Use of Whole Exome Sequencing for the Identification of Ito-Based Arrhythmia Mechanism and Therapy

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Abstract

Background: Identified genetic variants are insufficient to explain all cases of inherited arrhythmia. We tested whether the integration of whole exome sequencing with well-established clinical, translational, and basic science platforms could provide rapid and novel insight into human arrhythmia pathophysiology and disease treatment. Methods and Results: We report a proband with recurrent ventricular fibrillation, resistant to standard therapeutic interventions. Using whole-exome sequencing, we identified a variant in a previously unidentified exon of the dipeptidyl aminopeptidase-like protein-6 (DPP6) gene. This variant is the first identified coding mutation in DPP6 and augments cardiac repolarizing current (Ito) causing pathological changes in Ito and action potential morphology. We designed a therapeutic regimen incorporating dalfampridine to target Ito. Dalfampridine, approved for multiple sclerosis, normalized the ECG and reduced arrhythmia burden in the proband by >90-fold. This was combined with cilostazol to accelerate the heart rate to minimize the reverse-rate dependence of augmented Ito. Conclusions: We describe a novel arrhythmia mechanism and therapeutic approach to ameliorate the disease. Specifically, we identify the first coding variant of DPP6 in human ventricular fibrillation. These findings illustrate the power of genetic approaches for the elucidation and treatment of disease when carefully integrated with clinical and basic/translational research teams.

Figures

  • Figure 1. Abnormal repolarization and ventricular fibrillation. A, Presenting ECG with right bundle-branch block; J-point elevation (arrows) <2 mV in leads V1 to V3; thus, not consistent with Brugada pattern. B, Ectopy with short-coupled PVCs. C, Short-coupled PVC initiates VF requiring ICD shock in the presence of quinidine. Over 16 months, proband had 168 appropriate ICD discharges for VF ( 8.6 per month). ICD indicates implantable cardioverter-defibrillator; VF, ventricular fibrillation; PVC, premature ventricular contraction.
  • Figure 2. Negative response to procainamide challenge. Leads V1 through V3 are shown at baseline and every 5 minutes after procainamide treatment. Note the absence of dynamic changes in morphology, lack of transition from saddleback to coved type morphology in lead V2, and no elevation of the J point.
  • Figure 3. Identification of novel splice form of DPP6 (DPP6-T). A, Human DPP6 (red, novel exon 5B). B and C, Domain organization of canonical DPP6 and truncated DPP6-T (red=novel residues). D, DPP6-T A>G variant. E, Confirmation of DPP6-T transcript from human heart (primers denoted by arrows in Panel C). F, DPP6-T expression in human heart by using DPP6-T Ig (F409 in Figure 2C). G, Relative DPP6 and DPP6-T expression in human heart chambers (n=4; *P<0.05 compared with LV). DPP6 indicates dipeptidyl aminopeptidase-like protein-6; LA, left atrium; LV, left ventricle; MW, molecular weight; RA, right atrium; RV, right ventricle.
  • Figure 4. Validation of DPP6-T–specific affinity-purified antibody. A polyclonal DPP6-T antibody was designed and generated. A, In vitro translated DPP6-T, but not DPP6, was recognized by anti–DPP6-T Ig. B, HEK cells were transfected with myc-tagged DPP6 and either myc-tagged DPP6-T or myc-tagged DPP6-T H332R. Cell lysates were immunoprecipitated by using anti-myc Ig and blotted with the DPP6-T Ig. The DPP6-T Ig specifically recognizes the DPP6-T and DPP6-T H332R with no reactivity against canonical DPP6. DPP6 indicates dipeptidyl aminopeptidase-like protein-6.
  • Figure 5. DPP6-T H332R displays increased Kv4.3 binding relative to DPP6-T. A and B, Coimmunoprecipitations of Kv4.3 and DPP6-T from detergent-soluble human left ventricle lysates. C, Purified GST-Kv4.3, but not GST alone associates with DPP6-T in pull-down experiments from detergent-soluble human left ventricular lysates. D and E, Purified GST-Kv4.3, but not GST, associates with radiolabeled DPP6-T and DPP6-T H332R. However, GST-Kv4.3 shows >2-fold increase in relative binding activity for DPP6-T H332R vs DPP6-T when corrected for input and protein loading (n=3; *P<0.05 compared to wild-type). Input lanes in top panel of (D) were taken from same gel at different exposure times. DPP6 indicates dipeptidyl aminopeptidase-like protein-6; WT, wild-type.
  • Figure 6. DPP6-T+ Kv4.3 displays augmented Ito compared with DPP6+ Kv4.3. A and B, DPP6-T H332R +Kv4.3 shows 7-fold increase in peak Ito compared with WT DPP6-T (n=6/condition; *P<0.05 compared with Kv4.3; #P<0.05 compared with Kv4.3+DPP6-T). DPP6 indicates dipeptidyl aminopeptidase-like protein6; WT, wild-type.
  • Figure 7. Ito-directed therapies are predicted to reduce repolarization dispersion and arrhythmia burden. A and B, Computer-simulated human endocardial (Endo) and epicardial (Epi) action potentials associated with DPP6-T and DPP6-T H332R. C, Simulated therapeutic intervention involving modest Ito block combined with modest increase in pacing rate. D, Simulated difference in endocardial and epicardial DAPD. Mathematical modeling predicts that Ito reduction combined with pacing reduces proarrhythmic dispersion in repolarization for H332R. DPP6 indicates dipeptidyl aminopeptidase-like protein-6; WT, wild-type; DAPD, action potential duration.
  • Figure 8. Ito-directed therapies reduce repolarization dispersion and arrhythmia burden. A, Proband ECG following dalfampridine and cilostazol. Note altered terminal portion of QRS complex and lower J point vs Figure 1A. B and C, ICD discharge events following initial event. Ablations noted with arrows. VF events reduced >90-fold since initiation of Ito-targeted therapy (*P<0.05 compared to approach lacking dalfampridine/cilostazol). The patient’s ICD has fired twice following the new therapeutic approach; both events occurred when patient was nonadherent with the dalfampridine/cilostazol regimen. ICD indicates implantable cardioverter-defibrillator; VF, ventricular fibrillation.

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Sturm, A. C., Kline, C. F., Glynn, P., Johnson, B. L., Curran, J., Kilic, A., … Mohler, P. J. (2015). Use of Whole Exome Sequencing for the Identification of Ito-Based Arrhythmia Mechanism and Therapy. Journal of the American Heart Association, 4(5). https://doi.org/10.1161/JAHA.114.001762

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