Figure 6

CYS1 variant c.318 + 5G > A caused abnormal splicing of RNA expressed from a transfected minigene in human renal cells. (A) Experimental minigene splicing constructs were generated by cloning the following CYS1 sequences between vector exons A and B: 20 bp of CYS1 exon 1 3’ sequence including either the WT or the c.318 + 5G > A patient-derived variant splice donor site; then 200 bp of adjacent intron1 sequence; then CYS1 sequence consisting of the entire CYS1 exon 2 (53 bp) flanked by 200 bp of adjacent CYS1 intronic sequences. Minigene constructs were transfected into human renal collecting duct (CD-hTERT) cells⁸⁵. Normal splice donor (Do) and acceptor (Ac) sites are indicated by arrows. Forward (Fwd) and reverse (Rev) primers corresponding to sequences in vector exons A and B, respectively, were used in RT-PCR. The position of the c.318 + 5G > A patient-derived variant is indicated with a red X. (B) RT-PCR reaction products (using Fwd & Rev primer pair) from CD-hTERT RNA following transfection with the indicated plasmids. Amplicons were resolved by agarose gel electrophoresis. RT+, addition of reverse transcriptase; RT-, negative control reactions lacking reverse transcriptase. CD-hTERT indicates template RNA from untransfected cells used as a negative control. NTC, no template control. The vector positive control generated an amplicon of approximately 200 bp, as expected (purple asterisk). CYS1-ex2 and CYS1-ex1DNR-ex2 generated amplicons of the expected 250 bp size (red asterisks). In contrast, the c.318 + 5G > A variant showed loss of the 250 bp amplicon and the appearance of a novel, shorter amplicon (blue asterisk). The identities of the bands marked with asterisks were confirmed by cloning of excised DNA and subsequent restriction and sequence analysis (Supplementary Data). (C) RT-PCR amplicon composition and size (bp) corresponding to splicing products generated from transfected minigene constructs amplified using the Fwd and Rev primer pair (shown in A). Outcomes for the variant construct, assuming an effect of the CYS1 variant on splicing, could not be predicted a priori. The composition of the splicing product shown here is from the actual sequence of the cloned amplicon (see Supplementary Data). Sequences of cloned splice product amplicons from all other constructs corresponded precisely to anticipated outcomes.