Elsevier

Heart Rhythm

Volume 2, Issue 11, November 2005, Pages 1218-1223
Heart Rhythm

Original-clinical genetic
Targeted mutational analysis of ankyrin-B in 541 consecutive, unrelated patients referred for long QT syndrome genetic testing and 200 healthy subjects

https://doi.org/10.1016/j.hrthm.2005.07.026Get rights and content

Background

Mutations in ANK2-encoded ankyrin-B underlie long QT syndrome type 4 (LQT4) and various other dysrhythmia phenotypes.

Objectives

The purpose of this study was to determine the prevalence and spectrum of ankyrin-B mutations in a large cohort of unrelated patients referred for LQTS genetic testing and among healthy control subjects.

Methods

Between August 1997 and July 2004, 541 consecutive, unrelated patients (358 females, average age at diagnosis 24 years, average QTc 482 ms) were referred to Mayo Clinic’s Sudden Death Genomics Laboratory for comprehensive mutational analysis of the five cardiac channel genes implicated in LQTS: KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6). Based on this prior analysis, 269 of 541 cases lacked an identifiable mutation (genotype negative). In this study, targeted mutational analysis of 10 ANK2 exons (36,37,39–46) encoding the critical C-terminal regulatory domain or implicated previously as hosting pathogenic mutations was performed on genomic DNA from 541 patients and 200 control subjects using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing.

Results

Overall, 14 distinct nonsynonymous variants (10 novel) were observed in 9 (3.3%) of 269 genotype-negative LQTS patients, 5 (1.8%) of 272 genotype-positive LQTS cases, 4 (4%) of 100 white controls, and 9 (9%) of 100 black controls. Four variants found in controls (L1622I, T1626N, R1788W, and E1813K) were implicated previously as LQT4-associated mutations and displayed functional perturbations in vitro. All genotype-negative LQTS cases hosting ANK2 variants had been diagnosed as “atypical” or “borderline” cases, most presenting with normal QTc, nonexertional syncope, U waves, and/or sinus bradycardia.

Conclusion

Nonsynonymous ankyrin-B variants were detected in nearly 3% of unrelated LQTS patients and nearly 7% of healthy control subjects. Genotype-negative LQTS patients with a single ANK2 variant displayed nonexertional syncope, U waves, sinus bradycardia, and extracardiac findings. Whether the identification of previously reported functionally significant variants residing in 2% of apparently healthy subjects suggests proarrhythmic potential or potential misclassification warrants further scrutiny.

Introduction

Long QT syndrome (LQTS) is a disorder of cardiac repolarization often denoted by a prolonged QT interval that can precipitate syncope, seizures, and/or sudden death via its trademark dysrhythmia of torsades de pointes.1 LQTS affects approximately 1 in 5,000 persons. Since 1995, congenital LQTS has been understood as a cardiac channelopathy with mutations involving KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) responsible for approximately 65% to 75% of clinically robust LQTS.2, 3, 4, 5, 6, 7 In addition, the pathogenic basis for a small minority of patients is explained by mutations involving either the KCNE1-encoded (LQT5) or KCNE2-encoded (LQT6) beta subunits of the IKs and IKr potassium channels, respectively.8, 9 Mutations involving the KCNJ2-encoded Kir2.1 potassium channel cause approximately 50% of Andersen Tawil syndrome (ATS, also annotated by some as LQT7).10 In 2003, mutations in ANK2, which encodes the membrane adaptor protein ankyrin-B, were established as the pathogenic basis for the previously elusive chromosome 4-linked LQTS locus (LQT4).11, 12, 13 This finding established ankyrin-B as the first noncardiac channel form of LQTS, extending the discipline from channelopathies to electropathies.

Ankyrin-B is a member of a large family of proteins that link various integral membrane proteins to the spectrin-based cytoskeleton. Proteins that belong to the ankyrin family are closely related and often coexpressed in the same cell types, but each isoform of the family has distinct functions. Ankyrin-B, a 220-kDa isoform originally named for its presence in the brain, plays a critical role in anchoring the Na/K-ATPase, Na/Ca exchanger, and inositol 1,4,5-triphosphate (InsP3) receptor at the T-tubule/sarcoplasmic reticulum membrane sites in cardiomyocytes.13, 14 The C-terminal regulatory domain in ankyrin-B is divergent from other isoforms and is vital to functional roles specific to cardiomyocytes.13, 14 Although ankyrin-B is also expressed in the brain, no neurologic phenotype has been established in human patients with ANK2 mutations. Ankyrin-B (−/−) mice exhibit a number of neurologic defects, including hypoplasia of the corpus callosum and pyramidal tracts, dilated ventricles, and extensive degeneration of the optic nerve.15

The first human mutation in ANK2 was identified in the large French kindred in which the chromosome 4 LQTS locus originally was described in 1995.12 The mutation, E1425G, is localized to the spectrin-binding domain of ankyrin-B. In addition to QT prolongation, affected family members displayed many cardiac phenotypes somewhat atypical for LQTS, including severe sinus bradycardia, polyphasic T waves, and atrial fibrillation.11

Since then, Mohler et al16 completed a study of a large cohort of patients diagnosed with various arrhythmia disorders, which revealed eight unrelated patients hosting putative arrhythmia associated ankyrin-B mutations.16 In addition to E1425G, the study identified four additional novel mutations: L1622I, T1626N, R1788W, and E1813K, all of which were localized to the ankyrin-B C-terminal regulatory domain, absent in controls, and shown to be functionally significant with an in vitro rescue assay. These ANK2-genotype positive patients had diverse clinical presentations.16

In this study, we sought to determine the spectrum and prevalence of ANK2 mutations and potentially relevant nonsynonymous variants in a large cohort of unrelated patients referred explicitly for LQTS genetic testing and among 200 unrelated, reportedly healthy control subjects and to define the phenotype(s) associated with ANK2-positive cases.

Section snippets

Study participants

Between August 1997 and July 2004, 541 consecutive, unrelated patients (358 females) were referred to the Sudden Death Genomics Laboratory at Mayo Clinic, Rochester, Minnesota, for LQTS genetic testing (average age at diagnosis = 24 ± 16 years; average QTc = 482 ± 57 ms).4 Following receiving written consent for this Mayo Foundation Institutional Review Board-approved protocol, DNA was extracted from peripheral blood lymphocytes using the Purgene DNA extraction kit (Gentra, Inc., Minneapolis,

Results

Overall, targeted ANK2 mutational analysis involving exons 36, 37, and 39–46 revealed a total of 14 (10 novel) distinct nonsynonymous ankyrin-B variants. Six of the 14 variants were found in LQTS cases only, 3 of the 14 distinct variants were found among LQTS cases and controls, and 5 of the 14 unique variants were found in controls only. Figure 2 shows the location of these variants on the linear topology of ankyrin-B. All 14 were missense variants and were conserved across species. These

Discussion

Loss of ankyrin-B function has been identified as the pathogenic mechanism mediating LQT4.11, 16 Here we report the overall observation of 9 (7 novel) nonsynonymous variants in nearly 3% of unrelated patients referred explicitly for LQTS genetic testing. Ankyrin-B variants were found in 3.3% of genotype-negative and 1.8% of genotype-positive LQTS cases. Six of the nine variants found in our LQTS cohort were not found in controls, localized to functionally significant domains, and may represent

Conclusion

The precise pathogenic association between ankyrin-B variants and arrhythmogenic phenotype are unclear. In this study, putative pathogenic mutations were seen in approximately 1% of patients with LQTS, whereas nearly 7% of normal control subjects also hosted nonsynonymous ankyrin-B variants. Whether the identification of previously reported functionally significant variants residing in 2% of apparently healthy subjects suggests proarrhythmic potential or potential misclassification warrants

Acknowledgments

We express our gratitude to the study participants and to Carla Haglund, study coordinator of Mayo Clinic’s Sudden Death Genomics Laboratory.

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  • Cited by (41)

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      However, the assumption that variants residing within such a gene cannot impact clinical decision making may be flawed. For example, unless current estimates of LQTS/LQTS subtype prevalence represent vast underestimates, functional rare variants in KCNE2 (i.e. p.Thr10Met-KCNE2, p.Met54Thr-KCNE2, and p.Ile57Thr-KCNE2) [44,48] are simply observed too frequently in public exomes to support even a weakly penetrant, but self-sufficient, role in the pathogenesis of a LQTS subtype widely believed to account for ≤1% of LQTS cases [26,36,49]. However, each of these KCNE2 rare variants have been observed in individuals who exhibited a prolonged QT phenotype, including severe manifestations such as TdP and SCD, in the presence of additional endogenous and exogenous risk factors [44].

    • Defective interactions of protein partner with ion channels and transporters as alternative mechanisms of membrane channelopathies

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      Further analysis revealed a single ANK2 missense variant, A4274G, resulting in E1425G [58]. Since this initial discovery, a number of additional ankyrin-B human gene variants have been identified [59–63]. Patients with ANK2 loss-of-function variants may display sinus node dysfunction, atrial fibrillation, polymorphic ventricular arrhythmias, and/or conduction defects [58,60,61].

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    Dr. Ackerman’s research program is supported by the Dr. Scholl Foundation, the CJ Foundation for SIDS, a Clinical Scientist Development Award from the Doris Duke Charitable Foundation, an Established Investigator Award from the American Heart Association, and RO1 (HD42569) from the National Institutes of Health.

    J. Sherman and D.J. Tester are co-equal first authors.

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