Aggtaag

* Normal donor site HBA2 IVS2

AGGTgAG

** Alternative donor site HBA2 exon 1

gGGTAAG

Donor site HBA2 IVS2 after deletion

AGGctcc

Figure 1.25 Sequence diversity and alternative splicing. (A) A variant resulting in p thalassaemia modulates alternative splicing: the G to A nucleotide substitution at IVS position 1 in HBB results in two abnormally spliced RNAs (HbVar ID 884; HBB:c.315 + 1G>A; OMIM 141900.0348). (B) A structural haemoglobin variant, Hb E, in which a G to A substitution activates a cryptic donor site and both mildly reduces p globin synthesis and results in alternative splicing (HbVar ID 227; HBB:c.79G>A; OMIM 141900.0071). (C) A pentanucleotide deletion modulates splicing of HBA2. The normal exonic structure of HBA2 is shown together with sequence spanning the 3' half of exon 1 and start of intron 1. A pentanucleotide deletion (TGAGG) (HBA2:c.95+2_95+6deltGAGG) abolishes the normal donor site at the exon/intron boundary and leads to activation of an alternative site within exon 1.

CACGC and was associated with a ten-fold reduction in gene expression (Orkin et al. 1982a; Treisman et al. 1983). The same nucleotide position was found to be the site of a C to T substitution (HBB:c.-137C>T) which reduced gene expression by half (Kulozik et al. 1991); a change in the neighbouring nucleotide from C to T (HBB:C.-138C>T) similarly reduced gene expression (Orkin et al. 1984).

Analysis of gene regulation within the globin loci has provided many important fundamental insights into the molecular basis for the control of gene expression, notably the role of more distant upstream regulatory elements (Higgs and Wood 2008b). For example, patients with thalassaemia were identified in whom the structural genes were intact but upstream elements deleted. Hatton and colleagues described a 62 kb deletion (aaRA) linked to an a thalassaemia phenotype which was associated with a marked reduction in a globin gene expression (Fig. 1.26) (Hatton et al. 1990). At least 14 similar deletions have been described within the a globin locus of variable size but i nvolving up stream elements, described as 'multispecies conserved sequences' (MCSs) as they are conserved across species; co-localization with erythroid specific DNase I hypersensitive sites was also noted (Higgs and Wood 2008b).

DNase I hypersensitive sites are regions of DNA more susceptible to cleavage by the enzyme DNase I due to the more open chromatin conformation associated with regulatory elements. One MCS in particular, MCS-R2 (also known as HS-40), was shown to enhance a globin expression on its own; deletion of this site reduced expression to less than 5% of normal, while if all MCSs are deleted there is complete silencing of a globin gene transcription. Notably, deletions or insertions of intervening sequence between the upstream elements and gene promoters did not affect expression. MCSs appear to play a critical role in recruitment of the RNA polymerase enzyme and general transcription factors. Similar effects were noted in the p globin locus when particular upstream DNase I hypersensitive sites were lost due to deletions (Kioussis et al. 1983; Driscoll et al. 1989).

Human Mar. 2006 chr16:1-220,000 (220 000 bp)

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