Targeted next-generation sequencing detects novel gene–phenotype associations and expands the mutational spectrum in cardiomyopathies

<div><p>Cardiomyopathies are a heterogeneous group of primary diseases of the myocardium, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC), with higher morbidity and mortality. These diseases are genetically diverse and associated with rare mutations in a large number of genes, many of which overlap among the phenotypes. To better investigate the genetic overlap between these three phenotypes and to identify new genotype–phenotype correlations, we designed a custom gene panel consisting of 115 genes known to be associated with cardiomyopathic phenotypes and channelopathies. A cohort of 38 unrelated patients, 16 affected by DCM, 14 by HCM and 8 by ARVC, was recruited for the study on the basis of more severe phenotypes and family history of cardiomyopathy and/or sudden death. We detected a total of 142 rare variants in 40 genes, and all patients were found to be carriers of at least one rare variant. Twenty-eight of the 142 rare variants were also predicted as potentially pathogenic variants and found in 26 patients. In 23 out of 38 patients, we found at least one novel potential gene–phenotype association. In particular, we detected three variants in <i>OBSCN</i> gene in ARVC patients, four variants in <i>ANK2</i> gene and two variants in <i>DLG1</i>, <i>TRPM4</i>, and <i>AKAP9</i> genes in DCM patients, two variants in <i>PSEN2</i> gene and four variants in <i>AKAP9</i> gene in HCM patients. Overall, our results confirmed that cardiomyopathic patients could carry multiple rare gene variants; in addition, our investigation of the genetic overlap among cardiomyopathies revealed new gene–phenotype associations. Furthermore, as our study confirms, data obtained using targeted next-generation sequencing could provide a remarkable contribution to the molecular diagnosis of cardiomyopathies, early identification of patients at risk for arrhythmia development, and better clinical management of cardiomyopathic patients.</p></div

Distribution of specific and overlapping genes carrying rare variants identified in this study, among the three cardiomyopathic phenotypes.

<p>Distribution of specific and overlapping genes carrying rare variants identified in this study, among the three cardiomyopathic phenotypes.</p

New gene–phenotype associations and number of novel gene rare variants for each cardiomyopathic phenotype.

<p>New gene–phenotype associations and number of novel gene rare variants for each cardiomyopathic phenotype.</p

Genetic profile of each cardiomyopathic patient carrying potentially pathogenic rare variants.

<p>Genetic profile of each cardiomyopathic patient carrying potentially pathogenic rare variants.</p

Family pedigree and mutation identification.

<p>(A) Electropherogram of the <i>LMNA</i> gene variant. (B) The 7 duplicated amino acids (LLNSKEA) are highly conserved among <i>LMNA</i> gene homologs in vertebrates. (C) Family pedigree of the index patient with the novel <i>LMNA</i> gene mutation.</p

Clinical characteristics of family members according to <i>LMNA</i> mutation carrier status.

<p>Mean values ± standard deviation or absolute frequencies and percentage of patients.</p><p>AF = atrial fibrillation; AVB = atrioventricular block; BMI = body mass index; CMRI = cardiac magnetic resonance imaging; DBP = diastolic blood pressure; Echo = echocardiography; LGE = late gadolinium enhancement; LVEDD = left ventricular end-diastolic diameter; LVEF = left ventricular ejection fraction; NSVT = non-sustained ventricular tachycardia; PVCs = premature ventricular complexes; RVEF = right ventricular ejection fraction; RVOT = right ventricular outflow tract; SBP = systolic blood pressure; SVT = sustained ventricular tachycardia.</p><p>*RVOT value by echocardiography was available in 19 of 20 patients, 9 of whom were <i>LMNA</i> mutation-positive subjects.</p><p>†CMRI was performed in 15 subjects, 7 of whom were <i>LMNA</i> mutation carriers.</p><p>Clinical characteristics of family members according to <i>LMNA</i> mutation carrier status.</p

Clinical characteristics of <i>LMNA</i> mutation-positive family members.

<p>AF = atrial fibrillation; ARVC = arrhythmogenic right ventricular cardiomyopathy; AVB = atrioventricular block; CMR = cardiac magnetic resonance imaging; Com = comments; Echo = echocardiography; EF = ejection fraction; F = female; FU = Follow-up; Gen = Gender; HT = heart transplantation; ICD = implantable cardioverter-defibrillator; ILR = implantable loop recorder; LBBB = left bundle-branch block; LGE = late gadolinium enhancement; LV = left ventricle; LVEDD = left ventricular end-diastolic diameter; M = male; m = minor; Mj = major; np = not performed; NSVT = non-sustained ventricular tachycardia; PM = pacemaker; PVCs = premature ventricular complexes; RBBB = right bundle-branch block; RV = right ventricle; RVOT = right ventricular outflow tract; SB = sinus bradycardia; Sub = Subject; SVT = sustained ventricular tachycardia; TFC = Task Force criteria.</p><p>*at diagnosis/clinical presentation (years);</p><p>**Morphology of NSVT was determinable in 4 of 6 <i>LMNA</i> mutation-positive family members having NSVT;</p><p>***for ARVC diagnosis Mj/m.</p><p>Clinical characteristics of <i>LMNA</i> mutation-positive family members.</p

Clinical characteristics of index patient and <i>LMNA</i> mutation-positive family members.

<p>Index patient’s electrocardiogram (ECG) showing, at clinical presentation, (A) sinus rhythm, first-degree AV block, and PVC of LBBB morphology and, 13 years later, (B) complete AV block. (C) Asystole documented on implantable loop recorder memory (subject III-8). (D) Sustained VT detected on telemetry monitoring, effectively terminated by internal ICD shock (subject III-1). (E) Episodes of non-sustained VT with LBBB morphology and inferior axis (subject III-5) on 12-lead Holter monitoring. (E) Episodes of non-sustained VT with RBBB morphology and superior axis (subject IV-2) on 12-lead Holter monitoring.</p

CMR imaging of <i>LMNA</i> mutation-positive family members.

<p>(A) Short-axis cine (a) and LGE sequences in the Short-axis (b) and 2-chambers long-axis views (c). Bulging (arrow in a) and LGE of the RV free wall (arrowheads in b). Linear midwall LGE is localized at the interventricular septum and LV inferior wall (arrowheads in c) (subject III-5). (B) and (C) LGE sequences in the short axis (a) and 4-chambers long axis views (b). LGE with linear midwall pattern is shown on the LV inferior wall and basal interventricular septum (arrowheads) (subjects III-8, and IV-2).</p

Analysis of nuclear envelope integrity under stressing condition in LMNA transfected HL-1 cells.

<p>Nuclear WT LMNA (A) and DUP LMNA (B) signals in control (CTR) and stressing conditions (Hyperosmolarity, Hypoxia, Oxidative stress). The merged signals of LMNA proteins and the RFP nuclear marker are shown in the insets. Confocal XY planar projections are depicted in each experimental condition.</p