Introduction
DCM is characterized as left ventricular (LV) or biventricular systolic dysfunction and dilatation, and there are no abnormal loading conditions or coronary artery disease [
1]. The frequency of the disease varies from 1:250 to 1:2.500 and is the most common cause of heart failure (HF) in the young and the leading cause of heart transplantation worldwide [
2]. The disease progression is related to various factors, such as toxic damage, delayed initiation of medical treatment, and the presence of an adverse genetic background [
3]. Genetic evaluation has become an important integral part of patient care in cardiomyopathies, and can help predict prognosis. There are many genes and alleles related to DCM, so detailed genetic testing encompasses ever-increasing gene panels [
4]. The most detected mutations in DCM are truncating variants in
TTN [
4,
5]. However, the spectrum of genes related to DCM is broad, and our knowledge of the natural history of genetic DCM is poor [
6]. Failure to find genetic variants does not necessarily mean that there is no genetic etiology to the DCM [
7]. Most diagnostic panels contain a variety of genes whose contribution to DCM is not entirely clear. Therefore, newly notified genes have uncertain molecular or clinical significance [
8]. The effort to determine a relationship between gene variants of unknown significance and a phenotype of the disease is important not only for cardiovascular research but also for the clinical genetics of patients and their families [
9]. Cardiac MRI and echocardiography are the most used imaging methods in the diagnosis of DCM. However, the phenotype of DCM can be diverse, that is why the evaluation of the whole-heart myocardial mechanics and morphometrics is important.
In this work, we aim to evaluate whether the various variants in cardiac-related genes are associated with changes in the phenotype of the whole-heart myocardial mechanics or morphometrics in patients with nonischemic dilated cardiomyopathy (NIDCM).
Discussion
Our research findings have unveiled potential links between variations in specific cardiac genes and cardiac function or morphometrics parameters among individuals with DCM. It was revealed that five genes appear to have an impact on myocardial mechanics and morphometrics in an indirect manner and have not been previously linked to DCM. These findings differ from the conventional Genome-wide Association Studies (GWAS), making direct comparisons with other research challenging.
We did not observe significant associations with classical cardiomyopathy genes. This is particularly unexpected considering that variants in genes encoding sarcomere proteins would seemingly exert the greatest influence on the clinical progression of the condition. Most of these genes, such as TTN gene, are rather large and accumulated a substantial amount of mutations during evolution, mostly in non-conserved regions. It is plausible that these mutations elicit minimal adverse effects.
Moreover, LOX, GATAD1, and RASA1 genes variants were related to better cardiac function and morphometrics parameters, which might be unexpected because mutations with worse clinical outcomes should be less common in the population. It might be that these variants alone do not influence disease development enough to be selective in human evolution.
Furthermore, lysyl oxidase (LOX) is a protein that includes a family of five copper-dependent enzymes (LOX and four LOX-like isoenzymes (LOXL1–4)) essential for extracellular matrix homeostasis and remodeling. LOX and LOXLs isoenzymes play an important role in the control of vascular homeostasis, remodeling, control of vascular stiffness, oxidative stress, and calcification [
16] as well as for the biogenesis of connective tissue matrices [
17]. Experimental models have described evidence of the disturbance of LOX/LOXLs activity and cardiovascular diseases [
18,
19]. Knock-out animal models have shown a relationship between changes in LOX gene and human aortic aneurysms and dissection [
20]. Moreover, LOX family proteins are associated with myocardial stiffness and disturbed LV function. LOX has been observed to be involved in the fibrosis that leads to end-stage DCM. Upregulation of LOX may lead to an imbalance of extracellular matrix degradation and synthesis, so it may participate in DCM and HF remodeling [
21]. Variants that decrease this upregulation might be protective. In our study 34 patients had predicted benign variant rs1800449 with frequency in gnomAD of 0,17. Previously, the variant was studied numerous times in association with various cancers and only once in association with cardiovascular disease—higher prevalence for ischaemic heart disease [
22]. Other variant was rs368947781 in only 2 patients (gnomAD 0,0001), no studies reported in the literature. Our results revealed the association of the
LOX gene variants with lesser myocardial damage. Variants in the
LOX gene were related to smaller ventricles volumes evaluated by 2D echocardiography or MRI. BNP concentration was also lower in cases with
LOX gene variants.
Our study results showed that the existence of the
GATAD1 gene was related to the better function of both ventricles and smaller dimensions of LV and LA. BNP concentration was also lower in patients with this gene. The
GATAD1 was first described as an ocular development-associated gene in 2002 [
23], which, as the name suggests, was studied in the association of eyes development, and in 2011 has been reported in one study as a possible cause for autosomal recessive DCM [
24]. Zebrafish knockout models developed phenotypes similar to HF in aged models or after induced stress [
25]. In our study, we found four different
GATAD1 gene variants, all reported in ClinVar as benign: rs10281879 (gnomAD 0,11;
n = 21, 2 homozygous cases) missense variant G54S reported in various associations; rs564747350 (gnomAD 0,0001;
n = 1) missense variant A202T with no reports in the literature; rs34768413 (gnomAD 0,017;
n = 2) missense variant R233W; rs139637606 (gnomAD 0,003;
n = 2). The function of this gene and its product is not well studied, so it is difficult to speculate about the implication of this gene in the development or modification of DCM.
RASA1 gene is described as associated with vascular malformation syndromes such as Klippel-Trenaunay-Weber syndrome, Sturgeon-Weber syndrome, vein of Galen aneurysmal malformation, etc.
RASA1 is a cytoplasm protein transported to the cell membrane upon increased intracellular Ca2 + concentrations. It participates in cell growth, proliferation, differentiation, and apoptosis. Naturally, this gene is extensively investigated in cancer development [
26]. Haploinsufficiency of
RASA1 increases in RAS-MAPK pathway signaling. The same pathway activation is a known mechanism for various RASopathies like Noonan, Costello, and other syndromes. These syndromes have a broad phenotypic spectrum, and the common manifestation is hypertrophic cardiomyopathy [
27]. The number of published cases is relatively small, so the wider phenotypic spectrum caused by variants in the
RASA1 gene remains unknown [
28]. In our study, the
RASA1 gene was related to the better function and smaller size of both ventricles and better function of atria. We found seven different variants across the
RASA1 gene. The rs111840875 (Ala99Val; gnomAD 0,03;
n = 7) has several records in ClinVar as benign and no literature reports in cardiovascular association. One record in ClinVar as a variant of unknown significance, and one report in literature in the case of capillary malformation-arteriovenous malformation [
28] were detected with variant rs373892264 (Gly156Val; gnomAD 0,03 < 0,01;
n = 1). Another variant was rs60835975 (gnomAD 0,03;
n = 10 cases), and one of them is in a homozygous state. The T nucleotide deletion variant is in the homopolymer region just before 11 exons with reports in ClinVar as benign. Other 4 variants are related to homopolymer region just before 14 exon: variant rs377722838 (gnomAD 0,12;
n = 22) and single cases of rs75512926 (gnomAD 0,02;
n = 1); rs747412034 (gnomAD 0,003;
n = 1) and 7 cases rs36000817 (gnomAD 0,064;
n = 7) with reports in ClinVar as bening and no reports in literature. These variants before 14 exons might be errors due to alignment, as these variants do not repeat in the same cases and are the same variation at the homopolymeric region. More studies are needed to evaluate the association of this gene with DCM.
The Ras family of small G proteins is composed of enzymes that hydrolyze GTP into GDP and is an important component of intracellular signal transduction. Three main human Ras genes are known: H-Ras, K-Ras, and N-Ras. Only a few studies have investigated the role of K-Ras in the heart, and it is related to cardiac cell proliferation [
29]. No previous data report
KRAS gene relationships with DCM pathogenesis. A lot of genes are offered from multiple commercial testing laboratories for the evaluation of DCM. However,
KRAS is one of the genes that activates the previously mentioned RAS-MAPK pathway and plays an important role in syndromic cardiomyopathy, such as Noonan syndrome, neuromuscular disease, and mitochondrial myopathies [
4]. In the
KRAS gene, we found a single variant rs1137282 c.519T > C, Asp173 = (gnomAD 0,19;
n = 28). This variant is often reported as benign in ClinVar and is extensively studied in cancer development. One study shows that this variant might increase the gene’s expressivity [
30], but it was tested in specific ethnic cell lines and might not be applicable in the European population. In our study, the presence of the
KRAS gene variants was associated with worse ventricular or atria function or dilatation of cardiac chambers.
KRIT1 gene germline pathogenic deleterious variants are causative for cerebral cavernous malformations [
31]. This gene belongs to the Ras family and regulates endothelial cell junction integrity, stabilizes cell-to-cell junctions, and participates in cell adhesion and migration. The loss of its function leads to increased beta-catenin signaling and abnormal vascular development [
32]. It is not clear whether variants of this gene could affect the manifestation of cardiomyopathies. However, we found 4 synonymous variants across our cases: rs149437256 (gnomAD 0,005;
n = 1); rs11542682 (gnomAD 0,09;
n = 24); rs143710815 (gnomAD 0,0037;
n = 1); rs200684252 (gnomAD < 0,001;
n = 1). All of them are recorded in ClinVar as benign. As in the case of the
KRAS gene, the presence of the
KRIT1 gene was associated with the enlargement of both ventricles.
Our findings are difficult to compare to other studies because we attempted to analyze all gene variants at simultaneously. Related studies with larger sample sizes can pick up single variants as better predictors for DCM. GWAS with 2719 cases found variants in
SLC6A6,
BAG3, and
HSPB7 genes [
33]. Other studies found different genes, such as
HCG22,
ZBTB17,
FRMD4B,
USP3,
TTN,
SLC39A8,
MLIP,
FLNC,
ALPK3, and
FHOD3 [
34‐
36]. Many of these studies use a p-value of 5*10
− 8, which allows us to find a stronger linkage, but many studies have different results, only repeating genes are
HCG22, BAG3, and
HSPB7. Applying new techniques, such as long-read sequences, allows alignment to more complete human genomes [
37] and having more uniform biobanks with control cases will allow us to better understand the heterogeneity of DCM.
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