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Molecular lesions in thalassaemias are complex. Of the two common types, majority of β thalassaemias are caused by point mutations, while most of the α thalassaemias result from gene deletions.

β Thalassaemias

There is a single β globin locus on each chromosome (number 11) and as humans are diploid there are two β genes. Normal structures of globin genes and globin synthesis have been considered earlier (see chapter on “Overview of physiology of blood”).

β thalassaemias are classified into two major types: β⁰ thalassaemia and β⁺ thalassaemia. β⁰ thalassaemia is characterised by complete absence of β chain synthesis (complete deficiency of β chains) while in β⁺ thalassaemia β chain synthesis is reduced but not completely lacking (partial deficiency of β chains). Usually individuals having one normal and one abnormal β globin gene have β thalassaemia minor while persons in whom both β globin genes are abnormal have β thalassaemia major.

β thalassaemia displays marked genetic heterogeneity with more than 200 molecular lesions having been reported. It has been observed, however, that in a particular population (e.g. Asian Indians, Mediterranean, Southeast Asian, American Blacks, etc.). Only a few β thalassaemia mutations are consistently and commonly found and account for 90% of the abnormal β thalassaemia genes (Table 4.2). Mutations frequent in Asian Indians are illustrated in Figure 4.13 and also shown in Box 4.5.

Box 4.5 Common β thalassaemia mutations in India

• Intron 1 position 5 (G→C)

A brief outline of mutations causing β thalassaemia is given below .

Mutations which affect transcription: Initiation and rate of transcription are regulated by the promoter region which is located immediately in front (upstream or 5’ end) of globin genes. Two highly conserved sequences in the promoter region, ATAAA and CACACCC, appear to be essential for efficient initiation of transcription of the β globin gene. Mutations affecting these promoter sequences cause reduction in globin gene transcription. As some amount of β globin is produced, patients develop β⁺ thalassaemia. Some of the mutations that affect transcription are shown in Figure 4.14.

Table 4.2: Common mutations causing β thalassaemia in different ethnic groups Asian Indians

• IVS-1 position 5 (G→C) Consensus splice site mutation

• 619 bp deletion

• Codon 8/9+G Frameshift mutation

• IVS-1 position 1 (G→T) Splice junction mutation

• Codons 41/42 (-TTCT) Frameshift mutation Southeast Asians

• IVS-2 position 654 (C→T) Cryptic splice site mutation

• Codons 41/42 (-TTCT) Frameshift mutation

• –28 A→G Promoter mutation

• Codon 17 A→T Nonsense codon mutation Mediterranean

• Codon 39 (CAG→TAG) Nonsense codon mutation

• IVS-1 position 1 (G→A) Splice junction mutation

• IVS-1 position 110 (G→A) Cryptic splice site mutation

• IVS-1 position 6 (T→C) Consensus splice site mutation

• IVS-2 position 745 (C→G) Cryptic splice site mutation African American

• –88 (C→T) Promoter mutation

• –29 (A→G) Promoter mutation

• Poly-A (AATAAA→AACAAA) RNA cleavage poly-A signal mutation

Figure 4.13: Schematic representation of β thalassaemia mutations frequently observed in Asian Indians

Mutations that affect splicing of RNA: Mutations that cause abnormal splicing are very common. Majority of them occur within introns but some of them affect exons.

Splicing mutations may alter the normal splice junction or may create alternative splice sites at abnormal locations.

Mutations altering normal splice junction: The GT and AG dinucleotides at the start (5’ splice site) and the end (3’ splice site) of introns respectively are obligatory for normal splicing. If mutations alter these splice sites then splicing fails to occur resulting in absence of β globin synthesis and formation of β⁰ thalassaemia alleles (Fig. 4.15).

Mutations creating alternate splicing sites at abnormal locations. They occur in introns or exons. A mutation in intron or exon produces an alternate splicing site so that some of the messenger RNAs are spliced at mutant site and some are spliced at normal site.

As shown in Figure 4.16, a mutation G→A at position 110 of intron 1 produces a new active splicing site AG. It has been shown in this case that 90% of splicing occurs at newly created abnormal site and about 10% at normal site. This results in severe β⁺ thalassaemia as abnormal splicing predominates.

Mutations in exons may also activate cryptic splice sites. An example is substitution G to A in codon 26 (GAG→AAG) of exon 1. This mutation has two effects: activation of cryptic splice site and formation of abnormal haemoglobin, HbE. A cryptic splice site is one, which resembles to some extent the normal splice site but is not used normally for splicing. A mutation in the cryptic site makes it an active splice site. Normal splicing of mRNA containing the G to A substitution in codon 26 of exon 1 leads to the formation of the abnormal haemoglobin, HbE. This mutation is associated with reduced production of mRNA (Fig. 4.17).

Mutations affecting consensus sequences: Apart from AG and GT dinucleotides, sequences surrounding the intron-exon boundaries are markedly similar. These Figure 4.14: Two examples of mutations affecting transcription in selected population groups are shown. Arrows indicate sites where one base is substituted by another (–88C→T and –87 C→G) By convention, nucleotides that are located 5´ to the gene are given minus number from the transcription start nucleotide. Both these mutations cause β⁺ thalassaemia

Figure 4.15: Mutations that alter normal splice junctions. Arrows indicate substitutions that alter the normal splice site. The mutations illustrated are intron 1 position 1 G→T (Asian Indians), intron 1 position 1 G→A (Mediterranean), and intron 2 position 1 G→A. All these mutations cause β⁰ thalassaemia

Figure 4.16: Mutation creating alternate splicing site in intron 1. Arrow (↑) shows mutant splicing site in intron 1 due to substitution G→A at position 110

Figure 4.17: Activation of cryptic splice site in exon 1 by mutation G→A in codon 26. This substitution causes (1) formation of an abnormal haemoglobin HbE, and (2) alteration of sequence in exon 1 which activates a cryptic splice site

sequences are highly conserved during evolution and are known as consensus sequences. Mutations in consensus sequences produce β thalassaemia of variable severity (Fig. 4.18).

Polyadenylation mutations: Mutations in polyadenylation sequence AATAAA at the 3’ end of the globin gene are associated with β⁺ thalassaemia (Fig. 4.19).

Mutations which lead to the formation of the chain termination codon: UAA, UAG and UGA are chain termination codons in β globin mRNA. Substitution of a single nucleotide in the coding sequence to create chain termination codon (nonsense mutation) will interrupt the translation of mRNA. This will generate non-functional fragments of β globin and cause β⁰ thalassaemia (Fig. 4.20).

Frameshift mutations: Reading frame comprises of sequentially arranged triplets of three bases. Each triplet codes for a specific amino acid. Mutations that delete or insert one, two, or more than three bases cause alteration in the sequence of the reading frame. This results in the formation of incorrect amino acids and secondly, at some place in the sequence, chain termination codon is formed which stops translation at that place (Fig. 4.21). These mutations cause β⁰ thalassaemia.

Deletions: 619-base pair deletion of the β globin gene is common in Asian Indians. It removes part of intron 2, exon 3, and some sequences 3’ to the globin gene (Fig. 4.22).

This deletion causes β⁰ thalassaemia in the homozygous state. Apart from this, gene deletions are rare in β thalassaemias.

Figure 4.18: Two mutations affecting consensus sequence shown are: (1) intron 1 position 5 (G→C) and (2) intron 1 position 5 (G→T). They cause β⁰ thalassaemia

Figure 4.19: Polyadenylation mutations

Figure 4.20: Mutation to termination codon. Nonsense mutation in codon 39 (C→T) prevalent in Mediterranean population is shown. This mutation causes formation of a stop codon TAG (UAG in mRNA) at the 39th codon which leads to premature termination of translation

Figure 4.21: Frameshift mutation in codon 16. A deletion C in codon 16 alters the reading frame and also creates a chain termination codon prematurely at codon 18

Figure 4.22: The Indian 619 bp deletion

Dominant thalassaemia: Recently, mutations have been identified in exon 3 of β globin gene that cause production of unstable globin chains. Unstable globin chains and unpaired chains precipitate in erythroblasts in bone marrow that leads to their premature destruction. This causes disease expression even in heterozygous state;

this is called as dominant thalassaemia.

α thalassaemias

There are two α gene loci on each chromosome 16 and since humans are diploid there are four α genes. The normal α globin haplotype is αα and the normal genotype is written as αα/αα.

α thalassaemias are classified into two types—α⁰ and α⁺ thalassaemias. In α⁰ thalassaemia there is total absence of α chain synthesis from one chromosome, while in α⁺ thalassaemia α chain synthesis from one chromosome is decreased but not absent.

Most cases of α thalassaemias result from gene deletions. Deletions which remove both α genes cause complete absence of α chain production from the affected chromosome (α⁰ thalassaemia). They are particularly common in Southeast Asia. Hydrops foetalis and HbH disease are largely restricted to Southeast Asia because of prevalence of cis α gene deletions (αα/- -). Deletion of one α globin gene out of the two is associated with α⁺ thalassaemia. However, α⁺ thalassaemias also result from mutations of α globin genes (non-deletional α⁺ thalassaemia). Various mutations giving rise to α⁺ thalassaemia include: (1) Mutations which cause aberrant splicing; (2) Mutations of chain terminator codon: Single base substitution in chain terminator codon results in, instead of termination of chain, continuation of translation until another chain terminator codon is encountered. This results in lengthening of α polypeptide chain. Two examples of this are Hb Constant Spring (chain terminating codon UAA is changed to CAA which codes for glutamine), and Hb Koya Dora (UAA →UCA which codes for serine); (3) Mutations which cause instability of α globin chain after translation. The newly produced α chains are rapidly degraded.

Since individuals in India most often carry αα/α- haplotype (α⁺), severe forms of α thalassaemia are rare.