A Novel Loss-of-Function Variant in the Chloride Ion Channel Gene Clcn2 Associates with Atrial Fibrillation

Figures

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  Figure 1. Genetic information. (A) Pedigree of the family with CLCN2- c.1041_1044del variant. Square: Male. Circle: Female. Black filled for AF affected individual. White filled for unaffected individual. Diagonal line for diseased individual. The presence of the variant is indicated with “+” for presence and “−” for absence. (B) Sanger chromatograms of the affected patients. (C) Schematic presentation of ClC-2 protein topology indicating position of frame shift mutation (red cross) and effect of mutation (truncated part in grey).

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  Figure 2. Relative expression of CLCN2 in tissue from the four heart chambers of healthy individuals (n = 7) analyzed by qPCR. YWHAZ and RPL13A were used as reference genes. LV – left ventricle; Endo - endocardium; myo - myocardium; epi – epicardium; RV – right ventricle; LA – left atrium; RA – right atrium.

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  Figure 3. Effect of ClC-2 variant on whole cell current. (a) Representative current traces of whole-cell voltage-clamp recordings from HEK293 cells transiently transfected with ClC-2 WT (left) or mutated ClC-2 (CLCN2- c.1041_1044del) (right). Currents were elicited by the voltage protocol shown. (b) Current voltage relationship of wild type (closed circles) and mutated ClC-2 (open circles), n = 9 and 10.

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  Figure 4. Effect of heterozygous expression of ClC-2 variant. (a) Representative current traces from empty Xenopus laevis oocytes and oocytes expressing ClC-2 WT, ClC-2 MUT or ClC-2-WT and ClC-2-MUT co-expressed (50% + 50%). Currents were elicited by the voltage protocol shown in the inset. (b) Current voltage relationship of empty oocytes (crosses), wild type (closed circles), mutated ClC-2 (open circles), and heterozygote expression (half-filled) n = 18, 40, 30, and 19, respectively). (c) Voltage-dependence of channel activation relationships from oocytes expressing wild type or co-expressing wild type and mutated channels. *WT vs. MUT; $WT vs. WT/MUT; £WT/MUT vs. MUT.

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  Figure 5. Western blot of HEK293 cells expressing WT or mutant channels shows lowered protein levels. (a) Representative western blot of protein extracts from HEK293 cells transfected with empty plasmid (lane 1), ClC-2 WT (lane 2) or ClC-2 MUT (lane 3). Lamin was used as a loading control. Bands representing ClC-2 or Lamin proteins are indicated by arrows. (b) Quantification of protein expression from three independent experiments.