This group of diseases has a particularly high frequency in a broad belt extending from the Mediterranean basin, Middle East, The Subcontinent all the way to the islands of the Pacific
Introduction (contd)
First recognized by Thomas B. Cooley in 1925
Pathological changes first described by Whipple and Bradford in 1936
Globin Chains
Tetrameric structure HbA α2β2
HbA2 α2δ2 Adults
HbF α2γ2
Hb Portland ζ2γ2
Hb Gower 1 ζ2ε2 Embryo
Hb Gower 2 α2ε2
Globin genes
β globin genes - Ch 11Spread over 60 kb and arranged in the order of
5’-ε-Gγ-Aγ-ψβ-δ-β-3’
α globin genes - Ch 16Arranged in the order of 5’-ζ'- ψζ –ψα1-ψα2-3’
Ψβ, ψζ and ψα are pseudogenes
Classification Clinical
Hydrops fetalis – Four gene deletion α-thalassemia
Thalassemia Major – transfusion dependent, homozygous β0-thalassemia or other combinations of β-thalassemia trait
Thalassemia intermediaThalassemia minor – β0-thalassemia trait, β+-
thalassemia trait, HPFH, ζ β-thalassemia trait, α0-thalassemia trait, α+-thalassemia trait
Classification (contd) Genetic α-Thalassemia
α0
α+
○ Deletion (-α)○ Non-deletion (αT)
β-Thalassemiaβ0
β+
Normal HbA2
Silent
Classification (contd) δβ-Thalassemia
(δβ)0
(Aγδβ)0
(δβ)+
γ-Thalassemia δ-Thalassemia
δ0
δ+
εγδβ-Thalassemia
Hereditary persistence of fetal hemoglobinDeletion (δβ)0 (Aγδβ)0
Non-deletion○ Linked to β-globin genes Gγβ+
Aγβ+
○ Unlinked to β-globin genes
β-Thalassemia The gene located on the short arm of Ch 11 Appropriate expression is dependent upon
LCR located 5 – 25 kb upstream Complete absence of β-globin chain β0
Largely variable reduction of β-globin output β+
More than 200 mutations have been found most of which are “point mutations”
Gene deletion is a rare cause of β-thalassemia except for the Sub-continent
Classes of β-thalassemia mutationsTranscription Deletions
Promoters
Processing of mRNA Splice junctionConsensus sequenceInternal IVSCryptic splice sites in exonsCleavage and polyadenylation siteCAP site
Translation NonsenseFrameshiftInitiation site
Post-translational instability Exon 3 mutationsOther unstable β-chains
Non deletional β-thalassemia(1) Transcription mutations Promoter mutations
Involve the TATA box and CACCC boxReduce the binding of RNA polymeraseReduce the rate of mRNA transcriptions by 20 –
30 %Result in β+-thalassemia and a mild
phenotype 5’ Untranslated region mutations
This is 50-nucleotide regionMutations result in a mild phenotype
(2) Mutations Affecting mRNA Processing Splice junction and consensus sequence
mutationsMutations of 5’-GT- and 3’-AG- completely
abolish normal splicing, thus β0-thalassemiaMisspliced RNA cannot be translatedEfficiency of normal splicing may be decreased by
mutations within the consensus sequences immediately adjacent to the splice junctions e.g. mutations at position 5 of IVS-1 severe β+-thalassemia phenotype
Cryptic site mutations in introns and exons2 cryptic splice site mutations identified in IVS-1
and four in IVS-2IVS-1-110 GAIVS-1-116 TGSevere β+- or β0-thalassemia phenotypeMutations at codons 19, 26 and 27 result in
abnormal hemoglobins e.g. cd 26 HbE (Glu Lys)
Poly (A) and other 3’ Untranslated region mutationsThe AAUAAA sequence represents a signal for
the cleavage and polyadenylation reaction.Polyadenylation is important in the stability of
mRNA and mutations in this region affect the efficacy of translation resulting in β+-thalassemia of mild severity
(3) Mutations Affecting mRNA Translation Initiation codon mutations
The initiator codon ATG codes for methionine and is a signal for starting translation
7 different point mutations identifiedβ0-thalassemia
Nonsense mutationsFormation of stop codons TAA, TAG or TGAPremature interruption of mRNA translationβ0-thalassemiaNonsense-mediated decay
Frameshift mutationsInsertion or deletion of one or a few nucleotides
alters the reading frame of the encoded mRNA starting at the site of mutation. The new reading frame results in a novel abnormal amino acid sequence and in a premature termination downstream.
β0-thalassemia
(4) Post-translational stability Nonsense mutation in exon 3 are not
subjected to nonsense-mediated decay and hence abnormal mRNA is translated thus leading to the formation of long, unstable β-globin gene products
This is the basis for dominant β-thalassemia β-thalassemia intermedia
Deletional β-Thalassemias
Several deletions affecting only the β-globin chain has been reported the most important of them is a 619 bp deletion removing the 3’ end of the β-globin chain and is common in Pakistan and India.
Phenotype of β0-thalassemia with unusually high levels of HbF and HbA2 in heterozygotes
Total deletion of β-cluster result in lack of any globin production and hence in (εGγ-Aγδβ)0
thalassemia
δβ-Thalassemia Much less common than β-thalassemia Some cases are due to deletions of β- and
δ- gobin genes, others are due to unequal crossing over between the homologous δ- and β- genes thus forming hybrid δβ genes called Lepore and βδ-genes called anti-Lepore
The Lepore Hb contains N-terminal amino acid sequence of the normal δ-chain and the C-terminal sequence of normal β-chain
Three variants of Hb Lepore have been described Boston or Washington (δ 87/β IVS-2-8)BaltimoreHollandia
Depending upon the point of transition from δ to β sequence
(δβ)0-thalassemia results from different length deletions of the δ- and β-globin genes
Based on the presence of one (Gγ-)or both (Gγ- and Aγ-) genes and synthesis of only Gγ- or both (Gγ- and Aγ-) globin genes, two groups of (δβ)0-thalassemia have been identifiedGγ(Aγδβ)0
GγAγ(δβ)0
Hereditary Persistence of Fetal Hemoglobin (HPFH)
Heterogenous group of diseases Little clinical importance but may change
the phenotype of β-hemoglobinopathies Some forms result from long deletions of β-
globin gene cluster. Homozygotes have 100 % HbF
Another type of HPFH results from point mutations in the promoter regions upstream from either the Gγ- or Aγ-globin genes which allows these to be active in adult life
The linked β-genes also remain active and hence are called Gγβ+ and Aγβ+ HPFH
The third type consists of low HbF and is called Swiss HPFH.
Genetic determinant seems to be located on Ch 6
β-Thalassemia Intermedia The term is used to describe patients with
the clinical picture of thalassemia which, although not transfusion dependent, is associated with much more severe degree of anemia than found in carriers
β-Thalassemia Intermedia Mild forms of β-thalassemia
Homozygosity for mild β+-thalassemia allelesCompound heterozygosity for two mild β+-
thalassemia allelesCompound heterozygosity for a mild and more
severe β-thalassemia allele
Inheritance of α- and β-thalassemiaβ+-thalassemia with α0-thalassemia (- -/αα) or α+-
thalassemia (- α/αα or - α/- α)β+-thalassemia with genotype of HbH disease
(- -/- α)
β-Thalassemia with elevated γ-chain synthesisHomozygous β-thalassemia with heterocellular
HPFHHomozygous β-thalassemia with Gγ or Aγ
promoter mutationsCompound heterozygosity for β-thalassemia and
deletion forms of HPFH
Compound heterozygosity for β-thalassemia and β- chain variantsHbE/ β-thalassemiaOther interactions with rare β-chain variants
Heterozygous β-thalassemia with triplicated α-chain genes (ααα)
Dominant forms of β-thalassemia Interactions of β and (δβ)+ or (δβ)0 -
thalassemia
The Pakistani perspective The five most common mutations
IVS 1-5 (G-C) (Most common in South Pakistan)Fr 8-9 (+G) (Most common in North Pakistan) del 619Fr 41-42 (-TTCT)IVS 1-1 (G-T)
These five constitute about 82 % of all the mutations
Phenotype-Genotype relationships Remarkable phenotypic variability Molecular basis for this diversity partly
understood Genetic modifiers of β-thalassemia
Primary mutationsSecondary reduce degree of imbalanceTertiary complications of disease
α-Thalassemia More common than β-thalassemia Located in telomeric region of Ch 16 α1 and α2 only differ in IVS-2 and in 3’ noncoding
region Level of transcription of α2 is two to three times
more than α1 i.e. α2 produces more α-globin than α1
The expression of α-globin genes is controlled by the sequences in and around the structural genes and by a region located 40 kb upstream called Hypersensitive site (HS)-40.
Deletional α-Thalassemia Nondeletional α-Thalassemia
Deletional α-Thalassemia Common cause of α-Thalassemia Mechanism
The α chains are embedded within 2 highly homologous regions extending approx 4 kb. 3 homologous subsegments (X, Y and Z) separated by non-homologous elements have been defined.
Reciprocal recombination between Z boxes which are 3.7 kb apart and between X boxes which are 4.2 kb apart gives rise to chromosomes with only one α gene
These are referred as –α3.7-kb rightward deletion and –α4.2-kb leftward deletion
Based on the exact location within the Z box where the crossover took place, the –α3.7-kb deletion is further divided into –α3.7 I, –α3.7 II and –α3.7 III
Deletions that remove all or part of the α-globin gene cluster including both α genes and sometimes the embryonic ζ gene result in α0-thalassemia
α0-thalassemia also occurs from deletions of the α-globin regulatory element HS-40 and twelve such deletions have been reported
Nondeletional α-Thalassemia Less common Constant Spring mutation is common in
South-East Asia Majority of the nondeletional mutants so far
reported occur in the α2 gene and have a severe effect on α-globin gene expression
Hb Constant Spring (α 142 TAA CAA, Stop Glu) results from change of stop codon to an amino acid resulting in very low amount of an α-chain variant elongated by 31 amino acids
Phenotype-Genotype relationships The homozygous states for the non-
deletional forms of α+-thalassemia often have a more severe phenotype than those for the deletion forms.