Fig. 2
Identification and functional analysis of NT5C2 mutation. a Pedigree of the consanguineous family and Sanger sequencing of NT5C2 splice site mutation. The inheritance pattern is consistent with an autosomal recessively segregated disorder. b Schematic illustration of NT5C2 protein and coding transcript. Upper panel, protein structure with depicted active sites (nucleophile and proton donor at positions 52 and 54, respectively), metal binding sites (Magnesium metal at positions 52, 54, 351), allosteric activators binding sites (positions 127, 154, 354, 436, 453), substrate binding region (position 202–210), and Asp/Glu-rich acidic region (position 549–561). G330 and S387 denote boundaries of the deleted region encoded by exon 14. Bottom panel, coding transcript with numbered exons and the location of all the reported NT5C2 mutations including the present study (red). Skipped exon 14 reported herein is highlighted yellow. c NT5C2 RNA splicing. RT-PCR using exon14-flanking primers (right diagram depicting exons as numbered boxes, introns as dashed lines, primers as half arrows, and donor splice site mutation as red circle) shows the absence of the wild-type (358 bp including exon 14) and the presence only of the mutant (187 bp lacking exon 14) band in the two affected, whereas both bands are present in the heterozygous cases. Results demonstrate skipping of exon 14 from mature NT5C2 transcript as a consequence of intron 14 donor splice site mutation, without alteration of transcriptional expression. Ctrl is control RNA from unrelated normal individual. Genomic DNA (gDNA) is used to confirm specificity of assay toward spliced RNA. GAPDH is housekeeping gene used for normalization. d Mutant NT5C2 lacking exon 14 is unstable. In-vitro expression analysis in HEK293 cells transfected with wild type (WT) or mutant (MT) NT5C2 fused to V5 tag. Note the marked low expression levels of mutant NT5C2-Δexon14-V5 (~64 kDa) compared to wild-type NT5C2-V5 (~70 kDa), indicating severe instability of mutant NT5C2 as a consequence of exon 14 skipping. Beta-actin (ACTB) is used as loading control. V, empty expression vector; M, mock; kDa, kilodalton