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.