Thalassaemia mutations within the 5′UTR of the human β‐globin gene disrupt transcription

Abstract

SummaryThe mechanisms by which mutations within the 5¢ untranslated region (UTR) of the human b-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human b-globin 5¢UTR, namely: +10()T), +22(G fi A), +33(C fi G) and +(40-43)()AAAC), which are present in patients with b-thalassaemia intermedia. Expression of these genes under the control of the b-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61AE6%, 68%, 85AE2% and 70AE6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3¢ end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10()T) and +33(C fi G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G fi A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.

Comparative analysis of secreted enzymes between the anthropophilic–zoophilic sister species Trichophyton tonsurans and Trichophyton equinum

“…These results are not surprising given that a relationship between the number of regulatory repeat elements and transcript copy number has been well described for other genes (Sgourou et al 2004;Lee et al 2008;Akhtar et al 2009). A similar correlation between Sub2 transcript copy number and microsatellite copy number in the 3 0 UTR appears to be present, however, all copy-number variations were not represented among the isolates studied and thus the association cannot be confirmed.…”

Disease-Associated Mutations That Alter the RNA Structural Ensemble

“…The SNP is not located near any transcription, translation start or stop sites (Figure 1A). A recent study demonstrated that the C33G mutation (replacing C33 with a G) has a negligible effect on mRNA transcriptional levels [33]. A possible cause for the disease state is therefore a conformational change in the RNA structure.…”

Increased activity of Diaphanous homolog 3 ( DIAPH3 )/ diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila

“…We demonstrate that the mutant 5′ UTR is sufficient to drive overexpression in a luciferase reporter vector, suggesting that this highly conserved sequence in the 5′ UTR of DIAPH3 may be important for its transcriptional regulation. Similar mutations in the 5′ UTR of the β-globin gene have been shown to result in thalassemia through a reduction of mRNA levels (12,13). Given the observed effect of overexpression, we postulate that the c.-172G > A mutation may decrease the binding of a transcriptional repressor.…”