Figure 5

Functional analysis of the -10T allele. (A) Representative chromatograms showing nucleotide substitution at position -10. (B) Carriers of the minor -10T allele with neurological manifestations (black box; n = 11) showed significantly decreased GLA mRNA expression levels in peripheral mononuclear blood cells compared to healthy non-carriers (white box; male: n = 8; female: n = 9). (C) GLA expression is significantly decreased in symptomatic homo-/hemizygous T allele carriers (n = 4) versus symptomatic CT carriers (n = 7). (D) Schematic representation of the GLA 5′-flanking region. (E) Transient transfection of GLA promoter constructs in EA.hy926 cells revealed two regions with significant transcriptional activity. (F-H) Insertion of the minor T allele into promoter constructs (black bars) resulted in a decreased transcriptional activity in SH-SY5Y (F), EA.hy926 (G) and THP-1 (H) cells. (I) EMSA with nuclear extract from EA.hy926 cells revealed one specific competable (non-allelic) band (upper arrow) and a slower migrating T allele-specific band (lower arrow). F: probe without extract and competitor. Data are given as mean ± SEM; LU: light units; Luc: luciferase.*p < 0.05; ***p < 0.001.