*Article* **Check the Need–Prevalence and Outcome after Transvenous Cardiac Implantable Electric Device Extraction without Reimplantation**

**Giuseppe D'Angelo 1,†, David Zweiker 1,2,3,\*,†, Nicolai Fierro 1, Alessandra Marzi 1, Gabriele Paglino 1, Simone Gulletta 1, Mario Matta 4, Francesco Melillo 5, Caterina Bisceglia 1, Luca Rosario Limite 1, Manuela Cireddu 1, Pasquale Vergara 1, Francesco Bosica 1, Giulio Falasconi 1, Luigi Pannone 1, Luigia Brugliera 6, Teresa Oloriz 7, Simone Sala 1, Andrea Radinovic 1, Francesca Baratto 1, Lorenzo Malatino 8, Giovanni Peretto 1, Kenzaburo Nakajima 1, Michael D. Spartalis 1, Antonio Frontera 1, Paolo Della Bella <sup>1</sup> and Patrizio Mazzone <sup>1</sup>**


**Abstract:** Background: after transvenous lead extraction (TLE) of cardiac implantable electric devices (CIEDs), some patients may not benefit from device reimplantation. This study sought to analyse predictors and long-term outcome of patients after TLE with vs. without reimplantation in a highvolume centre. Methods: all patients undergoing TLE at our centre between January 2010 and November 2015 were included into this analysis. Results: a total of 223 patients (median age 70 years, 22.0% female) were included into the study. Cardiac resynchronization therapy-defibrillator (CRT-D) was the most common device (40.4%) followed by pacemaker (PM) (31.4%), implantable cardioverterdefibrillator (ICD) (26.9%), and cardiac resynchronization therapy-PM (CRT-P) (1.4%). TLE was performed due to infection (55.6%), malfunction (35.9%), system upgrade (6.7%) or other causes (1.8%). In 14.8%, no reimplantation was performed after TLE. At a median follow-up of 41 months, no preventable arrhythmia-related events were documented in the no-reimplantation group, but 11.8% received a new CIED after 17–84 months. While there was no difference in short-term survival, five-year survival was significantly lower in the no-reimplantation group (78.3% vs. 94.7%, *p* = 0.014). Conclusions: in patients undergoing TLE, a re-evaluation of the indication for reimplantation is safe and effective. Reimplantation was not related to preventable arrhythmia events, but all-cause survival was lower.

**Keywords:** extraction; reimplantation; pacing; ICD; CRT

**Citation:** D'Angelo, G.; Zweiker, D.; Fierro, N.; Marzi, A.; Paglino, G.; Gulletta, S.; Matta, M.; Melillo, F.; Bisceglia, C.; Limite, L.R.; et al. Check the Need–Prevalence and Outcome after Transvenous Cardiac Implantable Electric Device Extraction without Reimplantation. *J. Clin. Med.* **2021**, *10*, 4043. https://doi.org/10.3390/ jcm10184043

Academic Editor: Michael Henein

Received: 5 August 2021 Accepted: 1 September 2021 Published: 7 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Cardiovascular implantable electronic devices (CIED) are increasingly used for the treatment of brady- and tachy-arrhythmic cardiomyopathies, leading to rising numbers of patients with CIEDs [1]. However, the incidence of CIED-related complications is not negligible and in some situations transvenous lead extraction (TLE) is indicated. Infection is the most feared complication, with an incidence of 1.9 per 1000 device-years [2], being responsible for relevant morbidity and potentially life-threatening complications [2,3]. Other indications for TLE include lead failure associated with adverse arrhythmic effects, vein stenosis/occlusion, presence of recalled leads, or facilitation of MRI conditionality. Furthermore, lead extraction may be considered after shared-decision making with the patient, for example during device upgrade [2].

TLE carries a non-negligible risk of procedure-related complications, such as cardiac tamponade, tricuspid valve regurgitation, embolization, vascular complications, and death [4]. Moreover, reimplantation of CIEDs after extraction puts the patient at risk of repeat infection or complications. For this reason, current guidelines recommend patients' re-evaluation after explant, aiming to identify patients strictly requiring device reimplantation and those who can benefit from a conservative management [2,3].

The aim of this study was to identify patients without reimplantation, to assess their long-term outcome compared to remaining patients and to document the risk of further device-related complications in reimplantation patients in a high-volume tertiary centre.

#### **2. Materials and Methods**

This study is a retrospective analysis of all patients undergoing TLE at the Department of Cardiac Electrophysiology and Arrhythmology, San Raffaele Hospital, Milan, Italy, between January 2010 and November 2015. The institutional ethics committee approved the analysis.

#### *2.1. TLE and Post-Procedural Management*

The indication of TLE was set according to current guidelines [2,3] after detailed discussion with the referring physician and the patient. All TLE procedures were performed in the electrophysiology laboratory under conscious sedation or general anaesthesia using a stepwise approach as described elsewhere [4]. All lead extractions were performed with standby cardiac surgery on-site. In case of pacemaker dependency, a standard activefixation lead was placed in the right ventricle and connected to a temporary pacemaker by the end of the procedure.

After the procedure, patients were treated in our arrhythmia unit with continuous ECG monitoring and transthoracic echocardiography was performed.

#### *2.2. Decision to Reimplant*

The decision to reimplant the CIED was based on the individual indication for new CIED implantation at time of TLE according to current international guidelines [5,6], taking into account the patient's history, clinical evaluation, frailty, Holter ECG, and echocardiogram. Second level exams, such as invasive electrophysiological study or cardiac magnetic resonance, were performed in selected patients, according to the clinical presentation. The patient's preference was especially taken into account if the indication for CIED implantation was unclear (e.g., IIb indication for implantation) or the patient strongly denied or favoured reimplantation. Pacing-dependent patients were always implanted a new CIED, but the type of device was also reassessed before reimplantation. The main indications for reimplant in pacemaker patients were intermittent or chronic high-grade AV block or symptomatic sick sinus syndrome. In patients with previous bradycardiatachycardia syndrome, the cardiac rhythm in the year before TLE was assessed from the CIED storage and a reimplant was omitted if the patients had been in stable atrial

fibrillation without episodes of bradycardia. In previous ICD patients, reimplantation was offered in patients with a history of sustained ventricular arrhythmia and a left ventricular ejection fraction below 35%. In patients with CRT, reimplant was recommended in patients with good response to CRT therapy.

The reimplantation was performed at the ipsilateral side, directly after the lead extraction, or after at least 7 days of antimicrobial therapy and negative blood cultures, for at least 72 h at the contralateral side in patients with device infection. In selected cases without the dependency of pacing, the reimplantation was performed during a second stay in hospital a few weeks after the index procedure.

#### *2.3. Follow-Up*

Following reimplantation or decision to discontinue device therapy, patients were discharged and followed thereafter in our clinic after one month and at a 6–12-month interval afterwards.

#### *2.4. Data Collection*

All patients receiving TLE at our centre were identified using the department's prospective TLE registry. Patients were excluded if the decision to reimplant was left to the referring centre and in case of in-hospital death before the decision was made. In case of multiple extractions, the first procedure was included as index procedure. Baseline, procedural and follow-up data, as well as complications, were retrieved from the hospital's information system. In case of missing follow-up, patients without reimplantation were additionally contacted via telephone.

#### *2.5. Endpoints*

Complete procedural success was defined as the removal of all targeted leads and all lead material from the vascular space without the occurrence of any permanently disabling complication or procedure-related death. Clinical success was defined as the removal of all targeted leads and lead material from the vascular space that could oppose a risk of perforation, embolic events, or perpetuation of infection, in the absence of complications. Failure of the procedure was defined as the inability to achieve either complete procedural or clinical success, or the occurrence of any permanently disabling complication, or procedurerelated death. Major complications were defined as outcomes that were life-threatening, resulting in significant or permanent disability or death, or required surgical intervention. Minor complications were defined as events related to the procedure that required medical intervention or minor surgery. Device-related complication at follow-up was defined any complication that was exclusively caused by the implanted device and required invasive interventions as result; pocket changes due to battery depletion were excluded.

#### *2.6. Statistics*

Patients were stratified into two groups based on reimplantation after TLE: All patients that received reimplantation during the index stay or were scheduled a reimplantation procedure at the time of discharge were summarised into the "reimplantation" group, whereas remaining patients formed the "no reimplantation" group. Continuous variables are reported as mean (standard deviation, SD) or median (interquartile range, IQR), and categorical variables as percentage (absolute number). Continuous data were compared by student's T test or Mann-Whitney U-test as appropriate; categorical variables were compared with Fisher's test. Multivariable analysis using logistic regression analysis was performed to assess the role of predictors of the absence of need for device reimplantation. Therefore, all baseline characteristics, as shown in Table 1 with a univariable *p* value < 0.1, were included. A two-tailed *p* value < 0.05 was considered statistically significant. All analyses were performed using R 4.0.5 (The R Project, Vienna, Austria).


**Table 1.** Baseline characteristics of the included population.

AV: atrioventricular; eGFR: estimated glomerular filtration rate calculated with CKD-EPI formula; LVEF: left ventricular ejection fraction; ICD: implantable cardioverter defibrillator; CMP: cardiomyopathy; CRT-D: cardiac resynchronization therapy-defibrillator; CRT-P: cardiac resynchronization therapy-pacemaker; PM: pacemaker; † secondary prevention as indication for primary CIED implantation in ICD and CRT-D patients (data available in 69% of cases).

#### **3. Results**

Out of 242 patients undergoing 246 TLE procedures during the observation period, 223 patients were included into the analysis. Remaining patients either died in hospital (*n* = 2) or the decision to reimplant was not documented or left to the referring hospital (*n* = 17).

#### *3.1. Baseline Characteristics and TLE Procedure*

Median age was 70 (IQR 58–76) years and 22.0% were female. Main comorbidities were reduced left ventricular ejection fraction (63.2%), arterial hypertension (53.4%), chronic kidney disease (37.7%), and atrial fibrillation (34.6%, Table 1).

Overall, 40.4% of patients had a CRT-D, 31.4% a single or dual chamber pacemaker, and 26.9% an ICD. Remaining patients had a CRT-P (1.4%).

Infection was the main reason of extraction (55.6%), which was present in the pocket in 41.3%, while systemic infection with active endocarditis was identified in 16.1%. Lead malfunction was the cause of extraction in 35.9% of patients, followed by device upgrade (6.7%) and other causes (1.8%, such as patient discomfort, Table 1). Out of 2.5 ± 0.9 present leads per patient, 2.2 ± 1.1 were planned to be explanted; an explant of the entire system was planned in 78.0%. Median lead age was 68 months with a total range of 0 to 327 months.

TLE was clinically successful in 99.6% of cases and removal was complete in 95.5%, utilizing advanced extraction tools in 35.9% of cases. Both major and minor complications occurred in 3.1% each. Further details can be found in Supplemental Table S1.

#### *3.2. Decision Not to Reimplant*

In 34 patients (14.8%), the decision not to reimplant the CIED was taken. This included 12 patients (36.4%) that previously had an ICD, 11 patients (33.3%) with CRT-D and 10 patients (30.3%) with a pacemaker. The decision was based on a negative electrophysiological study in 21.2%, restoration of LV function in 21.2%, absence of arrhythmia during continuous ECG monitoring in 18.2%, patients' preference in 12.1% and negative MRI in 6.1%. Persistent infection was another factor that played a role in the decision in 33.3% of cases. Another reason was negative electro-anatomical mapping in the presence of ARVD (*n* = 1, 3.0%). More details about patients that did not receive a reimplantation can be found in Supplemental Table S2.

In patients with reimplantation, a device upgrade was performed in 14.2%, while the device was downgraded in 9.0%. The reimplantation was performed mostly on the contralateral side (55.8%). In one case (0.5%), the reimplantation was performed with epicardial leads with the device in the abdomen.

#### *3.3. Factors Favouring No Reimplantation*

In patients without reimplantation, a reduced left ventricular ejection fraction was more prevalent (Table 1). Regarding the indication for CIED implantation, there were significant differences between groups, with sick sinus syndrome and inherited cardiac disease being more common in patients without reimplant. Device infection was significantly more common in this patient group (81.8% vs. 51.1%, *p* = 0.001), especially presence of endocarditis (33.3% vs. 13.2%, *p* = 0.008). In multivariable analysis, absence of ischemic cardiomyopathy (*p* = 0.047) and absence of AV block (*p* = 0.014) were significant predictors for absence of reimplantation, as well as high left-ventricular ejection fraction (*p* = 0.024, Table 2).

**Table 2.** Univariable and multivariable analysis assessing the role of clinical parameters in predicting the absence of reimplantation.


\*: *p* < 0.05; CIED: cardiac implantable electric device; CMP: cardiomyopathy; AV: atrioventricular; LVEF: left ventricular ejection fraction; OR: odds ratio.

#### *3.4. Follow-Up*

Median follow-up duration was 42 months with no differences between groups (Table 3). While cumulative one-year mortality in patients with vs. without reimplantation was similar (98.0% vs. 100.0%), five-year mortality was significantly higher in patients with reimplantation (94.7% vs. 78.3%, *p* = 0.014, Figure 1). Hospitalizations for device revision (in the reimplantation group) or late reimplantation (in the no-reimplantation group) were similar (11.1% vs. 12.1%, *p* = 0.771)


NaR: number at risk; \*: *p* < 0.05.

**Figure 1.** Cumulative survival of no-reimplantation vs. reimplantation groups.

In patients with reimplantation, device-related hospitalizations (excluding pocket changes due to battery depletion) occurred in 11.1% after an interval of 27 ± 25 months after the extraction procedure (with a range of 1 to 84 months). Reasons were lead failure necessitating repositioning (7.9%, mostly due to dislocation), pocket revision in imminent decubitus (2.1%), and generator recall (1.1%). There was one case of recurrent device infection in a female CRT-D patient that initially received extraction due of a 94-monthold fractured right ventricular lead. During follow-up after 77 months, she developed pocket dehiscence that progressed to pocket infection despite two surgical pocket revisions. Finally, a second CIED extraction procedure was performed in six patients (3.2%) a median of 42 months after the procedure.

In patients without reimplantation, 15.2% received an implantable loop recorder for detection of pauses in two patients (6.1%) with previous PM and for detection of ventricular arrhythmias in three patients (9.1%) with previous ICD. In total, four patients (12.1%) had a late reimplantation of their device; two patients received an ICD for ventricular arrhythmias and two patients received a CRT-D reimplantation for progressing heart failure. No repeat hospitalizations for invasive treatment of recurrent infection were documented (Supplemental Table S2).

#### **4. Discussion**

This analysis of consecutive patients undergoing TLE at our centre reveals that (1) prevention of reimplantation was possible in a significant proportion of patients undergoing TLE with a low risk of arrhythmia-related events; (2) baseline comorbidities and the primary indication for CIED implantation are the main predictors for device reimplantation; and (3) long-term mortality was higher in patients without reimplantation, but mostly due to non-cardiac causes.

This study shows that following a rigorous work-up, patients that do not profit from a CIED reimplantation can be identified with a low risk of complications due to undertreatment. In this analysis, in 15.2% of cases an immediate reimplantation was prevented. Due to the heterogeneity of clinical characteristics of patients undergoing TLE, we did not identify a "one size fits all" regimen to evaluate the need for reimplantation; instead, a patient-tailored approach was necessary, including the patient's clinical status and will, cardiac magnetic resonance imaging, electrophysiologic study, and loop recorder implantation. As a CIED implantation may has a significant impact on the patient's daily life [7], the patient's opinion has to be incorporated in the final decision; it played a major role in 14.8% of cases without reimplantation in this analysis. In multiple regression analysis, we identified the CIED indication and the current left-ventricular ejection fraction to be significantly correlated with the decision to reimplantation. Patients without high degree atrioventricular block were more likely to be discharged without a CIED, probably because other indications for PM implantation have a higher potential to resolve (e.g., permanent AF in patients with previous symptomatic brady-tachy-syndrome). Patients without reimplantation were less likely to suffer from ischemic cardiomyopathy and reduced left-ventricular ejection fraction, as these conditions represent a class I indication for ICD implantation according to current guidelines [5]. In the current literature, similar (14.3%) [8] or even higher rates (40.7%) [9] of patients without reimplantation after TLE can be found. Differences in reimplantation may be explained by the incorporation of patients receiving TLE for indications other than CIED infection in this analysis. Interestingly, we did not find patients that had no indication for CIED therapy at time of implantation in contrast to Döring et al., who reported a proportion of 27% in patients without reimplantation [9].

During follow-up, we fortunately did not document a signal towards events caused by missing reimplantation in the no-reimplant group and for 17 months, no reimplantation occurred. Furthermore, we did not document ongoing device complications leading to repeat interventions in this group. Considering long-term outcome, it is apparent that more than one in ten patients out of this group may still need a reimplantation, but many years after initial TLE. Therefore, medical checks at regular intervals may be good for these patients, which is indeed more difficult considering that they do not have a CIED anymore. Interestingly, hospitalizations for CIED revision in the reimplantation group and late CIED reimplantation in the no-reimplantation group were similar.

In the whole population, there was no reintervention due to recurrent CIED infection necessary; only one patient with previous lead failure developed device infection at followup. The low rate of CIED reinfection is consistent with previous literature [10]. However, a significant proportion of patients with device-reimplantation (7.9%) had to be hospitalised

for CIED revision, mostly due to lead dislocation. Therefore, a close follow-up may be beneficial in these patients.

While long-term survival was significantly lower in the no-reimplantation group, we did not identify deaths that may have been prevented by CIEDs. The reduced mortality in the no-reimplantation group was also seen in other studies dealing with reimplantation after TLE [8,9], but this effect is explained to be caused by older age and a high rate of non-cardiac deaths in the no-reimplantation group [9].

#### *Limitations*

While this study adds valuable evidence on the long-term outcome of patients undergoing TLE at a high-volume centre with vs. without reimplantation, it is subject to a few limitations: first, it is subject to bias (such as information bias) due to its retrospective nature. Despite rigorous investigation and telephonic contact of patients, some patients were lost to follow-up. Second, the low rate of patients may have led to underpowering of factors that explain no-reimplantation. Third, this analysis may not be extrapolated to other centres with a different volume and different TLE indications as well as procedures. Furthermore, no data about dependency on temporary pacing after TLE was available, as well as details of the primary CIED implantation (e.g., LVEF in CRT patients). Lastly, with the evolution of leadless pacing in the last years new concepts may allow the reimplantation of devices that previously was deemed too risky [11,12].

#### **5. Conclusions**

The prevention of reimplantation after TLE, after careful evaluation, is safe and does not lead to an increased rate of preventable arrhythmia-related events. The primary indication and current left-ventricular ejection fraction represent independent predictors for reimplantation. Patients without reimplantation experience reduced long-term survival compared to remaining patients at follow-up.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jcm10184043/s1, Table S1: Procedural outcome, Table S2: Individual patient characteristics of patients without reimplantation.

**Author Contributions:** Conceptualization, G.D.; methodology, G.D., D.Z., P.M.; validation, G.D., D.Z., N.F., A.M., G.P. (Gabriele Paglino), S.G., M.M., F.M., L.R.L., C.B., M.C., P.V., F.B. (Francesco Bosica), G.F., L.P., L.B., T.O., S.S., A.R., F.B. (Francesca Baratto), L.M., G.P. (Giovanni Peretto), K.N., M.D.S., A.F., P.D.B., P.M.; formal analysis, G.D., D.Z.; investigation, G.D., D.Z.; resources, G.D., P.D.B., P.M.; data curation, G.D., P.M., D.Z.; writing—original draft preparation, G.D., D.Z., A.F., P.D.B., P.M.; writing—review and editing, G.D., D.Z., N.F., A.M., G.P. (Gabriele Paglino), S.G., M.M., F.M., L.R.L., C.B., M.C., P.V., F.B. (Francesco Bosica), G.F., L.P., L.B., T.O., S.S., A.R., F.B. (Francesca Baratto), L.M., G.P. (Giovanni Peretto), K.N., M.D.S., A.F., P.D.B., P.M.; visualization, D.Z.; supervision, G.D., P.M.; project administration, G.D., P.M.; funding acquisition, P.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** According to the institution's ethical review board, no formal ethical review was necessary for the study due to its retrospective nature.

**Informed Consent Statement:** Patient consent was waived due to the study's retrospective nature.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** D.Z. received speaker honoraria and travel grants from Daiichi Sankyo and research grants from Boston Scientific. P.M. is a proctor for Cook Medical. All other authors report no conflict of interest whatsoever.

#### **References**


## *Article* **Clinical Features of LMNA-Related Cardiomyopathy in 18 Patients and Characterization of Two Novel Variants**

**Valentina Ferradini 1, Joseph Cosma 2, Fabiana Romeo 2, Claudia De Masi 1, Michela Murdocca 1, Paola Spitalieri 1, Sara Mannucci 1, Giovanni Parlapiano 1, Francesca Di Lorenzo 1, Annamaria Martino 3, Francesco Fedele 4, Leonardo Calò 3, Giuseppe Novelli 1,5,6, Federica Sangiuolo 1,\* and Ruggiero Mango <sup>2</sup>**


**Abstract:** Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed myocardial performance in the absence of hypertension, valvular, congenital, or ischemic heart disease. Mutations in LMNA gene, encoding for lamin A/C, account for 10% of familial DCM. LMNA-related cardiomyopathies are characterized by heterogeneous clinical manifestations that vary from a predominantly structural heart disease, mainly mild-to-moderate left ventricular (LV) dilatation associated or not with conduction system abnormalities, to highly pro-arrhythmic profiles where sudden cardiac death (SCD) occurs as the first manifestation of disease in an apparently normal heart. In the present study, we select, among 77 DCM families referred to our center for genetic counselling and molecular screening, 15 patient heterozygotes for LMNA variants. Segregation analysis in the relatives evidences other eight heterozygous patients. A genotype–phenotype correlation has been performed for symptomatic subjects. Lastly, we perform in vitro functional characterization of two novel LMNA variants using dermal fibroblasts obtained from three heterozygous patients, evidencing significant differences in terms of lamin expression and nuclear morphology. Due to the high risk of SCD that characterizes patients with lamin A/C cardiomyopathy, genetic testing for LMNA gene variants is highly recommended when there is suspicion of laminopathy.

**Keywords:** dilated cardiomyopathy (DCM); LMNA; lamin A; lamin C; next generation sequencing (NGS)

#### **1. Introduction**

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed myocardial performance in the absence of hypertension, valvular, congenital, or ischemic heart disease. Diverse aetiologies for DCM have been revealed, including genetic mutations, infections, inflammation, autoimmune diseases, exposure to toxins, and endocrine or neuromuscular causes [1]. As regards to genetic forms of DCM, more than 40 genes have been identified, causing defects in various cellular compartments and pathways such as the nuclear envelope, the contractile apparatus, the Z-disk, and calcium handling [2].

**Citation:** Ferradini, V.; Cosma, J.; Romeo, F.; De Masi, C.; Murdocca, M.; Spitalieri, P.; Mannucci, S.; Parlapiano, G.; Di Lorenzo, F.; Martino, A.; et al. Clinical Features of LMNA-Related Cardiomyopathy in 18 Patients and Characterization of Two Novel Variants. *J. Clin. Med.* **2021**, *10*, 5075. https://doi.org/10.3390/jcm10215075

Academic Editor: Michael Henein

Received: 9 September 2021 Accepted: 27 October 2021 Published: 29 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Mutations in LMNA (MIM 150330) gene, encoding for lamin A/C, account for 0.5–5% of DCM; however, its prevalence increases up to 10% in familial DCM and up to 33% in DCM associated to atrioventricular conduction disorders [3,4]. Lamin proteins form the nuclear lamina, a protein meshwork laying the inner surface of the nuclear envelope [5]. Lamin A and lamin C represents two isoforms encoded by a single gene (LMNA), located on chromosome 1q21.2-q21.3 [6]. Lamins, in addition to conferring cellular and nuclear integrity [7], are implicated in a plethora of crucial cellular functions, such as mechano-transduction, chromatin protection/organization, regulation of signaling, and gene expression [8,9]. To date, more than 500 LMNA variants have been reported [10] causing a wide variety of diseases and ranging from premature ageing to metabolic and skeletal muscle disorders [11,12].

LMNA-related cardiomyopathies are characterized by heterogeneous clinical manifestations that vary from a predominant structural heart disease, mainly mild-to-moderate left ventricular (LV) dilatation associated or not with conduction system abnormalities, to high pro-arrhythmic profile, where sudden cardiac death (SCD) occurs as first manifestation in an apparently normal heart [3]. Brady- and tachy-arrhythmias are a very common finding in lamin cardiomyopathies with conduction system disease commonly preceding the development of DCM by few years to a decade or more [13]. Moreover, supraventricular tachyarrhythmias (SVT) are generally more common than malignant ventricular arrhythmias (VA) at first clinical contact [14]. The mode of inheritance of cardiac laminopathies is autosomal dominant with an almost complete penetrance by the seventh decade [15–17]. Lamin cardiomyopathies are characterized by a poor prognosis and a high rate of major cardiac events, with the most aggressive clinical course [18].

In the present study, we report the genotype–phenotype correlation of 18 DCM patients evidenced heterozygotes for LMNA variants out of 77 referred to our Medical Genetics Unit. In three of them, functional analyses have been performed in order to validate the pathogenicity of two novel lamin variants detected during this work.

#### **2. Materials and Methods**

#### *2.1. Study Population*

Seventy-seven DCM patients and their relatives followed up at the Cardiology Unit of Policlinico Casilino (Rome, Italy) were genotyped at the Medical Genetics Unit of Tor Vergata Hospital; after genetic counselling and informed consent was signed, 15 probands evidenced heterozygotes for LMNA variants.

#### *2.2. Clinical and Instrumental Characterization*

Probands were defined as the first patients in a family referred for genetic testing due to a diagnosis of phenotypic DCM based on the Mestroni criteria for familial DCM [19]. The age of onset of symptoms or documented first traits of the disease was recorded. Family members who underwent genetic testing as part of family screening and had no reported cardiac symptoms at the time of the genetic testing were defined as genotype-positive phenotype-negative family members. Atrioventricular block by PR interval was assessed from a resting 12-lead electrocardiography (ECG). Arrhythmias (atrial and ventricular) were collected from a resting 12-lead ECG, exercise ECG, Holter monitoring and pacemaker, or implantable cardioverter defibrillator (ICD) monitoring. Ventricular arrhythmias were classified as non-sustained ventricular tachycardia (VT), defined as ≥3 consecutive ventricular beats with a rate ≥120/min lasting <30 s, or sustained VA, defined as VT with a rate ≥120/min lasting >30 s, ventricular fibrillation (VF), appropriate antitachycardia pacing (ATP) therapy, appropriate defibrillator shock therapy, and aborted cardiac arrest. Implantable cardioverter defibrillator and cardiac resynchronization therapy (CRT) interrogations were retrospectively reviewed and eventual therapies (ATP or defibrillator shock) recorded. Two-dimensional echocardiography was performed at the subject's first visit using the Vivid 7 or Vivid E9 system (GE Healthcare, Horten, Norway) and analyzed using commercially available software (EchoPACVR, GE). LV ejection fraction (EF) and

LV volumes were calculated from apical views using Simpson's biplane method. Left ventricular diameters were obtained from the parasternal long-axis view. When possible, patients underwent CMR at baseline by using a 1.5-T scanner (Philips Intera CV; Philips Healthcare, Best, The Netherlands) and a phased array cardiac receiver coil, according to standard acquisition protocols set by the Society for Cardiovascular Magnetic Resonance [20]. Electrocardiogram-gated, breath-hold steady-state free precession cine images were acquired in both the long- and the short-axis planes from the LV apex to the LV base. Images were subsequently analyzed offline by using a commercially available software (View Forum software, Version 5.1, Philips Healthcare, Best, The Netherlands). LV and RV end-diastolic diameters and volumes as well as end-systolic diameters and volumes, stroke volumes, EF, and left atrium area were calculated, in accordance with the Society of Cardiac Magnetic Resonance criteria [21], by using the Extended MR WorkSpace 2.6.3.4, 2012 Philips Medical System work-station. LV dilatation was diagnosed in the presence of indexed end-diastolic volumes >81 mL/m2 for men and >76 mL/m<sup>2</sup> for women, respectively [22].

#### *2.3. Genetic Analysis*

Genomic DNA was extracted from peripheral blood using EZ1 AdvancedXL (Qiagen), according to the manufacturer's instructions. After Qubit 2.0 quantification, NGS was performed (Ion Torrent S5 and Ion Chef System) using a Custom Panel for SCD (Supplementary Table S1), designed by Ion Ampliseq Designer (Thermo Fisher Scientific, Waltham, MA, USA). Results were analyzed with Ion Reporter and Integrated Genome Viewer (IGV). The interpretation of genetic variants was conducted by Human Gene Mutation Database (HGMD), VarSome, ClinVar, Exac, and GnomAD. Moreover, DANN and Genomic Evolutionary Rate Profiling (GERP) were used. Sanger sequencing was used to confirm genetic variants and segregation analysis.

#### *2.4. Fibroblasts Derivation from Skin Biopsy*

Primary skin fibroblasts were obtained by a skin punch biopsy from two healthy donors (WT) and three DCM patients, after written consent. Tissues were treated as already described [23], and after 15 days, primary culture of the derived human dermal fibroblasts (HDFs) was expanded and analyzed.

#### *2.5. Immunofluorescence*

HDFs were incubated with primary antibodies anti-Lamin A/C (N-18, sc-6215, Santa Cruz Biotechnology) and anti-prelamin A (C-20, sc-6214, Santa Cruz Biotechnology), as described [23]. Nuclei were counterstained with HOECHST (33342, Thermo Fisher Scientific, Waltham, MA, USA). Images have been acquired by fluorescence microscope (Zeiss Axioplan).

#### *2.6. Detection of Nuclear Abnormalities*

For every patient's fibroblast culture, at least 3 × 100 cells in different areas of the sample were evaluated using a Zeiss Axiplan fluorescence microscope, equipped with a 100× oil objective (Plan Apo, NA1.32). Different aspects of nuclear morphology were assessed: nuclear blebs (herniations), extensive lobulations, or donut-like invaginations of the nucleus; also, Lamin staining abnormalities were scored, including extranuclear staining and the presence of so-called honeycombs. Morphometric analysis of nuclei of HDFs WT and DCM has been performed on images from Zeiss Axiplan fluorescence microscope (Hoechst-stained nuclei), using the ImageJ processing software (http://rsbweb.nih.gov/ij/ (accessed on 20 May 2020)), by analyzing at least 10 field/sample or a minimum of 300 cells/sample. The following parameters have been analyzed by tracing nuclei and obtaining, from the ImageJ software, the following parameters: (i) nucleus area, (ii) nucleus circularity (with a value of 1.0 indicating a perfect circle), (iii) nucleus elongation (aspect ratio: the major axis over the minor axis of the fit ellipse), and (iv) nucleus roundness (the inverse of aspect ratio). The analyses have been performed on images from three different

experiments, and results have been reported as mean values ± SD (fold DM vs. WT). Statistical analyses have been assessed by using the Student's two-tailed *t*-test (\* *p* < 0.05 as statistically significant).

#### *2.7. Gene Expression Analysis*

After TRIzol extraction (Invitrogen; Life Technologies Corporation, Carlsbad, CA, USA), total RNAs of patients and controls HDFs were DNase I (RNase-free)-treated (Ambion; Life Technologies Corporation), reverse transcribed using the High-Capacity cDNA Archive kit (Life Technologies Corporation) and used in real-time reverse transcription (RT)–polymerase chain reaction (PCR). mRNAs levels were measured by SYBR Green chemistry (Life Technologies Corporation) using the following primers: Lamin A: forward 5 -ACTGGGGAAGAAGTGGCCAT-3 ; Lamin A: reverse 5 -GCTGCAGTGGGAGCCGT-3 ; Lamin C: forward 5 -AACTCCACTGGGGAAGAAG-3 ; Lamin C: reverse 5 -CATCTCCAT CCTCATGGTC-3 ; GAPDH: forward 5 -TTGCCCTCAACGACCACTTTG-3 ; GAPDH: reverse 5 -CACCCTGTTGCTGTAGCCAAATTC-3 . GAPDH was used as reference gene. WT value corresponds to the mean value of two wild type samples.

#### *2.8. Western Blot Assay*

Proteins were extracted from patients and controls fibroblasts by RIPA Lysis buffer and Western blot analysis performed with primary antibody for Lamin A/C (N-18, sc-6215, Santa Cruz Biotechnology), followed by Mouse anti-Goat IgG (PIERCE Biotechnology). The signals were scanned and quantified on the ImageQuant LAS 4000 system, after normalizing with f β-actin. WT value corresponds to the mean value of two wild type samples.

#### **3. Results**

#### *3.1. Lamin A/C Variants and Cardiac Phenotype among DCM Patients*

Among 77 DCM families, 11 different LMNA variants were found in 15 subjects (19.5%) and confirmed by Sanger sequencing. The segregation analysis in nineteen relatives evidenced eight heterozygotes for a total of 23 (Table 1). The remaining 62 patients evidenced heterozygotes for variants in *TTN*, *DSP*, *MYBPC3*, *MYH7,* and *SCN5A* genes.


**Table 1.** LMNA gene variants identified in this study.
