Genetic contributions to the expression of acquired causes of cardiac hypertrophy in non-ischemic sudden cardiac death victims

The contribution of genetic variants to non-ischemic sudden cardiac death (SCD) due to acquired myocardial diseases is unclear. We studied whether SCD victims with hypertension/obesity related hypertrophic myocardial disease harbor potentially disease associated gene variants. The Fingesture study has collected data from 5869 autopsy-verified SCD victims in Northern Finland. Among SCD victims, 740 (13%) had hypertension and/or obesity as the most likely explanation for myocardial disease with hypertrophy and fibrosis. We performed next generation sequencing using a panel of 174 cardiac genes for 151 such victims with the best quality of DNA. We used 48 patients with hypertension and hypertrophic heart as controls. Likely pathogenic variants were identified in 15 SCD victims (10%) and variants of uncertain significance (VUS) were observed in additional 43 SCD victims (28%). In controls, likely pathogenic variants were present in two subjects (4%; p = 0.21) and VUSs in 12 subjects (25%; p = 0.64). Among SCD victims, presence of potentially disease-related variants was associated with lower mean BMI and heart weight. Potentially disease related gene variants are common in non-ischemic SCD but further studies are required to determine specific contribution of rare genetic variants to the extent of acquired myocardial diseases leading to SCD.

www.nature.com/scientificreports/ In this study, our goal was to determine whether non-ischemic myocardial diseases with LVH/fibrosis at autopsy after SCD and hypertension and/or obesity as the apparent triggers for LVH, may associate with genetic variants in arrhythmia candidate genes.

Methods
The Fingesture study. The study population is derived from the Fingesture study, which has gathered autopsy and clinical record data from 5869 consecutive SCD victims from the Oulu University Hospital District, a defined geographical area in northern Finland since 1998. The detailed study protocol has been previously published 3 . Briefly, a medicolegal autopsy was performed on all SCD victims in the National Institute for Health and Welfare, Oulu, Finland and at the Department of Forensic Medicine, University of Oulu, Oulu, Finland, by experienced forensic pathologists, each performing over 100 autopsies/year, using contemporary guidelines for diagnosing the cause of death. Finnish law requires medicolegal autopsy to be performed if the death is not due to a previously identified disease, a victim who was not treated by physician during his/her last illness, or if the death was otherwise unexpected. Therefore, Finland has the highest autopsy rates following SCD in Western societies 7 and the Fingesture study includes a majority of victims of unexpected SCD (estimated to be 60% of all sudden deaths) 8 in the defined area. Meticulous cardiac investigations were performed on all victims, including macroscopic dissection and investigation of myocardium, coronary arteries and valves, and histological samples taken from 3 to 5 sections of the heart. All causes of SCD in the Fingesture study during 1998-2017 are provided in Fig. 1. SCD victims in the present study. We selected 151 non-ischemic SCD victims who were found to have non-specific LVH and fibrosis at autopsy without diagnostic criteria for HCM, such as myocyte disarray or asymmetric septal hypertrophy. The inclusion criteria included cardiac hypertrophy at autopsy determined by a heart weight greater than the predicted value based on body surface area. In addition to cardiac hypertrophy, a clinical history of hypertension and/or autopsy findings related to hypertension (e.g. sclerotic renal arterioles) were characteristic of subjects with a hypertensive etiology. Obesity-induced myocardial disease included obesity with a BMI > 30, with excessive epicardial fat with or without LV dilatation 3 . Study subjects had no clinical history of CAD and coronary arteries had no stenosis > 50% or active plaques at autopsy. Patients with developmental or acquired aortic stenosis were not included.
DNA samples of the SCD victims were isolated from formalin fixed and paraffin embedded myocardial tissue samples taken at autopsy. We carried out genetic studies in the 151 of 740 individuals (20%) with SCD who met the criteria for acquired LVH due to hypertension or obesity, and whose DNA passed the quality control for further analysis.
Control subjects. The control study population is from the OPERA (Oulu Project Elucidating Risk of Atherosclerosis) project, which is an epidemiological, population-based study designed to address the risk factors and disease endpoints of atherosclerotic cardiovascular disease. The study population has been previously described in detail 9 . The study population at baseline consisted of a hypertensive cohort (261 men and 258 women) and a control cohort (259 men and 267 women) from the city of Oulu, Finland. Subjects were 40-59 years old at the time of recruitment. Subjects were recruited during December 1990 to March 1993, and

Results
Mean age of SCD victims was 54 ± 10 years, 82% were male and 50% had prior cardiac disease diagnosis. The presumed etiological basis for LVH was hypertension in 78 of the SCD victims (52%), and obesity was considered the most likely cause for LVH in the remaining 73 subjects (48%). In total, potentially disease-related variants (likely pathogenic or VUS) were present in 57 (38%) of the 151 SCD subjects. All variants were heterozygous. Likely pathogenic variants were present in 15 (10%) subjects. Seventy-six non-synonymous variants with MAF < 0.01 in SCD victims were classified as likely benign. There was no difference in prevalence of associated genetic variants between hypertension-associated versus obesity-associated SCD victims (40% vs 36% respectively; p = 0.62). Most of the potentially disease related variants were in HCM, ACM and DCM-related genes. SCD victims with likely pathogenic or uncertain variants had lower mean BMI (30.3 ± 5.9 kg/m 2 vs. 33.4 ± 7.2 kg/m 2 ; p = 0.003) and heart weight (528 ± 104 g vs. 557 ± 97 g; p = 0.05) than those without variants, but there was no statistically significant difference in the prevalence of patchy/diffuse myocardial fibrosis. Characteristics of SCD victims are presented in Table 2.
Likely pathogenic variants in SCD victims. One 19 ). Two variants were also present in PMF subjects in our previous study (p.Met982Thr in MYH7 and p.Ala372Pro in PKP2) 10 . Also, p.Thr78Met in conserved residue of CAV3 is previously associated with functional channelopathy 20 . Two subjects carried the p.Thr78Met variant and another had a pre-mortem ECG with normal QT interval, normal QRS duration, and no Brugada pattern in right precordial leads. One subject carried p.Gln168His variant in LMNA, which has no population frequency in Finland, was predicted to be damaging by in silico algorithm and located close to previously established pathogenic LMNA variant p.Arg166Pro.

Variants of uncertain significance in SCD victims. Forty-nine (32%) SCD victims carried uncertain
variants. These variants were considered as potentially disease-associated, rather than as likely benign due to previous reports in cardiomyopathy patients, location in a mutational hot spot, and/or prediction to be damaging by in silico algorithms. Uncertain variants were predominantly missense by type but one was predicted to have effect on canonical splicing (c.11254 + 2T>C in TTN). We classified this variant as VUS because loss of function variants in TTN gene are relatively common in general population (1-2%), and disease causing variants locate typically in the A-band or in highly expressed exon whereas our variant did not 21  Control subjects. Two control subjects with hypertension and hypertrophic heart (4%) carried likely pathogenic variants; Gly853Argfs (c.2555dupT) in MYBPC3, and Arg219Ter (c.566C>T) in LDB3. These variants were classified as likely pathogenic due to their absence from, or a very low frequency in, the general population and predicted loss of function effect. Additional 12 (25%) control subjects carried uncertain variants, which are presented in Supplementary Table 2. One subject carried two uncertain variants. The differences in the prevalence of likely pathogenic variants or VUSs were not statistically significant between SCD victims and control subjects (Fig. 2).

Discussion
Our systematic collection of almost 6000 consecutive autopsied SCD victims have shown that non-ischemic causes of SCD are not limited to young victims and monogenic etiologies, but constitutes also notable burden of SCD cases in victims over 40 years with acquired etiologies. In this study, 10% of SCD victims with presumably acquired non-ischemic cardiac hypertrophy at autopsy harbored likely disease-related rare genetic variants, and additional 28% carried VUSs. Among 48 control subjects with hypertension-associated LVH over 20 years of follow-up, two carried likely pathogenic variant (4%), and additional 25% carried VUSs. Although the prevalence of likely disease-causing variants was less than one-half of that observed in the SCD victims, the difference was not statistically significant. Of interest, observed likely pathogenic variants in control subjects may also have an effect on the cardiac hypertrophy. Previous studies investigating the genetic background of SCD due to non-specific structural cardiac diseases have reported conflicting results. Recent study from Lahrouchi et al. reported likely pathogenic variant in 1/29 SCD victim with autopsy findings of uncertain significance, whereas Hertz et al. reported rare variants with likely functional effects in 29% of SCD victims with non-diagnostic structural changes at autopsy 22,23 . Variable results may be explainable by different MAF thresholds for variant analysis. In comparison to aforementioned studies, our study subjects were not restricted to young SCD victims and had hypertension/obesity related myocardial www.nature.com/scientificreports/ disease at autopsy. Although our study subjects were substantially younger than general SCD patients with CAD, they were on average older than those dying suddenly associate with a typical HCM phenotype 24 . SCD victims with possibly disease related variants had lower mean BMI and heart weight in comparison to those without. Lower BMI is understandable by the view that high mismatch between acquired factors and the magnitude of cardiac disease may denote genetic predisposition in the disease development. Reasons for lower heart weight are not that obvious. Taken into notice that there was no notable difference in the prevalence of substantial myocardial fibrosis at autopsy, lower heart weight in subjects with relevant variants may represent higher fibrosis/hypertrophy-ratio. This may be in concordant with previous studies which have suggested that patients with genetic predisposition to LVH (HCM) have more myocardial fibrosis that those with hypertensive LVH 25,26 .
Many potentially disease related variants in our study had conflicting interpretations of their pathogenicity in ClinVar. These variants have properties that support pathogenic nature (e.g. descriptions in disease patients, in silico predictions, conservative residues) and their conflicting interpretation is mostly due to relatively high MAF, which is inconsistent with inherited cardiomyopathies, and non-definitive causal association of suspected genes with HCM, DCM or ACM. However, our SCD victims did not present with these cardiomyopathies, but rather with acquired myocardial diseases that had common structural abnormalities. Inherited cardiomyopathies are common in young SCD victims, but the overall incidence of SCD in young people is low. As seen in the Fingesture study, inherited cardiomyopathies only account for a minority of SCD cases due to non-ischemic myocardial diseases in general population (Fig. 1). Moreover, the genetic underpinnings of non-ischemic SCDs are probably much more diverse than the very rare highly pathogenic variants that cause Mendelian diseases.
Altogether, we assume that the variants in our study may not be the main cause of myocardial diseases, but rather a contributing factor in primarily acquired diseases. This hypothesis is supported by previous studies which have shown that family history of sudden death is a significant risk factor for sudden death also in middle-age and beyond, although cardiac diseases leading to SCD in this age group are primarily considered to be acquired 27,28 . According to a recent study from Khera et al., very rare disease-causing variants are associated with only 2.5% of SCDs in general population 29 , which is unlikely to explain the inherited predisposition to SCDs in general population alone. The concept of disease-modifying polymorphisms has gained attention as an explanation to the variable SCD risk in patients with the same cardiac disease. Evidence for this has been published among patients with long QT syndrome 30 . To our knowledge, the present study is the first to investigate the role of rare genetic variants in SCDs that are due to acquired non-ischemic myocardial diseases. Although the difference in the prevalence of suspected variants was not statistically significant, disease-modifying rare gene variants in acquired myocardial diseases is potentially an important field for further research, especially because such cardiac diseases may be preventable with appropriate pharmacological treatments and lifestyle interventions.

Limitations
First limitation is the absence of evidence regarding the co-segregation between the variants and the disease among first-degree family members. It is unreasonable to establish causality between variants and the disease solely based on the NGS study and current data regarding variant pathogenicity is incomplete. Our NGS sequencing method was neither able to detect copy number variation. Also, detection of myocardial fibrosis was based on subjective evaluation of macroscopic dissection and histological samples taken from 3 to 5 sections of the heart, which may not always be sufficient to detect scattered mild fibrosis. Although the Fingesture study includes the www.nature.com/scientificreports/ highest number of autopsy confirmed SCD cases in the world to our knowledge, we cannot be fully assured in every case that the SCD was actually due to the disease. Our study population was too small to generate statistically significant results, and further studies are needed to assess more precisely the impact of rare genetic variants on the occurrence of SCD among patients with hypertension and/or obesity related hypertrophic heart disease.

Conclusions
Potentially disease-related rare variants in myocardial structure encoding genes are common among nonischemic SCD victims with hypertension and/or obesity related hypertrophy and fibrosis at autopsy. Variants were mainly observed in genes related to HCM, ACM and DCM. Subjects with potentially disease-related variants had lower mean BMI and heart weight without significant difference in myocardial fibrosis. Taken into notice that such variants were also present in a small number of subjects with hypertension and cardiac hypertrophy, the results require further investigation as genetic modification may be responsible for the pattern of disease observed at autopsy in subjects with LVH associated with hypertension or obesity. The clinical relevance of this hypothesis lies in the importance of post-mortem investigations and in the guidance and treatment of genetically vulnerable subjects and their family members.

Data availability
All data generated or analysed during this study are included in this published article and its Supplementary Information files.