**4. Discussion**

Over the past few decades, technological advances in genetic sequencing have allowed to perform genetic testing worldwide. The number of genetic variants to analyse has increased massively [49] and so has the complexity of its interpretation. Achieving a reliable classification is crucial [36], especially in controversial entities like LVNC. LVNC is the most recently described cardiomyopathy and broadening the knowledge of its genetics field is an absolute necessity. Genetic yield is really variable depending on the reported series and very few papers have tried to compare LVNC phenotype with or without an identifiable genetic cause. Moreover, genetic variants classification performed before ACMG-AMP criteria [37] should be interpreted with caution. In addition, most studies included LVNC patients diagnosed only based on TTE criteria.

A study performed about 10 years ago, found a genetic "mutation" in 17.5% of their TTE-based cohort [40]. The generic yield improved to 29% in a study of 63 isolated LVNC diagnosed by TTE [50] and to 41% in another TTE based study [29]. However, no differences in clinical phenotypes between positive and negative pathogenic variants' carriers were found in either of these two studies.

In 2017, an interesting study in children classified variants according to ACMG [37] with a genetic yield of only 9% and, unfortunately, no comparison between carriers and non-carriers were done [18]. Wang et al. also analysed a childhood cohort with a higher genetic yield [38%] describing a poorer prognosis in pathogenic variants carriers (earlier age of onset and lower LVEF) than those without pathogenic variants [41]. Another German study of 68 index LVCN patients diagnosed by TTE reported a 38% genetic yield and described worse clinical outcomes in patients with pathogenic variants in LMNA and RBM20. In their cohort, TTN variants were the most frequent cause for LVNC and they associated TTN truncating variants with LVNC phenotype [51].

In our study, genetic testing identified a genetic cause in up to 34.2% of patients, a percentage within the expected ranges according to previously reported series. However, better understanding of VUS may improve this yield. Thanks to genetic screening not only 22 relatives at risk were identified, but also 27 relatives could be discharged. LVNC have been accepted to be a possible inherited condition. Therefore, we believe that genetic screening should be strongly recommended, like in any other kind inherited cardiomyopathy. Moreover, in this entity, genetic testing may be useful not only for family screening, but also to help in differential diagnosis with hypertrabeculation mimicking LVNC. In fact, if genetic testing had only been performed in those with moderate to severe LV dysfunction in follow-up, genetic yield would have improved to 75% (9/12).

The strength of our cohort relies not only in the genetic variants' classification based on ACMG-AMP guidelines [37], but also on the patient's selection to achieve a LNVC cohort with a solid diagnosis. Carriers of VUS (group 3) were not included in any comparison group, as its classification may easily change into LP or LB as genetic knowledge improves. However, interesting differences between carriers of P/LP variants (group 1) and non-carriers (group 2) were found. Family history of cardiomyopathies is mainly found in group 1, and possible trigger factors were only identified in group 2. In fact, trigger factors may explain the hypertrabeculation in one in every three patients from group 2. However, the most stunning finding was the LVEF evolution in follow-up (Figure 3). LVEF showed a tendency to improve in time in group 2, contrary to the tendency to worsen in group 1. Although these results should be interpreted with caution, genetic results could represent a predictor factor for LVEF evolution, especially if a trigger factor had been identified. In our cohort, this information could have been useful, for example, to delay the ICD implant decision in patients from group 2, whose LVEF improved with optimal medical therapy. Besides, all carries of TTN variants presented a fluctuant LVEF. On the other hand, CVAs are present in the same proportion in both groups, highlighting the importance of emboli risk assessment in all LVNC phenotypes.

We believe that our data reinforce the hypothesis that there are different ways to reach the same final phenotype, whether as congenital heart defect or as an inherited cardiomyopathy or as an acquired one. Although in this study we included LVNC patients based on the same criteria, they had different etiologies. It seems obvious that patients from group 1 have an inherited kind of cardiomyopathy. However, the presence of trigger factors in 1/3 of patients from group 2 support the hypothesis that LVNC phenotype could also be an acquired cardiomyopathy [4,9–11]. On the basis of an apparently normal heart with an underlying predisposition, the developing of hypertrabeculation leading to LVNC phenotype may (or may not) start to manifest at a certain age. Although prognostic differences in these different scenarios may be found (highlighting the role of genetic testing and LVEF evolution), clinicians should be aware of possible related complications like CVA in all LVNC phenotypes.

In addition, our data also supports the theory that LVNC could actually been underdiagnosed previously, even in autopsies [13,21,44,52]. In our series, TTE misdiagnosed LVNC in the first place in up to 45% of LVNC cohort. No wonder why the diagnostic based on imaging criteria is still a real challenge and an ongoing debate.

Furthermore, APE has played a critical role in helping to describe and classify LVNC as a novel cardiomyopathy and to settle its diagnostic criteria [17,19]. Nevertheless, contrary to HCM, there are no large case series available [44,53] and deep histopathological characterization is missing. HCM is known for its disarray, hypertrophy of myocytes and possible fibrosis [54]. This hypertrophy of myocytes is maximal at subendocardium [55,56] and nuclei can be enlarged, presenting nuclear pleomorphism and hyperchromasia [54]. In LVNC, APE data have been focused in compaction/non-compaction layers description and quantification [4,44], neglecting meticulous histopathological description [4,57]. Fibrosis areas considered secondary to ischemia due to microvascular dysfunction are described in some studies [58–60]. Burke et al. [4,44] did not found any difference between isolated LVNC and associated with CHD. Although Jenni et al. [17] claimed that no disarray was present, a recent transplantation series found it in one LVNC patient [59]. Proper myocyte description beyond isolated cases is also missing [59,61,62], and no genetic data are reported in any series. For this reason, the APE description of our sample, although small, is important. According to previous data, fibrosis and one case of necrosis were identified, but no inflammatory pattern. However, the most important finding was the cellular hypertrophy of myocytes, present in all the studied hearts. Moreover, its distribution is not uniform, being more pronounced in the non-compacted layer. However, the most striking feature was the irregular nuclear shape (Figure 4). In addition, the available genetic data, with a good yield in these patients with severe phenotype of LVNC, are really interesting. Although the described histopathological findings are present in the eight hearts, two of them intriguingly present a LP variant in a gene that encodes a component of the nuclear lamina, which determines nuclear shape and size.

#### **5. Limitations**

Family screening was not available for all relatives. All genetic variants identified in this cohort were reviewed by two biologists and two cardiologists trained in cardiogenetics, according to current published guidelines and available data [37]. Despite these efforts, some variants may be reclassified as additional data become available. Family screening for TTN variants from patients 11 and 12 are still pending due to the COVID-19 pandemic. The genetic testing yield may also improve as gene panels continue to expand and better classification of VUS is achieved. Further investigation in genetics and histopathological exam, expanding the series number, is definitely necessary to draw further conclusions.
