A mutation in the 5′ untranslated region of the human α‐galactosidase A gene in high‐activity variants inhibits specific protein binding

Abstract

Recently, normal individuals were identified who had high levels of plasma ¢x-galactosidase A activity and a G to A transition in the 5' untranslated (5' UT) region of the ol-galactosidase A gene. Electrophoretic mobility shift assays revealed that the wild-type sequence at the site of this mutation complexed with specific nuclear proteins. A standard NF-KB site competed with the 5' UT site for formation of these DNA-protein complexes. Complex formation was inhibited by the transition mutation. Therefore, the wild-type site might down-modulate expression of the o~-galactosidase A gene from this 5' untranslated region, which includes a previously described protein-binding site for another family of sequence-specific DNA-binding proteins, methylated DNA-binding protein.

The Modulatory Effects of the Polymorphisms in GLA 5′-Untranslated Region Upon Gene Expression Are Cell-Type Specific

“…One of the most common research applications of chimeric genetic reporter systems is in the analysis of cis-acting elements, like gene promoters; however, to the best of our knowledge, the functional consequences of the 5 0 UTR SNPs upon GLA gene expression have never been assayed in this manner. Overall, our results are consistent with the working hypothesis, based on observations in vivo that some of the minor 5 0 UTR SNPs alleles significantly affect aGal activity levels (Saifudeen et al 1995;Fitzmaurice et al 1997;Oliveira et al 2008a, b), and provide indirect evidence that the human GLA 5 0 UTR indeed contains sequences that are involved in the regulation of gene expression. These data also demonstrate, for the first time, that the 5 0 UTRdependent modulation of GLA gene expression may vary among different cell types.…”

“…Because they were neither translated nor part of the mRNA Kozak consensus sequence for translation initiation, they were regarded as biologically neutral, but subsequent studies showed that the c.-30A allele was associated with increased aGal activity in plasma (Saifudeen et al 1995) and the c.-10T allele with decreased aGal activity in leukocytes (Oliveira et al 2008a, b), as compared to the corresponding WT alleles. Since the GLA 5 0 UTR c.-10 and c.-30 positions are within binding sites respectively for the MDBP and the NFkB and Ets families of TF, the effect of those two nucleotide transitions upon aGal expression in vivo might be mediated by modulation of transcriptional activity.…”

“…ncbi.nlm.nih.gov/nuccore/NM_000169.2; National Library of Medicine, Bethesda, MD, USA). Three single nucleotide polymorphisms (SNPs) respectively identified by reference numbers rs2071225, rs3027585 and rs3027584 at the NCBI SNP database (dbSNP, http://www.ncbi.nlm.nih.gov/snp/), which result from cytosine-to-thymine (C>T) or adenineto-guanine (G>A) transitions at cDNA nucleotide positions c.-10(C>T), c.-12(G>A) and c.-30(G>A), counting backwards from the translation initiation codon, are relatively common in several ethnically different populations (Davies et al 1993;Saifudeen et al 1995;Wu et al 2011;Ferri et al 2012), including the Portuguese (Oliveira et al 2008a). The dbSNP lists three additional GLA 5 0 UTR SNPs, at positions c.-8(C>G), c.-18(T>C) and c.-105(A>G), respectively identified as rs371291716, rs545597063 and rs3027583, but these variants have never been reported in the Portuguese population.…”

“…Compared to the wild-type (WT) allele, the c.-30A allele is associated with increased plasma aGal activity (Saifudeen et al 1995;Fitzmaurice et al 1997), the c.-10T allele is associated with decreased activity of aGal in leukocytes (Oliveira et al 2008a, b) and the c.-44T allele might be associated with increased plasma aGal activity (Baptista et al 2010). Contrastingly, the SNP c.-12(G>A) seems to have no effect upon GLA gene expression (Oliveira et al 2008a).…”

“…Because the GLA 5 0 UTR contains a binding site for methylated DNA-binding protein (MDBP)/regulatory factor X (RFX) transcription factor family (Zhang et al 1990;Samac et al 1998), as well as partially overlapping binding motifs for the nuclear factor kappa-B (NFkB) and E-26 (Ets) transcriptional regulatory factors (Saifudeen et al 1995;Fitzmaurice et al 1997), it has been previously suggested that protein ligands to these sites might be involved in the regulation of GLA gene expression (Saifudeen et al 1995;Fitzmaurice et al 1997). Since the SNPs c.-10(C>T) and c.-30(G>A) involve nucleotides respectively located at each of those binding sites, they might act as polymorphic modulators of GLA gene expression, with possible clinical relevance particularly in males carrying GLA sequence variants associated with high REA.…”