RNA splicing (Box 1.20) may be modulated by DNA sequence diversity in a number of ways including by changes in regulatory sequences (for example exonic splicing enhancers), splice donor and acceptor sequences, or through activation of cryptic splice sites (Section 11.6) (Pagani and Baralle 2004; Wang and Cooper 2007). A notable example of this is seen with a G to A nucleotide substitution at position 1 of the second intron of HBB (intervening sequence 2, IVS2) in which the conserved dinucleotide GT at the 5' splice site is changed to AT (Fig. 1.25A). When such an event was analysed based on DNA sequence from a fetus with P° thalassemia, significant changes in alternative splicing were seen with insertion of the initial 47 bp of IVS2 between exons 2 and 3, together with a less abundant RNA in which exon 1 was spliced directly onto exon 3 (Treisman et al. 1982).
Hb E is a structural variant of haemoglobin that is very common in South East Asia (with estimates of 30 million carriers in the region) in which there is a G to A substitution (HBB:c.79G>A) resulting in an amino acid substitution, Glu26Lys, due to the change in codon sequence from GAG to AAG (Fig. 1.25B). Hb E was found to be associated with a slight reduction in amounts of p globin synthesis and production of small amounts of an alternatively spliced transcript (Traeger et al. 1980; Orkin et al. 1982b). The PE allele had a 'thalassaemic nature': the basis for this lay in alternative splicing as the nucleotide substitution activated a cryptic donor site in exon 1. The intronic sequence IVS1 was seen to be excised more slowly than normal and a small amount of alternatively spliced transcript involving the cryptic donor site was observed (Fig. 1.25B). This issue becomes significant if an individual inherits a p thal-assaemia allele together with Hb E when severe transfusion-dependent disease may result.
A further example involving splicing is provided by a pentanucleotide deletion (TGAGG) at the 5' donor sequence at the start of intron 1 in HBA2 which was found to result in a thalassaemia due to effects on splicing (Fig. 1.25C) (Orkin et al. 1981; Felber et al. 1982). This variant significantly disrupted the normal donor sequence, notably the invariant GT sequence, abolishing use of that site and leading to activation of a new donor consensus site 49 nt upstream within exon 1. This results in truncation of the mRNA such that a normal globin chain cannot be encoded; the alternative splice site itself, however, functions well with no unspliced or alternatively spliced RNA apparent.
Other DNA sequence variants leading to a+ thalassaemia are recognized that involve RNA processing and translation. The sequence AAUAAA on the RNA molecule some 11-30 nt from the terminal poly(A) tract plays an important role in 3' RNA processing, evidence for which was provided by an A to G nucleotide substitution in HBA2 (HBA2:c*+94A>G) that changes the DNA
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