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Fig. 2 | BMC Nephrology

Fig. 2

From: Use of patient derived urine renal epithelial cells to confirm pathogenicity of PKHD1 alleles

Fig. 2

ARPKD synonymous variant c.6900C > T promotes alternate splicing of exon 43 above basal level. a Schematic of human PKHD1 exon 42 and exon 43, that illustrates the alternate splicing of exon 43, promoted by the variant c.6900C > T (in white), leading to a splicing product devoid of the first 47 nucleotides of exon 43. b RT-PCR on cDNA isolated from wild type control URECs and ARPKD URECs lysates demonstrates alternate splicing of exon 43 in both cell lines. c Schematic of alternate splicing products of human PKHD1 around exon 43 predicted to occur on the basis of Sanger sequencing of colony PCR products after T cloning of URECs RT-PCR products. Position of each transcript-specific primer set is specified within this schematic. Transcript A contains a full-length exon 43 (131 nt), transcript B contains a shorter exon 43, devoid of the first 47 nucleotides, transcript C is completely devoid of exon 43 and transcript D contains a full-length exon 43 and a portion (124 nt) of intron 43–44. d PKHD1 gene expression analysis of control and ARPKD URECs. Expression level of PKHD1, as measured by using a set of primers that do not differentiate between different splicing products around exon 43, are reduced by 30% in ARPKD URECs compared to the wild type control. Levels of transcript A, containing full-length exon 43, are reduced by 60% in ARPKD URECs compared to the wild type control. The bars represent the mean values from the expression levels in two different wild type control UREC lines and the expression levels in ARPKD URECs, respectively. Values are normalised to wild type controls mean values. e Expression analysis of the different transcript isoforms (transcript B, C and D) around exon 43 of PKHD1, normalized to the expression levels of transcript A, in control and ARPKD URECs. Transcript B is upregulated 1.5-fold in ARPKD URECs compared to wild type. Expression levels of transcript B and C are low (20% or less of the expression levels of transcript A) in both control and ARPKD URECs. The bars represent the mean values from the expression levels in two different wild type control UREC lines and the expression levels in ARPKD URECs, respectively

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