case_images: PMC9745090_01
case_images: Fetal echocardiography to determine bradycardia. (A) M-mode echocardiography demonstrated an atrial rate of 135 beats per minute before treatment. (B) Doppler demonstrated a ventricular rate of 68 beats per minute before treatment and indicated a 2:1 AV block. (C) ECG presented a 2:1 transduction atrioventricular block postnatally. (D) ECG revealed a significant prolonged QTc interval after birth.
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case_images: F1
case_images: The ECGs assessments of the parents presented normal performance (Supplementary Figure S1). The atrial rate was 135 beats/min, while the ventricular rate was 68 beats/min (Figures 1A and B), and the cardiovascular system profile was normal (10/10 points). An ECG of the neonate demonstrated a 2:1 transduction AVB (Figure 1C). One week later, the ECG revealed a significantly prolonged QTc interval (498-534 ms, Figure 1D).
case_images: Clinical manifestations in Holters. (A) Transit ventricular tachycardia was identified. (B) Holter demonstrated a paired premature ventricular contraction.
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case_images: F2
case_images: Moreover, Holter monitoring demonstrated transit ventricular tachycardia (Figure 2A) and doublet premature ventricular contractions (Figure 2B).
case_images: Effects of KCNH2 c.1868C>A mutation on molecular protein structure. (A) Family pedigree tree of this proband. (B) Summary of current reports on the individuals of KCNH2 and its predicted damages on the molecular function of the c.1868C>A variant. (C) SWISS-MODEL to predict the variant's wild type and mutated protein crystal structure, and the variant impaired a large proportion of molecular structure. (D) Ensemble variance between the wild type and mutant protein structure. (E,G) SWISS-MODEL to predict the variant's wild-type and mutated protein crystal structures using 5k7l.1.A template, and structural changes were identified. (F) Ramachandran plots of wild-type KCNH2. (H) Ramachandran plots of KCNH2 with the p.T623N variant.
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case_images: F3
case_images: The family pedigree tree of this proband had been presented in (Figure 3A). To our knowledge, KCNH2 c.21868C>A has never before been reported in the 1000G and ExAC databases (Figure 3B). MutationTaster and Polyphen-2 were used with the R software to predict the pathogenicity of KCNH2 c.1868C>A and assess the effects of this mutation on the protein structure (Figure 3B). As there were KCNH2 protein crystal structure data available, analysis was performed using SWISS-MODEL (https://swissmodel.expasy.org/) with the 5k7l.1.A template (Figures 3E,G). The capability of the protein structure was estimated using Ramachandran plots (Figures 3F,H). The differences between wild-type and KCNH2 p.T623N mutant crystal structures were estimated by ensemble variance (Figures 3C,D). Ramachandran plots indicated that amino acid positions were altered (Figures 3F,H). Rebuilding the molecular structure based on a 5k7l.1.A template resulted in the identification of residue changes at amino acid position 623 (Figures 3E,G).
case_images: Functional validation of KCNH2 c.1868C>A mutation on cardiomyocyte. (A) Western blotting demonstrated the similar expression of KCNH2 protein between wild type and mutation. (B) Immunofluorescence staining revealed the successful infection of KCNH2 overexpression plasmids in cardiomyocytes. (C) Calcium transit waves between KCNH2-wt and KCNH2-mut plasmids transfected cardiomyocytes. (D) The increased duration of time to peak and decay50 was identified in KCNH2-mut cardiomyocytes. RFP, red fluorescence protein; SAA, sarcomeric alpha-actinin. **, p < 0.01; ****, p < 0.0001.
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case_images: PMC9745090_01_fgene-13-1010078-g004.jpg
case_images: F4
case_images: Tablets (cOmplete, 4693124001). The expression of KNCH2 was assessed by Western Blotting of mScarlet (Figure 4A). Then, immunofluorescence was performed to demonstrate the expression of transfected KCNH2 (mScarlet), while cardiomyocytes were stained with SAA (Figure 4B). Tyrode solution (140 mM NaCl; 4 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2; 10 mM Glucose; and 5 mM HEPES, pH 7.4, adjusted with NaOH) to remove the excess dye for 20 min. All image data were acquired using an FV3000 confocal microscope (Figure 4C), which demonstrated the prolonged duration of pgl3-KCNH2-mut-P2A-mScarlet infected NMVMs both in time to peak and decay50 (Figure 4D). Thus, this experiment validated the impaired KCNH2 function-carrying c.1868C>A allele.