A cryptic splice-altering KCNQ1 variant in trans with R259L leading to Jervell and Lange-Nielsen syndrome

Figures

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  Fig. 1. QTc value of the index patient. The QT interval corrected for heart rate (QTc) was measured in lead II from 12-lead electrocardiograms.

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  Fig. 2. NGS data visualization and identification of KCNQ1 variants. Graphics output of the IGV alignments of NGS reads from the index patient at two KCNQ1 genomic positions leading to the identification of the pathogenic variant c.776 G > T in exon 5 (variant #1 in a) and a previously undescribed variant c.1686−9 T > C in intron 13 of the KCNQ1 gene (variant #8 in b). A unique tag (#) ID number was assigned to each identified variant. The figure was assembled using scalable vector graphic files exported from the IGV application. NGS reads (horizontal grey bars) alignments to the reference genome sequence (RefSeq NC_000011.9) are displayed at two scales, 2800 bp and a zoomed version at 80 bp. Both nucleotide variants #1 and #8 were detected in the heterozygous state and the percentage of reads corresponding to each alternative nucleotide are shown with round brackets. Coverage graphs are shown with grey vertical bars and the coverage range is indicated in square brackets. Exon (E)–intron (i) structure of each displayed KCNQ1 regions are represented with blue boxes and lines, respectively. The complete descriptions of the ten KCNQ1 variants identified in the index patient’s genomic DNA by…

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  Fig. 3. Parental origin of the pathogenic c.776 G > T and the candidate c.1686−9 T > C KCNQ1 variants identified in the index patient. DNA sequencing chromatograms of the KCNQ1 gene exon 5 (left) and intron 13 (right) from the index patient (a), father (b), and mother (c). The index patient’s variant c.776 G > T in exon 5 was detected in his mother, whereas the c.1686−9 T > C variant in intron 13 was detected in his father.

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  Fig. 4. Family pedigree of the index patient. Available KCNQ1 genotype and clinical data are shown. Arrow indicates the index patient.

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  Fig. 5. Generation and characterization of patient-specific KCNQ1 minigene constructs. a Structure of the 2078-bp KCNQ1 minigene fragment from exon 13 (E13) to 15 (E15), which includes the c.1686−9 T > C variant in intron 13, that was amplified from the patient genomic DNA and cloned in-frame to both the FLAG (F) and Myc (M) epitope tags of the expression vector. b Identification of positive minigene constructs by agarose electrophoresis of plasmid DNA isolated from 22 ampicillin-resistant bacterial clones. Plasmids with bold numbers, displaying reduced electrophoretic mobility as compared to the empty vector (EV), were identified as positives (containing the insert) and sequenced. Plasmids with the reference T or alternative C nucleotide, corresponding to the variant c.1686−9 T > C, were classified as REF or MUT, respectively, as indicated on the top of each lane. c DNA sequencing chromatogram of REF 1517 and MUT 1521 plasmids. The arrows mark the position of the affected nucleotide. d KCNQ1 variants identified by Sanger sequencing of 19 positive plasmids. For each variant, the presence of the reference (−) or the alternative (+) nucleotide in each plasmid sequence is indicated.…

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  Fig. 6. RT-PCR analysis of HeLa cells transfected with KCNQ1 c.1686−9 T > C minigene plasmids. The agarose gel electrophoresis of minigene-derived KCNQ1 and the endogenous RPL19 RT-PCR products from two transfection experiments are shown in a and b using the indicated reference (REF) or mutant (MUT) KCNQ1 minigene plasmids (as described in Fig. 5). Amplification of KCNQ1 cDNA was performed with a forward primer located in E13 and a reverse primer in the vector-derived Myc epitope. Each RT-PCR lane (1–14) corresponds to a cell culture well processed independently. The amplification of RPL19 was used as a cDNA normalization control. NTR nontransfected cells, L DNA ladder. c DNA sequencing chromatograms of the longer 263-bp KCNQ1 band corresponding to the normal mRNA including exons 13–14–15 and the shorter 216-bp band with the complete skipping of exon 14 (ΔE14 KCNQ1). The deduced amino acid sequence is shown below the nucleotide sequence, as displayed by the Chromas software. The skipping of E14 introduces a frameshift (FS) at the start of E15. Full-size RT-PCR gel images are presented in Supplementary Fig. 5.

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  Fig. 7. Western blot analysis of HeLa cells transfected with KCNQ1 c.1686−9 T > C minigene plasmids. KCNQ1 Reference (REF) or Mutant (MUT) minigene plasmids (PL) were transfected into HeLa cells (in triplicated wells) as indicated. Cell lysates, derived from the same experiment and processed in parallel, were analyzed by western blotting with mouse monoclonal anti-FLAG (a) or anti-Myc (b) antibodies 48 h after transfection. NTR nontransfected cells, AB membrane stained with Amido Black 10B after immunostaining. c Structure of minigene-derived KCNQ1 and ΔE14 KCNQ1 proteins as predicted by sequence analysis of the corresponding cDNAs. F FLAG epitope, M Myc epitope, FS Frameshift, PTC premature termination codon.

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  Fig. 8. RT-PCR analysis of endogenous expression of KCNQ1 in the blood of the index patient’s parents. a Agarose gel electrophoresis of RT-PCR products of blood RNA isolated from the index patient’s mother (lane 1) and father (lane 2). Amplification of KCNQ1 cDNA was performed with a forward primer located in E13 and a reverse primer in E16. Negative controls were loaded in lane 3 (-RT) and 4 (nontemplate). The upper bands (254 bp) correspond to normal KCNQ1 mRNA and the lower one (207 bp) to the ΔE14 KCNQ1 aberrant mRNA. Below are shown the Sanger sequencing chromatograms of the 254 bp (b) and 207 bp (c) RT-PCR bands from the index patient’s father. The exon-exon junctions are marked with vertical dotted lines, and the number of each exon is indicated (E13-E16). The deduced amino acid sequence is shown below the nucleotide sequence, revealing the frameshift at the start of E15 that was created by the skipping of E14. Full-size RT-PCR image is shown in Supplementary Fig. 8.