A novel stop-gain pathogenic variant in the KCNQ1 gene causing long QT syndrome 1

An Iranian family recruited in this study, had been referred to Rajaie Cardiovascular Medical and Research Center, Tehran, Iran, for the experience of recurrent syncope of its children. The proband was a 10-year-old boy with a 3-year-old brother (Fig. 1A-, III-1 & III-2, respectively). The parents were unrelated and healthy: the father was 41 years old, and the mother was 34. Routine cardiovascular examinations, including echocardiography, 12-lead ECGs, exercise tests, and 24-h Holter monitoring, were carried out for all the family members, affected or healthy. The patients (Fig. 1A, III-1 & III-2) underwent an electrophysiological examination with a 2-electrode voltage−clamp amplifier (TEC10CD, NPI Electronics, Tamm, Germany) featuring KCl-filled electrodes of about 0.8 MU resistance [18]. The currents were measured at room temperature by applying bath solutions, including 10 mM of HEPES (pH 7.2), 105 mM of NaCl, 1.8 mM of CaCl2, and 10 mM of KCl. The Bazett formula was used for calculating the heart rate QT intervals. The study was performed due to the Declaration of Helsinki and was approved by the Ethics Committee of Rajaie Cardiovascular Medical and Research Center (IR.RHC.REC.1401.054). Informed consent was achieved from all participants and from the parents of participants below 16-year-old.

Blood samples of family members were obtained. Genomic DNA extraction was performed using the DNSol Midi Kit (Roche: Product No. 50072012). Whole-exome sequencing was done on the proband (Fig. 1A, III-I) at Macrogen (Amsterdam, the Netherlands). Enrichment and capture of all exones were carried out using SureSelect Exon V7 Library Prep Kit. Exome was sequenced on an Illumina HiSeq 6000 machine based on the manufacturer’s protocol. A read quality value of greater than 20 and a depth of greater than 7 was used for the next steps. The quality of the reads was surveyed with FastQC. The clean reads were aligned to the reference genome (UCSC Genome Browser, hg19) applying the Burrows–Wheeler Aligner (BWA-MEM v.07.17) [3]. Insertion and/or deletion and single-nucleotide polymorphism were called with the aid of the Genome Analysis Toolkit (GATK, v.4.1.4.1) [6]. Annotation of determined variants was done by ANNOVAR [15] and filtered according to the Exome Aggregation Consortium (ExAC), the 1000 Genomes Project, Exome-Sequencing Project ESP6500, and the Genome Aggregation Database (gnomAD) minor allele frequency (MAF) of 0.001. The candidate variants were investigated with bioinformatics tools, consisting of MutationTaster (www.​mutationtaster.​org), CADD (cadd.gs.washington.edu), PROVEAN (provean.jcvi.org), SIFT (https://​sift.​bii.​a-star.​edu.​sg), and PolyPhen-2 (genetics.bwh.harvard.edu/pph2) according to the 2015 guidelines of the American College of Medical Genetics and Genomics (ACMG) [10]. Moreover, the conservation of the variants regions were analyzed using the GERP +  + score and CLUSTALW Web Server (https://​www.​genome.​jp/​tools-bin/​clustalw) by comparing the amino acid sequences of different species.

Primer pair was designed using the Primer3 v.04.0 (http://​bioinfo.​ut.​ee/​primer3-0.​4.​0/​) with the sequences: forward: 5ʹ-TGCTCTTTGTTGACGACCA-3′ and reverse: 5′-AGCGTGGAAGTGCCCTCT-3′. PCR was carried out on a SimpliAmp Thermal Cycler (Thermo Fisher Scientific) with 1.5 mmol/L of MgCl2, 10 pmol/L of the primers, 200 mmol/L of dNTP, 100 ng DNA, and 1 U of Taq DNA polymerase (Amplicon, UK). Thereafter, incubation at 95 °C for 5 min and 35 amplification cycles (30 s at 95 °C, 30 s at 62 °C, and 30 s at 72 °C) was applied. The products of the PCR were sequenced on an ABI Sequencer 3500XL PE (Applied Biosystems), and the sequences were surveyed with CodonCode Aligner (v.7.1.2) (Fig. 1B).

The proband (Fig. 1A, III-1) had suffered 2 syncope episodes without stress in the past year. Sinus rhythm, infrequent premature atrial contractions, and a prolonged QT interval in some leads were detected in his Holter recording (Fig. 2A, C). The minimum Schwartz score [2] was 3.5, indicating the high probability of LQT syndromes.

Concerning the family history, the younger brother (Fig. 1A, III-2) also had suffered syncope and exhibited similar symptoms. The mother (Fig. 1A, II-4) had experienced chest pain and dyspnea in the preceding 5 months (no notable ECG manifestations). The father (Fig. 1A, II-3) had no history of arrhythmia or syncope, with no signs of heart disease or aberrant ECGs in his clinical examinations.

Whole-exome sequencing was performed on the proband (Fig. 1A, III-1) to discover the causative variant. A novel stop-gain pathogenic variant, c.968G > A, was found in the seventh exon of KCNQ1. This variant substituted tryptophan at site 323 for a stop codon, proposed to cause a premature KCNQ1 truncated protein and/or nonsense-mediated KCNQ1 mRNA decay. Notably, the variant has not yet been reported either in the 1000 Genomes Project, ExAc, gnomAD, HGMD, and ClinVar or in publications. According to the ACMG, c.968G > A was determined as a pathogenic variant (criteria: PVS1, PM2, PP1, and PP4). This nonsense variant was considered the cause of the disease by SIFT, PolyPhen-2, PROVEAN, FATHMM, and GERP +  + . The CADD Phred score was 41. The variant was confirmed in the proband (Fig. 1A, III-2) by PCR and Sanger sequencing in the heterozygous state. It was also detected in the proband’s affected brother (Fig. 1A, III-2) and the suspected mother (Fig. 1A, II-4) as a heterozygote. The father (Fig. 1A, II-3) had a normal sequence at this position (Fig. 1B). DNA from the other pedigree members was unavailable. A schematic secondary structure of the KCNQ1 protein is presented in Fig. 1C. In addition, based on the CLUSTALW results, tryptophan323 was located in the conserved part of the KCNQ1 protein (Fig. 1D).