Next Article in Journal
Molecular Signaling Effects behind the Spontaneous Soleus Muscle Activity Induced by 7-Day Rat Hindlimb Suspension
Previous Article in Journal
Advancements of CRISPR-Mediated Base Editing in Crops and Potential Applications in Populus
Previous Article in Special Issue
Antioxidants and azd0156 Rescue Inflammatory Response in Autophagy-Impaired Macrophages
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 15
10.3390/ijms25158315
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification of Variants of Uncertain Significance in the Genes Associated with Thoracic Aortic Disease in Russian Patients with Nonsyndromic Sporadic Subtypes of the Disorder

by
Irina A. Goncharova
1,
Sofia A. Shipulina
1,
Aleksei A. Sleptcov
1,
Aleksei A. Zarubin
1,
Nail R. Valiakhmetov
1,
Dmitry S. Panfilov
2,
Evgeniya V. Lelik
2,
Viktor V. Saushkin
2,
Boris N. Kozlov
2,
Ludmila P. Nazarenko
1 and
Maria S. Nazarenko
1,*
1
Research Institute of Medical Genetics, Tomsk National Research Medical Center, Russian Academy of Sciences, 10 Ushaika Str., Tomsk 634050, Russia
2
Cardiology Research Institute, Tomsk National Research Medical Center, Russian Academy of Sciences, 111a Kievskaya Str., Tomsk 634012, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(15), 8315; https://doi.org/10.3390/ijms25158315
Submission received: 20 June 2024 / Revised: 22 July 2024 / Accepted: 26 July 2024 / Published: 30 July 2024
(This article belongs to the Special Issue Molecular Mechanisms, Pathogenesis, and Novel Therapeutic Strategies in Cardiovascular Disease)
Browse Figures
Versions Notes

Abstract

:
Nonsyndromic sporadic thoracic aortic aneurysm (nssTAA) is characterized by diverse genetic variants that may vary in different populations. Our aim was to identify clinically relevant variants in genes implicated in hereditary aneurysms in Russian patients with nssTAA. Forty-one patients with nssTAA without dissection were analyzed. Using massive parallel sequencing, we searched for variants in exons of 53 known disease-causing genes. Patients were found to have no (likely) pathogenic variants in the genes of hereditary TAA. Six variants of uncertain significance (VUSs) were identified in four (9.8%) patients. Three VUSs [FBN1 c.7841C>T (p.Ala2614Val), COL3A1 c.2498A>T (p.Lys833Ile), and MYH11 c.4993C>T (p.Arg1665Cys)] are located in genes with “definitive” disease association (ClinGen). The remaining variants are in “potentially diagnostic” genes or genes with experimental evidence of disease association [NOTCH1 c.964G>A (p.Val322Met), COL4A5 c.953C>G (p.Pro318Arg), and PLOD3 c.833G>A (p.Gly278Asp)]. Russian patients with nssTAA without dissection examined in this study have ≥1 VUSs in six known genes of hereditary TAA (FBN1, COL3A1, MYH11, NOTCH1, COL4A5, or PLOD3). Experimental studies expanded genetic testing, and clinical examination of patients and first/second-degree relatives may shift VUSs to the pathogenic (benign) category or to a new class of rare “predisposing” low-penetrance variants causing the pathology if combined with other risk factors.

Graphical Abstract

1. Introduction

It is known that thoracic aortic aneurysm/dissection (TAAD) has a significant genetic component, with 20% of patients having a genetic cause of the disease [1]. The remaining 80% of cases have neither a family history of aortic disease nor causal pathogenic variants in the genes implicated in hereditary aneurysms and are considered sporadic subtypes of the disorder. The sporadic subtype is frequently defined as a multifactorial pathology because, along with genetic causes, it is characterized by the presence of a wide range of risk factors [2]. This subtype is characterized by high heterogeneity involving diverse genetic variants: from very rare with high penetrance to variants common in the population that were identified in genome-wide association studies (GWASs) and posing a lower risk of the disease [1,2,3,4].
Studies on nonsyndromic sporadic TAAD (nssTAAD) have shown the presence of rare pathogenic variants and common single-nucleotide polymorphisms (SNPs) in genes associated with Marfan syndrome (FBN1) and Loeys–Dietz syndrome (TGFBR1 or TGFBR2) as well as rare copy number variants (CNVs) affecting MYH11, ELN, or TGFB2, whose mutations correlate with familial types of TAAD [1,5,6,7,8]. In patients with sporadic thoracic aortic dissection, rare pathogenic variants and CNVs have been identified in hereditary-aneurysm genes, including FBN1, MYH11, EFEMP2, TGFBR2, FBN2, COL3A1, and MYLK, and 28% of the patients have >1 variant of unknown significance (VUS) in the genes of hereditary aneurysms; this occurrence is significantly higher than in a control group [9,10,11].
A substantial number of molecular genetic studies combine TAAD together into one group because dissection is considered a natural consequence of an aneurysm [2,3,12,13,14,15,16]. Nevertheless, it has been reported that 60% of patients with type A dissection (according to the Stanford classification) have a diameter of <5.5 cm, and type B dissections occur without significant dilatation of the aorta and are thus suggestive of a possibly different molecular pathogenesis of aneurysm and dissection of the thoracic aorta [2,4]. Studies specifically examining ascending aortic aneurysm without dissection are rare [17].
There are several guidelines for the management of patients with syndromic or familial forms of TAAD, including genetic testing for mutations in certain genes [18,19]. At the same time, patients with nssTAAD have also clinically relevant variants in the genes of hereditary aneurysms, and some relatives of patients with aortic aneurysm have pathological changes in thoracic aorta images that can be discovered in a long-term (more than 10 years) follow-up. For example, in “sporadic” patients, this sign may indicate a de novo mutation, which subsequently may be classified as the etiology. Hence, guidelines for the management of patients with nssTAAD are being developed actively. These guidelines include recommendations for thoracic aorta visualization and genetic testing not only for patients but also for first- and second-degree relatives [2,20].
Currently, massive parallel sequencing is the most common technology for molecular genetic testing of patients with TAAD. Targeted panels for TAAD diagnostics include different numbers of genes for hereditary aneurysms in different ethnic groups [20,21,22,23]. In the Russian Federation, very few genetic studies, such as an analysis of specific exons of genes ACTA2, NOTCH1, and FBN1 in patients with Marfan syndrome, have been performed on patients with thoracic aortic aneurysm [24,25,26].
There are also few articles that describe molecular genetic testing of patients with TAAD by exome/clinical exome sequencing [27,28,29]. The results indicate that exome sequencing is an effective approach to the identification of a genetic cause of TAAD and can be used in genetic counseling of patients and their families. Moreover, 10% of patients with syndromic and nonsyndromic thoracic aortic aneurysms share some mutations [20].
Genetic studies often combine familial and sporadic cases as well as aneurysms and aortic dissections; however, it is possible that each category has a unique spectrum of clinically significant variants. Besides, it is probable that there is an ethnic specificity for mutations in the genes of hereditary aneurysms [30].
Thus, the purpose of this study was to identify by massively parallel sequencing clinically relevant genetic variants in the genes of hereditary aneurysms in Russian patients with nssTAA without dissection.

2. Results

The study involved 41 Russian patients with sporadic ascending aortic nondissecting aneurysm. None of the patients were diagnosed with syndromic subtypes or had a family history of aortic dilatation or congenital connective tissue dysplasia. The majority of patients were males (65.8%) (Table 1).
The average age of the study group was 54 (48; 64) years and did not differ significantly between male and female participants (p = 0.559). The majority of patients (61.0%) had a bicuspid aortic valve. Arterial hypertension (64.3%), obesity (42.9%), hyperlipidemia (23.8%) and type 2 diabetes mellitus (T2DM) (7.1%) were also found in the study group. The prevalence of these risk factors did not differ between the male and female patients (Table 1). Coronary artery disease (CAD), angina pectoris, or myocardial infarction were found in 30.9% of patients and were more prevalent among males (p = 0.039). Atherosclerosis of coronary, carotid, renal or femoral arteries was detected in 50% of the patients and was represented mostly by coronary atherosclerosis (more often observed in males) (Table 1).
By computed tomography, we assessed the degree of aortic wall and coronary artery (CA) calcification in 26 patients with subsequent calculation of the Agatston index [31]. The average Ca-score in the group was as follows: CA, 71.2; aorta, 107. The Ca-score was higher in males than in females, although the difference was not statistically significant (Table 1).
As a result of clinical exome sequencing and identification of rare genetic variants in 53 genes of syndromic and hereditary TAA subtypes in Russian patients with nssTAA without dissection, no pathogenic or likely pathogenic variants were identified according to the American College of Medical Genetics (ACMG) classification. The 45 identified variants were classified as follows: 34 benign, 5 likely benign, and 6 VUSs (Supplementary Table S1).
Because no pathogenic or likely pathogenic variants were found in the study population, we provide a more detailed description of these VUSs (and their carriers) (Table 2). These variants may be associated with the pathology in question, but at present, this cause–effect relation is not proven. A detailed description of patients who are not VUS carriers is presented in Supplementary Table S3.
All identified VUSs are missense variants and were detected in genes of different categories according to the classification based on the strength of association with hereditary thoracic aortic aneurysms (HTAA) [13]: genes FBN1, COL3A1, and MYH11 are in the first category (A) with a “definitive” effect of pathogenic variant on the pathology; NOTCH1 belongs to the second category (B): “potentially diagnostic” genes that may be the cause of thoracic aortic dilatation but mostly correlate with other clinical signs; COL4A5 is affiliated with the third category (C): genes with limited evidence of an effect on HTAA; and PLOD3 is in the fourth category (D): genes associated with diseases of the thoracic aorta according to experimental studies with no clinical evidence of their involvement (Table 2).
Patient #54 (male, 48 years old, diameter of the mid-ascending aorta = 50 mm) was found to have three VUSs (in FBN1, COL3A1, and PLOD3 genes; Table 2; Figure 1). Among the risk factors for TAAD and other cardiovascular diseases (CVDs), the patient received a diagnosis of arterial hypertension (Table 2). Additionally, he had a diagnosis of urolithiasis with microlith formation in the renal cavitary system of both kidneys.
FBN1 (NC_000015.10) c.7841C>T (p.Ala2614Val) has never been previously identified in patients with TAAD. Potential pathogenicity or deleterious score of the variant was examined based on predictions of several in silico tools available at VarSome and CADD (PHRED; Table 2). Only the MutPred predictor available at VarSome assessed this variant as pathogenic, while nine other prediction scales assessed FBN1 c.7841C>T as a VUS. Fifteen in silico tools available at VarSome evaluated FBN1 c.7841C>T as benign. According to the CADD (PHRED) scale, this variant is pathogenic (23.6). Another variant—FBN1 c.7841C>A p.(Ala2614Asp)—has been described previously at the same position of the gene (chr15:48415746) and was also classified as a VUS according to the VarSome pathogenicity scale (there is no information on allele frequency and disease association).
The second VUS, COL3A1 (NC_000002.12) c.2498A>T (p.Lys833Ile), has not been described before, and its relation to this pathology is also unknown (Table 2). The VarSome pathogenicity prediction scale classifies this variant as pathogenic based on eight predictors (12: VUS, 3: benign). CADD (PHRED) also classified this variant as pathogenic (28.5). According to ClinVar, rs371344739 (COL3A1 c.2498A>G, p.Lys833Arg, ID 199701) has previously been described at this position (chr2:189003007). The frequency of this VUS is described for populations in gnomAD (1.1 × 10−4).
The third VUS, PLOD3 (NC_000007.14) c.833G>A (rs1041461490, p.Gly278Asp), is registered in gnomAD (6.8 × 10−6), RUSeq (5.9 × 10−4), and ClinVar (ID 1386463), but there is no information about its association with relevant pathologies (Table 2). CADD (PHRED) classifies this variant as pathogenic (25.6), although VarSome predictors classified this variant as pathogenic (11) or a VUS (10).
Patient #43 (male, 72 years old, diameter of the mid-ascending aorta = 69 mm) was found to have a VUS: MYH11 (NC_000016.10) c.4993C>T (rs768569707, p.Arg1665Cys) (Figure 2A). Along with nssTAA, this patient received a diagnosis of arterial hypertension, hyperlipidemia, atherosclerosis of coronary and carotid arteries, CAD, and chronic cerebral ischemia (Table 2). This patient had advanced coronary and aortic calcification (Agatston indices 423 and 937, respectively). Multicystic kidney, varicose veins, chronic gastritis, esophagitis, and osteochondrosis of the cervical spine were identified.
The frequency of the rs768569707 variant is 4.4 × 10−5 in gnomAD (Table 2). According to the VarSome pathogenicity prediction scale, this variant is pathogenic judging by 11 predictors (9: VUS and 1: benign); it is also categorized as pathogenic by the CADD (PHRED) scale (32.0). According to ClinVar, this variant (ID 405480) was previously reported in both patients with TAA and in children with left ventricular noncompaction, which is a hereditary type of cardiomyopathy [32].
Patient #13 (male, 58 years old, diameter of the mid-ascending aorta = 45 mm) has a VUS: COL4A5 (NC_000023.11) c.953C>G (chrX:108582900, rs1449979085, p.Pro318Arg) (Figure 2B). This patient received a diagnosis of aortic root aneurysm, bicuspid aortic valve, arterial hypertension, hyperlipidemia, and obesity (Table 2). This patient also had stage 3b chronic kidney disease (glomerular filtration rate = 52 mL/min/1.73 m2).
The frequency of rs1449979085 variant is 8.3 × 10−6 among all populations in gnomAD (Table 2). According to VarSome, this variant is pathogenic based on three predictors (4: VUS, 16: benign); according to CADD (PHRED), this variant is closer to pathogenic (16.9). The VUS identified in patient #13 is described in ClinVar (ID 2485585), but information about its association with relevant pathologies, including TAA, is absent in the literature and publicly available databases.
VUS NOTCH1 (NC_000009.12) c.964G>A (p.Val322Met) was identified in patient #36 (female, 67 years old, diameter of the mid-ascending aorta = 62 mm) (Figure 2C). She also received a diagnosis of dilation of the descending aorta, hypertension, paroxysmal tachycardia, CAD, carotid atherosclerosis, and chronic cerebral ischemia (Table 2). Low calcification of coronary arteries (Agatston index 49) and high calcification of the aorta (Agatston index 237) were detected.
Variant c.964G>A NOTCH1 was detected with a frequency of 2.1 × 10−6 in gnomAD and is not registered in RUSeq population databases (Table 2). Among VarSome pathogenicity prediction scales, eight predictors classify this variant as pathogenic (10: VUS, 1: benign). Judging by CADD (PHRED), this variant is pathogenic (25.5). Previously, another VUS (c.964C>G, rs1317299348, p.Val322Leu) has been described in the NCBI database for these genome coordinates (chr9:136518726); however, there is no evidence that this variant is associated with a relevant disease. For NOTCH1 c.964G>A, identified in the present study, there is no information about an association with any pathology either, including TAAD.

3. Discussion

In the current study, we identified variants of uncertain significance in Russian patients with nonsyndromic sporadic ascending aortic nondissecting aneurysm by massive parallel sequencing of genes associated with TAAD.
No pathogenic or likely pathogenic variants were found in the genes of thoracic aortic aneurysms. Nonetheless, we identified six VUSs. Of these, three VUSs [FBN1 c.7841C>T (p.Ala2614Val), COL3A1 c.2498A>T (p.Lys833Ile), and MYH11 c.4993C>T (p.Arg1665Cys)] are in genes with a “definitive” effect of pathogenic variants on the pathology in question. Pathogenic variants in the FBN1 gene are the main cause of Marfan syndrome (OMIM: 154700). Pathogenic variants in COL3A1 are associated with vascular-type Ehlers–Danlos syndrome and with aneurysms of the aorta and other arteries (OMIM: 130050) [33]. Pathogenic variants in MYH11 have been described in individuals having TAAD with patent ductus arteriosus (PDA) [34].
The remaining VUSs [NOTCH1 c.964G>A (p.Val322Met), COL4A5 c.953C>G (p.Pro318Arg), and PLOD3 c.833G>A (p.Gly278Asp)] belong to genes with less evidence of the impact on TAAD. Nevertheless, pathogenic variants in the COL4A5 gene can cause Alport syndrome (OMIM: 301050): a condition characterized by kidney disease, hearing loss, eye anomalies, and TAAD [35]. To date, data have been accumulating on the associations of PLOD3 variants with connective tissue disease and its vascular complications as well as familial and sporadic vascular aneurysms in some populations [11,36,37]. It was demonstrated recently that NOTCH1 mutations are associated with nssTAA in the absence of the bicuspid aortic valve [38].
The protein products of genes containing a VUS in patients with nssTAA without dissection are mainly structural components of the extracellular matrix (FBN1, COL3A1, and COL4A5). The other genes encode a major contractile protein of smooth muscle cells (MYH11), a transmembrane receptor of Notch signaling (NOTCH1), and an enzyme responsible for the stability of intermolecular collagen crosslinks (PLOD3). Of note, we did not find disease-causing variants in genes of the TGF-β signaling pathway, which plays a major role in TAAD pathogenesis [14]. This may in part be explained by our small sample size.
In our study, 9.8% of patients with nssTAA without dissection had ≥1 VUSs. Our results are in line with previous papers about mixed groups of TAAD patients (syndromic, familial, and sporadic cases) [3,21]. Nonetheless, the proportion of individuals with at least one observed VUS has been reported to be the highest (28%) in a subgroup of sporadic aortic dissection [9]. Moreover, in our work, all the identified VUSs were missense variants, consistent with a report on French patients with nonsyndromic thoracic aortic aneurysms and dissections, where the disease was associated with only missense variants in the FBN1 gene [3].
Confirmation of a VUS’s relevance to sporadic ascending aortic aneurysm requires not only experimental studies but also clinical examination and genetic testing of first/second-degree relatives, especially for the TAAD cases with concomitant mutations in other genes. Further investigation is needed to address the family verification of the pathogenicity of these variants.
As new research results emerge confirming functional significance of genetic variants, pathogenicity criteria undergo changes, and variants may shift from one category to another [39]. On the other hand, some experimental research indicates that even VUSs that do not change their pathogenicity status with time can increase the risk of thoracic aortic aneurysm if combined with other genetic and nongenetic risk factors [40]. Therefore, we should keep patients with a VUS under observation including periodic imaging of the aorta starting from when the genetic variant was identified until the diagnosis is finally confirmed or refuted [19].
Genetic architecture of complex diseases (including nssTAA) is characterized by a combination of rare and common variants of genes. For some TAAD-causing genes, rare variants and common SNPs are associated not only with aortic aneurysm but also with other CVDs. For example, it has been shown that some variants of the NOTCH1 gene are associated with aortic valve calcification [41]. A deletion affecting the MYH11 gene has been identified in a family with pseudoxanthoma elasticum: a Mendelian disease featuring calcification of elastic fibers of the skin, arteries, and retina [42]. Common variants of the MYH11 gene are associated with CAD and high blood pressure [43], and SNPs in the FBN1 gene correlate with thoracic aortic aneurysm, coronary artery dissection, and blood pressure, according to the GWAS Catalog.
It is worth noting that in our study, patient #54 with three VUSs (in genes FBN1, COL3A1, and PLOD3) has only one CVD risk factor: arterial hypertension. VUSs in MYH11 and NOTCH1 were identified in two other patients here (#43 and #36), who have high calcification of coronary arteries and/or ascending aorta. Further investigation into both VUSs and analysis of common variants in the patients in combination with a deep clinical examination should help to identify relevant genotype–TAAD phenotype relations followed by the translation of these findings to clinical practice.
This study had several limitations. Firstly, the sample size was not large enough. Secondly, VUSs need further clinical family-based and functional validation. Thirdly, we analyzed only 53 genes associated with hereditary thoracic aortic aneurysm. However, there could be variants in other genes, that were not analyzed in this study. Exome or genome sequencing can help to overcome these shortcomings in a future study.
Despite the existing limitations of the study (small study group size), it may be reasonable to speculate that nssTAA without dissection shares the same molecular genetic mechanisms with syndromic and hereditary forms of TAAD. It has been shown that along with pathogenic and likely pathogenic variants, patients with sporadic aneurysm are characterized by accumulation of VUS, and GWAS studies have identified common variants in hereditary TAA and other genes [2,44].
Conclusion. The sporadic form is characterized by a unique genetic pattern, including different variants of a wide range of genes. Some of these variants may later be classified as pathogenic or benign, and some may be defined as a new class of rare “predisposing” low-penetrance variants, leading to the development of pathology in combination with other risk factors.
Future perspectives. To effectively reduce morbidity and mortality from thoracic aortic aneurysm/dissection in the future, it is necessary to identify this unique genetic pattern in patients with sporadic ascending aortic aneurysms. This task requires advanced genetic testing on an elaborated gene panel consisting of not only the 11 “causal” genes [2], but also genes that are assumed to be involved in hereditary and syndromic aneurysms. For some of these potential genes, their importance in the development of the pathology has not yet been confirmed by clinical studies. Confirmation of VUS significance in the development of sporadic ascending aortic aneurysm requires not only experimental studies, but also genetic testing of first/second-degree relatives. Moreover, we should provide clinical supervision for patients with potential causative variants including periodic imaging of the aorta starting from the moment when the genetic variant was identified until the diagnosis is finally confirmed or dismissed. Functional studies of VUSs, and population-level data with accurate phenotyping, will improve variant classification and reduce uncertainties for clinical implications of these variants.

4. Materials and Methods

4.1. Patient Characteristics

The study included patients with an aneurysm of the ascending aorta. All patients underwent open surgery at the Tomsk Cardiology Research Institute in the period from 2020 to 2023. All the candidates received a diagnosis of sporadic TAAD without any family history of aortic disease (their detailed characteristics are given in the Results Section 2). Patients were divided by sex (male/female) according to one assigned at birth.
Eligibility criteria were as follows: dilatation of the ascending aorta > 5.5 cm without aortic valve dysfunction or dilatation of the ascending aorta > 4.5 cm in the presence of severe aortic valve stenosis or insufficiency (grade 2+). Patients with aortic dissection/rupture, false aortic aneurysms, and patients requiring repeated interventions in the proximal thoracic aorta were excluded from the analysis.
Multidetector computed tomography (MDCT) was the basic method for diagnosis in patients with nssTAAD. The dimensions of the aorta were determined by MDCT perpendicular to the longitudinal axis, capturing the wall at several levels in accordance with national recommendations [45]. Image analysis and evaluation were performed independently by two experienced clinicians. MDCT of the aorta was performed on a 64-slice GE Discovery NM/CT 570C tomograph (GE Healthcare, Milwaukee, WI, USA) with the following parameters: 200–400 mA and 100–120 kW. Images were reconstructed with a slice thickness of 0.65 mm. A contrast agent was injected at a rate of 5 mL/s through the cubital vein by an automatic injector. The total volume of contrast was calculated on the basis of 1 mL per kilogram of body weight. To better visualize the aortic root and ascending part and to exclude artifacts, an ECG-synchronized analysis was performed from the bifurcation of the carotid arteries to the diaphragm. The resulting images were processed in Advantage Workstation 4.3 (GE Healthcare, USA).
Comorbidities were confirmed based on medical history and instrumental examination, including duplex ultrasonography of carotid, subclavian, vertebral, and femoral arteries; echocardiography; and coronary angiography. All patients underwent primary medical and genetic counseling, which included family history taking with pedigree construction, physical examination, and tests for congenital connective tissue dysplasia (assessed using Marfan and Beighton scores) [46,47].

4.2. Virtual Gene Panel Analysis

We searched for rare genetic variants (minor allele frequency < 1%) located in the exons of 53 genes associated with hereditary thoracic aortic aneurysm. The selection of genes was based on the data present in the ClinGen database (Appendix A) and in the literature (Supplementary Table S2) [10,13,17,29,48,49].

4.3. Clinical Exome Sequencing

DNA was isolated from peripheral blood leukocytes by phenol–chloroform extraction [50]. DNA was dissolved in TE Buffer, Tris-EDTA, 1 x Solution, pH 8.0. The quality and quantity of the DNA were checked by means of a Nanodrop 8000 spectrophotometer (Thermo, Waltham, MA, USA), a Qubit 3.0 fluorimeter (Thermo, USA), and a 1% LE agarose gel with 1× Tris-Acetate-EDTA (TAE) Buffer, pH 8.0.
DNA libraries were prepared using the Clinical Exome Solution Kit (Sophia Genetics, Lausanne, Switzerland), KAPA Library Amplification Kit KK2620 (Roche, Munich, Germany), and Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) according to the Clinical Exome Solution protocol (Sophia Genetics, Switzerland). The libraries were then quantified on a Qubit 3.0 fluorimeter (Thermo, Waltham, MA, USA) and a Bioanalyzer 2100 capillary electrophoresis system (Agilent, Santa Clara, CA, USA).
Sequencing was performed on the NextSeq 500 Illumina platform (Illumina, San Diego, CA, USA) with NextSeq 500/550 Mid Output Kit v.2 (300 cycles in 150 bp paired-end mode). Between 97% and 98.1% of the sequence reads were then successfully aligned to the reference genome (GRCh38) using Illumina DRAGEN Bio-IT. Exons of 53 target genes, representing 55% of the total exon length, had a minimum coverage of 30× in 79.3% (ranging between 77.87% and 80.4%) of cases, with an average coverage of 66.5×.
Variant annotation was performed in ANNOVAR. Classification of identified variants by degree of pathogenicity was carried out according to the American College of Medical Genetics (ACMG) standards and guidelines for the interpretation of sequence variants. For in silico pathogenicity prediction, we used VarSome, The Human Genomics Community tool (Appendix A). This is an automated classifier of genetic variants by degree of pathogenicity based on several in silico tools, including CADD, Polyphen2 HDIV, Polyphen2 HVAR, DEOGEN2, and PROVEAN. The frequency of VUS was determined using gnomAD (v4.1.0) and RUSeq Browser (Appendix A).

4.4. Sanger Sequencing

Validation of clinically relevant variants identified in genes FBN1, MYH11, COL3A1, COL4A5, NOTCH1, and PLOD3 was performed by bidirectional Sanger Sequence analysis on a 3730xl DNA Analyzer (Applied Biosystems, Norwalk, CT, USA). PCR amplification was performed using ready-to-use reaction mix HS-Taq PCR-Color (2×) (Biolabmix, Novosibirsk, Russia) with the primers specified in Table 3.
The specific primers were designed according to the hg38 genome assembly by means of the Primer Quest Tool and Primer Design and Search Tool (Appendix A).
The following reagents were used for purification of PCR products: thermolabile exonuclease Exo I (Thermo Scientific, Waltham, MA, USA), shrimp alkaline phosphatase (Thermo Scientific, USA), and deionized water.
The subsequent sequencing reaction was carried out using the BigDye Sequencing Kit v3.1 (Thermo Scientific, USA), 5× sequencing buffer, a forward or reverse primer, PCR product, and deionized water. Sequencing reaction products were purified with the D-Pure Reagent Kit (Nijmegen, The Netherlands). Sanger DNA sequencing result visualization and analysis were performed in the UGENE software (v42.0, Unipro, Moscow, Russia) [51].

4.5. Statistics

The normality of distribution was checked using the Shapiro–Wilk test. Group comparison was performed using non-parametric tests (Mann–Whitney). Results are presented as median, 1st and 3rd quartiles (Me (Q1; Q3)). For the analysis of qualitative parameters, Fisher’s exact test was applied. Using the PVR package in R, we also calculated the minimum effect size that can be detected at a power of 0.8. Differences were considered significant at p < 0.05.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25158315/s1.

Author Contributions

Conceptualization, I.A.G. and M.S.N.; methodology, I.A.G., A.A.S. and M.S.N.; software, A.A.Z.; data curation, I.A.G., S.A.S., D.S.P., E.V.L. and M.S.N.; formal analysis, I.A.G., S.A.S. and M.S.N.; investigation, I.A.G., S.A.S., N.R.V., A.A.S. and M.S.N.; writing—original draft, I.A.G.; writing—review and editing, I.A.G., M.S.N., S.A.S., A.A.Z., A.A.S., N.R.V., D.S.P., E.V.L., V.V.S., L.P.N. and B.N.K.; visualization, V.V.S., I.A.G., S.A.S., A.A.S. and M.S.N.; supervision, M.S.N.; funding acquisition, I.A.G., L.P.N. and M.S.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Russian Science Foundation, grant No. 22-25-00701, https://rscf.ru/en/project/22-25-00701/ (accessed on 29 July 2024).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP) and approved by the Local Biomedical Ethics Committee at the Research Institute of Medical Genetics, Tomsk National Research Medical Center (protocol #13, 15 November 2021) and by the Local Biomedical Ethics Committee at the Cardiology Research Institute of Tomsk National Research Medical Center (protocol #213, 12 May 2021).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Dataset available upon request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

References

  1. Cecchi, A.C.; Drake, M.; Campos, C.; Howitt, J.; Medina, J.; Damrauer, S.M.; Shalhub, S.; Milewicz, D.M. Aortic Dissection Collaborative. Current state and future directions of genomic medicine in aortic dissection: A path to prevention and personalized care. Semin. Vasc. Surg. 2022, 35, 51–59. [Google Scholar] [CrossRef] [PubMed]
  2. Milewicz, D.M.; Guo, D.; Hostetler, E.; Marin, I.; Pinard, A.C.; Cecchi, A.C. Update on the genetic risk for thoracic aortic aneurysms and acute aortic dissections: Implications for clinical care. J. Cardiovasc. Surg. 2021, 62, 203–210. [Google Scholar] [CrossRef] [PubMed]
  3. Arnaud, P.; Hanna, N.; Benarroch, L.; Aubart, M.; Bal, L.; Bouvagnet, P.; Busa, T.; Dulac, Y.; Dupuis-Girod, S.; Edouard, T.; et al. Genetic diversity and pathogenic variants as possible predictors of severity in a French sample of nonsyndromic heritable thoracic aortic aneurysms and dissections (nshTAAD). Genet. Med. 2019, 21, 2015–2024. [Google Scholar] [CrossRef] [PubMed]
  4. Zhou, Z.; Cecchi, A.C.; Prakash, S.K.; Milewicz, D.M. Risk factors for thoracic aortic dissection. Genes 2022, 13, 1814. [Google Scholar] [CrossRef] [PubMed]
  5. Prakash, S.K.; LeMaire, S.A.; Guo, D.C.; Russell, L.; Regalado, E.S.; Golabbakhsh, H.; Johnson, R.J.; Safi, H.J.; Estrera, A.L.; Coselli, J.S.; et al. Rare copy number variants disrupt genes regulating vascular smooth muscle cell adhesion and contractility in sporadic thoracic aortic aneurysms and dissections. Am. J. Hum. Genet. 2010, 87, 743–756. [Google Scholar] [CrossRef] [PubMed]
  6. Prakash, S.; Kuang, S.Q.; GenTAC Registry Investigators; Regalado, E.; Guo, D.; Milewicz, D. Recurrent Rare Genomic Copy Number Variants and Bicuspid Aortic Valve Are Enriched in Early Onset Thoracic Aortic Aneurysms and Dissections. PLoS ONE 2016, 11, e0153543. [Google Scholar] [CrossRef] [PubMed]
  7. LeMaire, S.A.; McDonald, M.L.; Guo, D.C.; Russell, L.; Miller, C.C.; Johnson, R.J.; Bekheirnia, M.R.; Franco, L.M.; Nguyen, M.; Pyeritz, R.E.; et al. Genome-wide association study identifies a susceptibility locus for thoracic aortic aneurysms and aortic dissections spanning FBN1 at 15q21.1. Nat. Genet. 2011, 43, 996–1000. [Google Scholar] [CrossRef]
  8. Meester, J.A.N.; Hebert, A.; Loeys, B.L. Structural genomic variants in thoracic aortic disease. Curr. Opin. Cardiol. 2023, 38, 157–161. [Google Scholar] [CrossRef] [PubMed]
  9. Guo, D.C.; Hostetler, E.M.; Fan, Y.; Kulmacz, R.J.; Zhang, D.; GenTAC Investigators; Nickerson, D.A.; Leal, S.M.; LeMaire, S.A.; Regalado, E.S.; et al. Heritable Thoracic Aortic Disease Genes in Sporadic Aortic Dissection. J. Am. Coll. Cardiol. 2017, 70, 2728–2730. [Google Scholar] [CrossRef] [PubMed]
  10. Wang, Z.; Zhuang, X.; Chen, B.; Wen, J.; Peng, F.; Liu, X.; Wei, M. 99-Case Study of Sporadic Aortic Dissection by Whole Exome Sequencing Indicated Novel Disease-Associated Genes and Variants in Chinese Population. Biomed. Res. Int. 2020, 2020, 7857043. [Google Scholar] [CrossRef]
  11. Chen, Z.R.; Bao, M.H.; Wang, X.Y.; Yang, Y.M.; Huang, B.; Han, Z.L.; Cai, J.; Fan, X.H. Genetic variants in Chinese patients with sporadic Stanford type A aortic dissection. J. Thorac. Dis. 2021, 13, 4008–4022. [Google Scholar] [CrossRef] [PubMed]
  12. Brownstein, A.J.; Kostiuk, V.; Ziganshin, B.A.; Zafar, M.A.; Kuivaniemi, H.; Body, S.C.; Bale, A.E.; Elefteriades, J.A. Genes Associated with Thoracic Aortic Aneurysm and Dissection: 2018 Update and Clinical Implications. Aorta 2018, 6, 13–20. [Google Scholar] [CrossRef] [PubMed]
  13. Renard, M.; Francis, C.; Ghosh, R.; Scott, A.F.; Witmer, P.D.; Adès, L.C.; Andelfinger, G.U.; Arnaud, P.; Boileau, C.; Callewaert, B.L.; et al. Clinical Validity of Genes for Heritable Thoracic Aortic Aneurysm and Dissection. J. Am. Coll. Cardiol. 2018, 72, 605–615. [Google Scholar] [CrossRef] [PubMed]
  14. Ostberg, N.P.; Zafar, M.A.; Ziganshin, B.A.; Elefteriades, J.A. The Genetics of Thoracic Aortic Aneurysms and Dissection: A Clinical Perspective. Biomolecules. 2020, 10, 182. [Google Scholar] [CrossRef] [PubMed]
  15. Guo, R.; Du, P.; Pei, Y.; Yang, J.; Li, S.; Chang, S.; Sun, H.; He, X.; Dong, J.; Zhou, J.; et al. Whole-Exome Sequencing Identified Genes Responsible for Thoracic Aortic Aneurysms and Dissections in three Chinese Families. Front. Genet. 2022, 13, 910932. [Google Scholar] [CrossRef]
  16. Krywanczyk, A.; Rodriguez, E.R.; Tan, C.D.; Gilson, T. Thoracic aortic aneurysm and dissection: Review and recommendations for evaluation. Am. J. Forensic Med. Pathol. 2023, 44, 69–76. [Google Scholar] [CrossRef] [PubMed]
  17. Li, Y.; Gao, S.; Han, Y.; Song, L.; Kong, Y.; Jiao, Y.; Huang, S.; Du, J.; Li, Y. Variants of Focal Adhesion Scaffold Genes Cause Thoracic Aortic Aneurysm. Circ. Res. 2021, 128, 8–23. [Google Scholar] [CrossRef] [PubMed]
  18. De Backer, J.; Bondue, A.; Budts, W.; Evangelista, A.; Gallego, P.; Jondeau, G.; Loeys, B.; Peña, M.L.; Teixido-Tura, G.; van de Laar, I.; et al. Genetic counselling and testing in adults with congenital heart disease: A consensus document of the ESC Working Group of Grown-Up Congenital Heart Disease, the ESC Working Group on Aorta and Peripheral Vascular Disease and the European Society of Human Genetics. Eur. J. Prev. Cardiol. 2020, 27, 1423–1435. [Google Scholar] [PubMed]
  19. Isselbacher, E.M.; Preventza, O.; Hamilton Black, J.; Augoustides, J.G.; Beck, A.W.; Bolen, M.A.; Braverman, A.C.; Bray, B.E.; Brown-Zimmerman, M.M.; Chen, E.P.; et al. 2022 ACC/AHA Guideline for the Diagnosis and Management of Aortic Disease: A Report of the American Heart Association/American College of Cardiology Joint Committee on Clinical Practice Guidelines. Circulation 2022, 146, e334–e482. [Google Scholar] [CrossRef] [PubMed]
  20. Mariscalco, G.; Debiec, R.; Elefteriades, J.A.; Samani, N.J.; Murphy, G.J. Systematic review of studies that have evaluated screening tests in relatives of patients affected by nonsyndromic thoracic aortic disease. J. Am. Heart Assoc. 2018, 7, e009302. [Google Scholar] [CrossRef] [PubMed]
  21. Weerakkody, R.; Ross, D.; Parry, D.A.; Ziganshin, B.; Vandrovcova, J.; Gampawar, P.; Abdullah, A.; Biggs, J.; Dumfarth, J.; Ibrahim, Y.; et al. Targeted genetic analysis in a large cohort of familial and sporadic cases of aneurysm or dissection of the thoracic aorta. Genet. Med. 2018, 20, 1414–1422. [Google Scholar] [CrossRef] [PubMed]
  22. Zheng, J.; Guo, J.; Huang, L.; Wu, Q.; Yin, K.; Wang, L.; Zhang, T.; Quan, L.; Zhao, Q.; Cheng, J. Genetic diagnosis of acute aortic dissection in South China Han population using next-generation sequencing. Int. J. Legal Med. 2018, 132, 1273–1280. [Google Scholar] [CrossRef] [PubMed]
  23. Duan, Y.; Xiong, J.; Lai, Z.; Zhong, Y.; Tian, C.; Du, Z.; Luo, Z.; Yu, J.; Li, W.; Xu, W.; et al. Analysis of the genetic contribution to thoracic aortic aneurysm or dissection in a prospective cohort of patients with familial and sporadic cases in East China. Orphanet J. Rare Dis. 2023, 18, 251. [Google Scholar] [CrossRef] [PubMed]
  24. Semyachkina, A.N.; Adyan, T.A.; Kharabadze, M.N.; Novikov, P.V.; Polyakov, A.V. Clinical and genetic characteristics of Russian Marfan patients. Russ. J. Genet. 2015, 51, 695–701. [Google Scholar] [CrossRef]
  25. Zhukov, V.A.; Zhernakov, A.I.; Gavrilyuk, N.D.; Pinaev, A.G.; Andronov, E.E.; Uspensky, V.E.; Irtyuga, O.B.; Moiseeva, O.M. Polymorphism analysis of the ACTA2 gene by pyrosequencing in patients with non-familial thoracic aortic aneurysms. Mol. Med. 2016, 14, 45–49. [Google Scholar]
  26. Irtyuga, O.B.; Freilikhman, O.A.; Krivonosov, D.S.; Malashicheva, A.B.; Tarnovskaya, S.I.; Uspensky, V.E.; Gordeev, M.L.; Rotar, O.P.; Kostareva, A.A.; Moiseeva, O.M. Role of the NOTCH1 gene in formation of aortic aneurysm. Russ. J. Cardiol. 2018, 23, 53–59. [Google Scholar] [CrossRef]
  27. Schubert, J.A.; Landis, B.J.; Shikany, A.R.; Hinton, R.B.; Ware, S.M. Clinically relevant variants identified in thoracic aortic aneurysm patients by research exome sequencing. Am. J. Med. Genet. A 2016, 170, 1288–1294. [Google Scholar] [CrossRef] [PubMed]
  28. Zhao, L.-Y.; Song, J.; Liu, Y.; Song, C.-X.; Yi, C. Mapping the epigenetic modifications of DNA and RNA. Protein Cell 2020, 11, 792–808. [Google Scholar] [CrossRef] [PubMed]
  29. Elbitar, S.; Renard, M.; Arnaud, P.; Hanna, N.; Jacob, M.P.; Guo, D.C.; Tsutsui, K.; Gross, M.S.; Kessler, K.; Tosolini, L.; et al. Pathogenic variants in THSD4, encoding the ADAMTS-like 6 protein, predispose to inherited thoracic aortic aneurysm. Genet. Med. 2021, 23, 111–122. [Google Scholar] [CrossRef] [PubMed]
  30. Ke, T.; Han, M.; Zhao, M.; Wang, Q.K.; Zhang, H.; Zhao, Y.; Ruan, X.; Li, H.; Xu, C.; Sun, T. Alpha-actin-2 mutations in Chinese patients with a non-syndromatic thoracic aortic aneurysm. BMC Med. Genet. 2016, 17, 45. [Google Scholar] [CrossRef]
  31. Tashima, Y.; Iwakoshi, S.; Inoue, T.; Nakamura, N.; Sano, T.; Kimura, N.; Inoue, T.; Adachi, K.; Yamaguchi, A. Aortic Agatston score correlates with the progression of acute type A aortic dissection. PLoS ONE 2022, 17, e0263881. [Google Scholar] [CrossRef] [PubMed]
  32. Hirono, K.; Hata, Y.; Miyao, N.; Okabe, M.; Takarada, S.; Nakaoka, H.; Ibuki, K.; Ozawa, S.; Origasa, H.; Nishida, N.; et al. LVNC study collaborates. Increased burden of ion channel gene variants is related to distinct phenotypes in pediatric patients with left ventricular noncompaction. Circ. Genom. Precis. Med. 2020, 13, e002940. [Google Scholar] [CrossRef] [PubMed]
  33. Lui, M.M.; Shadrina, M.; Gelb, B.D.; Kontorovich, A.R. Features of vascular Ehlers-Danlos syndrome among biobank participants harboring predicted high-risk COL3A1 genotypes. Circ. Genom. Precis. Med. 2023, 16, e003864. [Google Scholar] [CrossRef] [PubMed]
  34. Harakalova, M.; van der Smagt, J.; de Kovel, C.G.; Van’t Slot, R.; Poot, M.; Nijman, I.J.; Medic, J.; Joziasse, I.; Deckers, J.; Roos-Hesselink, J.W.; et al. Incomplete segregation of MYH11 variants with thoracic aortic aneurysms and dissections and patent ductus arteriosus. Eur. J. Hum. Genet. 2013, 21, 487–493. [Google Scholar] [CrossRef] [PubMed]
  35. Kashtan, C.E.; Segal, Y.; Flinter, F.; Makanjuola, D.; Gan, J.S.; Watnick, T. Aortic abnormalities in males with Alport syndrome. Nephrol. Dial. Transplant. 2010, 25, 3554–3560. [Google Scholar] [CrossRef] [PubMed]
  36. Ewans, L.J.; Colley, A.; Gaston-Massuet, C.; Gualtieri, A.; Cowley, M.J.; McCabe, M.J.; Anand, D.; Lachke, S.A.; Scietti, L.; Forneris, F.; et al. Pathogenic variants in PLOD3 result in a Stickler syndrome-like connective tissue disorder with vascular complications. J. Med. Genet. 2019, 56, 629–638. [Google Scholar] [CrossRef] [PubMed]
  37. Song, Y.; Lee, J.K.; Lee, J.O.; Kwon, B.; Seo, E.J.; Suh, D.C. Whole exome sequencing in patients with phenotypically associated familial intracranial aneurysm. Korean J. Radiol. 2022, 23, 101–111. [Google Scholar] [CrossRef]
  38. Torres-Juan, L.; Rico, Y.; Fortuny, E.; Pons, J.; Ramos, R.; Santos-Simarro, F.; Asensio, V.; Martinez, I.; Heine-Suñer, D. NOTCH1 gene as a novel cause of thoracic aortic aneurysm in patients with tricuspid aortic valve: Two cases reported. Int. J. Mol. Sci. 2023, 24, 8644. [Google Scholar] [CrossRef] [PubMed]
  39. Nykamp, K.; Anderson, M.; Powers, M.; Garcia, J.; Herrera, B.; Ho, Y.Y.; Kobayashi, Y.; Patil, N.; Thusberg, J.; Westbrook, M.; et al. Sherloc: A comprehensive refinement of the ACMG-AMP variant classification criteria. Genet. Med. 2017, 19, 1105–1117. [Google Scholar] [CrossRef] [PubMed]
  40. Kwartler, C.S.; Gong, L.; Chen, J.; Wang, S.; Kulmacz, R.; Duan, X.Y.; Janda, A.; Huang, J.; Kamm, K.E.; Stull, J.T.; et al. Variants of unknown significance in genes associated with heritable thoracic aortic disease can be low penetrant ‘risk variants’. Am. J. Hum. Genet. 2018, 103, 138–143. [Google Scholar] [CrossRef] [PubMed]
  41. Irtyuga, O.; Malashicheva, A.; Zhiduleva, E.; Freylikhman, O.; Rotar, O.; Bäck, M.; Tarnovskaya, S.; Kostareva, A.; Moiseeva, O. NOTCH1 mutations in aortic stenosis: Association with osteoprotegerin/RANK/RANKL. Biomed. Res. Int. 2017, 6917907. [Google Scholar] [CrossRef]
  42. Meloni, I.; Rubegni, P.; De Aloe, G.; Bruttini, M.; Pianigiani, E.; Cusano, R.; Seri, M.; Mondillo, S.; Federico, A.; Bardelli, A.M.; et al. Pseudoxanthoma elasticum: Point mutations in the ABCC6 gene and a large deletion including also ABCC1 and MYH11. Hum. Mutat. 2001, 18, 85. [Google Scholar] [CrossRef] [PubMed]
  43. Koyama, S.; Ito, K.; Terao, C.; Akiyama, M.; Horikoshi, M.; Momozawa, Y.; Matsunaga, H.; Ieki, H.; Ozaki, K.; Onouchi, Y.; et al. Population-specific and trans-ancestry genome-wide analyses identify distinct and shared genetic risk loci for coronary artery disease. Nat. Genet. 2020, 52, 1169–1177. [Google Scholar] [CrossRef]
  44. Klarin, D.; Devineni, P.; Sendamarai, A.K.; Angueira, A.R.; Graham, S.E.; Shen, Y.H.; Levin, M.G.; Pirruccello, J.P.; Surakka, I.; Karnam, P.R.; et al. Genome-wide association study of thoracic aortic aneurysm and dissection in the Million Veteran Program. Nat. Genet. 2023, 55, 1106–1115. [Google Scholar] [CrossRef]
  45. Hiratzka, L.F.; Bakris, G.L.; Beckman, J.A.; Bersin, R.M.; Carr, V.F.; Casey, D.E.; Eagle, K.A.; Hermann, L.K.; Isselbacher, E.M.; Kazerooni, E.A.; et al. 2010 ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM guidelines for the diagnosis and management of patients with Thoracic Aortic Disease: A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines, American Association for Thoracic Surgery, American College of Radiology, American Stroke Association, Society of Cardiovascular Anesthesiologists, Society for Cardiovascular Angiography and Interventions, Society of Interventional Radiology, Society of Thoracic Surgeons, and Society for Vascular Medicine. Circulation 2010, 121, e266–e369. [Google Scholar] [PubMed]
  46. Grahame, R.; Bird, H.A.; Child, A. The revised (Brighton 1998) criteria for the diagnosis of benign joint hypermobility syndrome (BJHS). J. Rheumatol. 2000, 27, 1777–1779. [Google Scholar]
  47. Loeys, B.L.; Dietz, H.C.; Braverman, A.C.; Callewaert, B.L.; De Backer, J.; Devereux, R.B.; Hilhorst-Hofstee, Y.; Jondeau, G.; Faivre, L.; Milewicz, D.M.; et al. The revised Ghent nosology for the Marfan syndrome. J. Med. Genet. 2010, 47, 476–485. [Google Scholar] [CrossRef]
  48. Rodrigues Bento, J.; Meester, J.; Luyckx, I.; Peeters, S.; Verstraeten, A.; Loeys, B. The genetics and typical traits of thoracic aortic aneurysm and dissection. Annu. Rev. Genom. Hum. Genet. 2022, 23, 223–253. [Google Scholar] [CrossRef]
  49. Hamandi, M.; Bolin, M.L.; Fan, J.; Lanfear, A.T.; Woolbert, S.K.; Baxter, R.D.; DiMaio, J.M.; Brinkman, W.T. A newly discovered genetic disorder associated with life-threatening aortic disease in a 6-year-old boy. J. Investig. Med. High. Impact Case Rep. 2020, 8, 2324709620909234. [Google Scholar] [CrossRef] [PubMed]
  50. Sambrook, J.; Russell, D.W. Purification of nucleic acids by extraction with phenol: Chloroform. CSH Protoc. 2006, 2006, pdb.prot4455. [Google Scholar]
  51. Okonechnikov, K.; Golosova, O.; Fursov, M.; UGENE team. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 2012, 28, 1166–1167. [Google Scholar] [CrossRef]
Ijms 25 08315 g001
Figure 1. Identification of the c.7841C>T:p.Ala2614Val variant in FBN1, the c.2498A>T:p.Lys833Ile variant in COL3A1, and variant c.833G>A:p.Gly278Asp in PLOD3 in patient #54 with aneurysm of the ascending aorta. (a) MDCT of the thoracic aorta. (b) Molecular genetic testing of the patient by clinical exome sequencing. (c) Sanger sequencing validation of the variants. Genetic variants are highlighted in yellow.
Figure 1. Identification of the c.7841C>T:p.Ala2614Val variant in FBN1, the c.2498A>T:p.Lys833Ile variant in COL3A1, and variant c.833G>A:p.Gly278Asp in PLOD3 in patient #54 with aneurysm of the ascending aorta. (a) MDCT of the thoracic aorta. (b) Molecular genetic testing of the patient by clinical exome sequencing. (c) Sanger sequencing validation of the variants. Genetic variants are highlighted in yellow.
Ijms 25 08315 g001
Ijms 25 08315 g002
Figure 2. Identification of VUSs in a patient with aneurysm of the ascending aorta. (A) MDCT of the thoracic aorta in patient #43, results of molecular genetic testing of the patient by clinical exome sequencing, and Sanger sequencing validation of the c.4993C>T:p.Arg1665Cys variant in MYH11. (B) MDCT of the thoracic aorta in patient #13, results of molecular genetic testing of the patient by clinical exome sequencing, and Sanger sequencing validation of the c.953C>G:p.Pro318Arg variant in COL4A5. (C) MDCT of the thoracic aorta in patient #36, results of molecular genetic testing of the patient by clinical exome sequencing, and Sanger sequencing validation of the c.964G>A:p.Val322Met variant in NOTCH1. Genetic variants are highlighted in yellow.
Figure 2. Identification of VUSs in a patient with aneurysm of the ascending aorta. (A) MDCT of the thoracic aorta in patient #43, results of molecular genetic testing of the patient by clinical exome sequencing, and Sanger sequencing validation of the c.4993C>T:p.Arg1665Cys variant in MYH11. (B) MDCT of the thoracic aorta in patient #13, results of molecular genetic testing of the patient by clinical exome sequencing, and Sanger sequencing validation of the c.953C>G:p.Pro318Arg variant in COL4A5. (C) MDCT of the thoracic aorta in patient #36, results of molecular genetic testing of the patient by clinical exome sequencing, and Sanger sequencing validation of the c.964G>A:p.Val322Met variant in NOTCH1. Genetic variants are highlighted in yellow.
Ijms 25 08315 g002
Table 1. Description of patients included in the study.
Table 1. Description of patients included in the study.
ParametersAll Patients (n = 41)Males (n = 27)Females (n = 14)p-Value
Age, years (Median (Q1; Q3))54(48; 64)58.0 (48.0; 62.5)53.0
(48.8; 68.5)		0.563
Mid-ascending aorta diameter,
mm (Median (Q1; Q3))		52 (49; 54)51.9 (46.5; 54.0)52.0
(50.3; 52.9)		0.535
Sinotubular junction diameter,
mm (Median (Q1; Q3))		42 (38.2; 46)44.0 (39.9; 52.3)39.0
(37.0; 40.9)		0.012
Proximal aortic arch diameter,
mm (Median (Q1; Q3))		39.5 (36; 42.5)39.5 (36.0; 41.8)39.5
(36.0; 43.0)		0.976
Bicuspid aortic valve,
n (%)		25 (61.0)14 (51.8)11 (78.6)0.105
BMI, kg/m2
(Median (Q1; Q3))		29 (26.4; 31.6)29 (27.3; 31.4)29.1 (23.6; 32.3)0.530
Arterial hypertension, n (%)27 (64.3)18 (64.3)9 (64.3)0.879
Hyperlipidemia, n (%)10 (23.8)7 (25.0)3 (21.4)0.751
Obesity (BMI (kg/m2) > 30), n (%)18 (42.9)12 (42.9)6 (42.9)1
T2DM or glucose intolerance, n (%)3 (7.1)2 (7.1)1 (7.1)1
Angina pectoris, CAD or MI, n (%)13 (30.9)12 (42.8)1 (7.1)0.039
Atherosclerosis, coronary, carotid, renal or femoral,
n (%)		21 (50)16 (59.2)4 (28.6)0.055
Calcification measurements
(number of patients)		26188-
Ca-score in coronary arteries
(Agatston index)		71.294.917.90.414
Ca-score in aorta (Agatston index)10712175.50.404
BMI: body–mass index, Ca-score: calcium score (Agatston index), MI: myocardial infarction, Q1: lower quartile, Q3: upper quartile, and p-value indicates the level of significance obtained from comparisons between men and women. The criterion for hyperlipidemia was the excess of optimal lipid values (LHD, LDL, triglycerides) depending on the risk profile of the patient. The diagnosis of arterial hypertension was based on stably elevated blood pressure at repeated (multiple) measurements on different days and according to the history of hypertension. Values p < 0.05 are highlighted in bold.
Table 2. Characteristics of patients with a VUS(s).
Table 2. Characteristics of patients with a VUS(s).
Characteristics IDPatient 13Patient 36Patient 43Patient 54
Age (years)58677248
Sexmfmm
Gene (category, (13))COL4A5 (C)NOTCH1 (B)MYH11 (A)FBN1 (A)COL3A1 (A)PLOD3 (D)
Genome coordinates (hg38/exon)chrX:
108582900/
exon 17		chr9:
136518726/
exon 6		chr16:
15719674/
exon 35		chr15:
48415746/
exon 64		chr2:
189003007/
exon 36		chr7:
101212888/
exon 8
Nucleotide:aminoacid changec.953C>G:
p.Pro318Arg		c.964G>A:
p.Val322Met		c.4993C>T:
p.Arg1665Cys		7841C>T:
p.Ala2614Val		c.2498A>T:
p.Lys833Ile		c.833G>A:
p.Gly278Asp
SNP ID
MAF *		rs1449979085
8.3 × 10−6/-		-
2.1 × 10−6/-		rs768569707
4.4 × 10−5/-		-
6.8 × 10−7/-		-
-		rs1041461490
6.8 × 10−6/5.9 × 10−4
In silico predictors **3/4/16
16.9		8/10/1
25.0		11/9/1
32.0		1/7/15
23.6		8/12/3
28.5		0/1/0
25.6
Aortic dimensions ***70/45/45/3436/62/45/3541/69/48/3645/50/35/30
Ca-score (Agatston index)Coronary arteriesno data494230
Aortano data2379370
Aortic valveBAVTAVTAVTAV
CADnoyesyesno
CVD risk factors (HTN/T2DM/HLD/O)yes/no/yes/yesyes/no/-/noyes/no/yes/noyes/no/no/no
AtherosclerosisnoCarA:up to 20%CorA: OMA 60%; LAD 40%; DA 40%; CarA: up to 20%no
BAV: bicuspid aortic valve, DA: diagonal branches, f: female, HLD: hyperlipidemia, HTN: hypertension, LAD: left anterior descending artery, m: male, MAF: minor allele frequency, O: obesity, OMA: obtuse marginal artery, SNP: single-nucleotide polymorphism, TAV: tricuspid aortic valve, CorA: coronary arteries, CarA: carotid arteries. On the basis of the strength of association with HTAAD, genes were classified into categories: A, definitive; B, strong; C, moderate; or D, limited [13]. MAF *—gnomAD total v. 4.1.0/RUSeq. In silico Predictors **—VarSome: Pathogenic/Uncertain/Benign; CADD (PHRED). Aortic dimensions ***—sinotubular junction/mid-ascending aorta/proximal aortic arch/arch (mm, according to MDCT).
Table 3. Details of the genes and primers used for Sanger sequencing.
Table 3. Details of the genes and primers used for Sanger sequencing.
GeneExonSequence
COL3A1
NC_000002.12		36F: 5′-GCTGAGAGATTGCTGTTG-3′
R: 5′-GGTGCTGAGATTCATACTTG-3′
FBN1
NC_000015.10		64F: 5′-GACAGCCACACAGGTAA-3′
R: 5′-CATAGCAAGAAGCCACATC-3′
MYH11
NC_000016.10		35F: 5′-CAGAGGAGGACGAAATGA-3′
R: 5′-TGTGCAAAGCTGAACTG-3′
COL4A5
NC_000023.11		17F: 5′-CCAGTATTCTCATTGCTTCTAT-3′
R: 5′-TATTTCTGCAACATGGACTG-3′
NOTCH1
NC_000009.12		6F: 5′-GGACACTCGCAGTAGAA-3′
R: 5′-TCCACAGAGCACAAAGA-3′
PLOD3
NC_000007.14		8F: 5′-GCTGGAAGATGCAACAC-3′
R: 5′-GGAAACGGTCCCACTAA-3′
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Goncharova, I.A.; Shipulina, S.A.; Sleptcov, A.A.; Zarubin, A.A.; Valiakhmetov, N.R.; Panfilov, D.S.; Lelik, E.V.; Saushkin, V.V.; Kozlov, B.N.; Nazarenko, L.P.; et al. Identification of Variants of Uncertain Significance in the Genes Associated with Thoracic Aortic Disease in Russian Patients with Nonsyndromic Sporadic Subtypes of the Disorder. Int. J. Mol. Sci. 2024, 25, 8315. https://doi.org/10.3390/ijms25158315

AMA Style

Goncharova IA, Shipulina SA, Sleptcov AA, Zarubin AA, Valiakhmetov NR, Panfilov DS, Lelik EV, Saushkin VV, Kozlov BN, Nazarenko LP, et al. Identification of Variants of Uncertain Significance in the Genes Associated with Thoracic Aortic Disease in Russian Patients with Nonsyndromic Sporadic Subtypes of the Disorder. International Journal of Molecular Sciences. 2024; 25(15):8315. https://doi.org/10.3390/ijms25158315

Chicago/Turabian Style

Goncharova, Irina A., Sofia A. Shipulina, Aleksei A. Sleptcov, Aleksei A. Zarubin, Nail R. Valiakhmetov, Dmitry S. Panfilov, Evgeniya V. Lelik, Viktor V. Saushkin, Boris N. Kozlov, Ludmila P. Nazarenko, and et al. 2024. "Identification of Variants of Uncertain Significance in the Genes Associated with Thoracic Aortic Disease in Russian Patients with Nonsyndromic Sporadic Subtypes of the Disorder" International Journal of Molecular Sciences 25, no. 15: 8315. https://doi.org/10.3390/ijms25158315

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop