Novel Variant in the ANK2 Membrane-Binding Domain Is Associated With Ankyrin-B Syndrome and Structural Heart Disease in a First Nations Population With a High Rate of Long QT Syndrome

Abstract

Background: Long QT syndrome confers susceptibility to ventricular arrhythmia, predisposing to syncope, seizures, and sudden death. While rare globally, long QT syndrome is ≈15× more common in First Nations of Northern British Columbia largely because of a known mutation in KCNQ1. However, 2 large multigenerational families were affected, but negative for the known mutation.

Methods and results: Long QT syndrome panel testing was carried out in the index case of each family, and clinical information was collected. Cascade genotyping was performed. Biochemical and myocyte-based assays were performed to evaluate the identified gene variant for loss-of-function activity. Index cases in these 2 families harbored a novel ANK2 c.1937C>T variant (p.S646F). An additional 16 carriers were identified, including 2 with structural heart disease: one with cardiomyopathy resulting in sudden death and the other with congenital heart disease. For all carriers of this variant, the average QTc was 475 ms (±40). Although ankyrin-B p.S646F is appropriately folded and expressed in bacteria, the mutant polypeptide displays reduced expression in cultured H9c2 cells and aberrant localization in primary cardiomyocytes. Furthermore, myocytes expressing ankyrin-B p.S646F lack normal membrane targeting of the ankyrin-binding partner, the Na/Ca exchanger. Thus, ankyrin-B p.S646F is a loss-of-function variant.

Conclusions: We identify the first disease-causing ANK2 variant localized to the membrane-binding domain resulting in reduced ankyrin-B expression and abnormal localization. Further study is warranted on the potential association of this variant with structural heart disease given the role of ANK2 in targeting and stabilization of key structural and signaling molecules in cardiac cells.

Keywords: Wolff–Parkinson–White syndrome; arrhythmia; genetic variation; heart rate; long QT syndrome.

Figure 1

Pedigrees of two Gitxsan families with the p.S646F variant. A) Family 1; B) Family 2. Legend denotes variant status and clinical features. Number below each individual represents manual QTc (ms) read blinded to mutation and clinical status.

Figure 2

Representative ECGs from four carriers of the p.S646F variant. A) Family 1, Indv 1: note U-waves; B) Family 1, Indv 3: note abnormal notching in V2; C) Family 2, Indv 10: note prolonged QTc; D) Family 2, Indv 11: note prolonged QTc

Figure 3

Ankyrin-B p.S646 is located within membrane-binding domain and highly conserved across species. A) Conservation of p.S646 across vertebrates. B) Ankyrin-B domain organization and location of p.S646F variant on membrane-binding domain.

Figure 4

Ankyrin-B MBD p.S646F protein is appropriately expressed and folded in vitro. A) SDS-PAGE shows the purified protein qualities of WT ankyrin-B MBD and p.S646F mutant. The molecular markers are shown on both sides. B-C) FPLC analytical gel filtration coupled with static light scattering profiles of WT and p.S646F show symmetric column behavior and nearly identical molecular weight. D) Circular Dichroism (CD) spectra of WT ankyrin-B MBD and p.S646F show the enrichment of helical structures. E) Urea denaturation-based assay of the stabilities of the WT ankyrin-B MBD and p.S646F mutant shows no obvious difference. F-G) Structural model shows that the p.S646 residue locates on the of the 19th ANK repeat on the outer surface of the ANK repeat solenoid (Protein Data bank ID code: 4 RLV).

Figure 5

Ankyrin-B p.S646F exhibits decreased post-translational stability in H9c2 cardiomyoblasts. A) Immunoblot of GFP-tagged ankyrin-B (AnkB; wild-type or p.S646F)-expressing H9c2 cell lysates 48 hours post-transfection probed with anti-GFP (upper panel) and anti-β-actin (lower panel). B) Quantification of GFP immunoreactivity revealed that ankyrin-B p.S646F expression is significantly reduced in comparison to the wild-type protein (n=3; *, p = 0.0408 by unpaired t-test). These data are representative of 3 separate independent experiments, each with n ≥ 3 independent biological replicates.

Figure 6

Ankyrin-B p.S646F is a loss-of-function variant in myocytes. A) GFP-ankyrin-B localizes to the membrane (arrows) of ankyrin-B^+/− neonatal cardiomyocytes. We observe similar localization of Na/Ca exchanger at the membrane of immature primary neonatal cardiomyocytes. Note: GFP localized primarily to nucleus in control experiments as expected (inset). B) GFP-ankyrin-B p.S646F is enriched in the perinuclear region of ankyrin-B^+/− neonatal cardiomyocytes. We observed similar localization of the Na/Ca exchanger in these myocytes (arrows). Scale bar equals 20 microns.

Figure 7

Model for novel disease-causing variant identified in the membrane-binding domain of the Ankyrin B protein (ANK2 c. 1937C>T p.Ser646Phe). NCX indicates Na/Ca exchanger; NKA, Na/K ATPase; IP3R, inositol 1,4,5-trisphosphate receptor; DHPR, dihydropyridine receptor.