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.
Subject(s)
Aortic Aneurysm, Thoracic , Genetic Predisposition to Disease , Humans , Female , Male , Russia/epidemiology , Aortic Aneurysm, Thoracic/genetics , Middle Aged , Adult , Myosin Heavy Chains/genetics , Fibrillin-1/genetics , Collagen Type III/genetics , Aged , Cardiac Myosins/genetics , High-Throughput Nucleotide Sequencing , Mutation , Genetic Variation , AdipokinesABSTRACT
Defects in the low-density lipoprotein receptor (LDLR) are associated with familial hypercholesterolemia (FH), manifested by atherosclerosis and cardiovascular disease. LDLR deficiency in hepatocytes leads to elevated blood cholesterol levels, which damage vascular cells, especially endothelial cells, through oxidative stress and inflammation. However, the distinctions between endothelial cells from individuals with normal and defective LDLR are not yet fully understood. In this study, we obtained and examined endothelial derivatives of induced pluripotent stem cells (iPSCs) generated previously from conditionally healthy donors and compound heterozygous FH patients carrying pathogenic LDLR alleles. In normal iPSC-derived endothelial cells (iPSC-ECs), we detected the LDLR protein predominantly in its mature form, whereas iPSC-ECs from FH patients have reduced levels of mature LDLR and show abolished low-density lipoprotein uptake. RNA-seq of mutant LDLR iPSC-ECs revealed a unique transcriptome profile with downregulated genes related to monocarboxylic acid transport, exocytosis, and cell adhesion, whereas upregulated signaling pathways were involved in cell secretion and leukocyte activation. Overall, these findings suggest that LDLR defects increase the susceptibility of endothelial cells to inflammation and oxidative stress. In combination with elevated extrinsic cholesterol levels, this may result in accelerated endothelial dysfunction, contributing to early progression of atherosclerosis and other cardiovascular pathologies associated with FH.
Subject(s)
Atherosclerosis , Hypercholesterolemia , Hyperlipoproteinemia Type II , Induced Pluripotent Stem Cells , Humans , Atherosclerosis/genetics , Cholesterol , Endothelial Cells , Hyperlipoproteinemia Type II/genetics , Inflammation/genetics , Lipoproteins, LDL , TranscriptomeABSTRACT
The LDLR locus has clinical significance for lipid metabolism, Mendelian familial hypercholesterolemia (FH), and common lipid metabolism-related diseases (coronary artery disease and Alzheimer's disease), but its intronic and structural variants are underinvestigated. The aim of this study was to design and validate a method for nearly complete sequencing of the LDLR gene using long-read Oxford Nanopore sequencing technology (ONT). Five PCR amplicons from LDLR of three patients with compound heterozygous FH were analyzed. We used standard workflows of EPI2ME Labs for variant calling. All rare missense and small deletion variants detected previously by massively parallel sequencing and Sanger sequencing were identified using ONT. One patient had a 6976 bp deletion (exons 15 and 16) that was detected by ONT with precisely located breakpoints between AluY and AluSx1. Trans-heterozygous associations between mutation c.530C>T and c.1054T>C, c.2141-966_2390-330del, and c.1327T>C, and between mutations c.1246C>T and c.940+3_940+6del of LDLR, were confirmed. We demonstrated the ability of ONT to phase variants, thereby enabling haplotype assignment for LDLR with personalized resolution. The ONT-based method was able to detect exonic variants with the additional benefit of intronic analysis in one run. This method can serve as an efficient and cost-effective tool for diagnosing FH and conducting research on extended LDLR haplotype reconstruction.
Subject(s)
Hyperlipoproteinemia Type II , Nanopores , Humans , Nucleotides , Phenotype , Mutation , Hyperlipoproteinemia Type II/genetics , Receptors, LDL/metabolismABSTRACT
Increasing evidence suggests that both coding and non-coding regions of sarcomeric protein genes can contribute to hypertrophic cardiomyopathy (HCM). Here, we introduce an experimental workflow (tested on four patients) for complete sequencing of the most common HCM genes (MYBPC3, MYH7, TPM1, TNNT2, and TNNI3) via long-range PCR, Oxford Nanopore Technology (ONT) sequencing, and bioinformatic analysis. We applied Illumina and Sanger sequencing to validate the results, FastQC, Qualimap, and MultiQC for quality evaluations, MiniMap2 to align data, Clair3 to call and phase variants, and Annovar's tools and CADD to assess pathogenicity of variants. We could not amplify the region encompassing exons 6-12 of MYBPC3. A higher sequencing error rate was observed with ONT (6.86-6.92%) than with Illumina technology (1.14-1.35%), mostly for small indels. Pathogenic variant p.Gln1233Ter and benign polymorphism p.Arg326Gln in MYBPC3 in a heterozygous state were found in one patient. We demonstrated the ability of ONT to phase single-nucleotide variants, enabling direct haplotype determination for genes TNNT2 and TPM1. These findings highlight the importance of long-range PCR efficiency, as well as lower accuracy of variant calling by ONT than by Illumina technology; these differences should be clarified prior to clinical application of the ONT method.
Subject(s)
Cardiomyopathy, Hypertrophic , Nanopore Sequencing , Humans , Carrier Proteins/genetics , Mutation , Cardiomyopathy, Hypertrophic/genetics , Troponin T/geneticsABSTRACT
Ring chromosome 8 (r(8)) is one of the least frequent ring chromosomes. Usually, maternal chromosome 8 forms a ring, which can be lost from cells due to mitotic instability. The 8q24 region contains the imprinted KCNK9 gene, which is expressed from the maternal allele. Heterozygous KCNK9 mutations are associated with the imprinting disorder Birk-Barel syndrome. Here, we report a 2.5-year-old boy with developmental delay, microcephaly, dysmorphic features, diffuse muscle hypotonia, feeding problems, motor alalia and noncoarse neurogenic type of disturbance of muscle electrogenesis, partially overlapping with Birk-Barel syndrome phenotype. Cytogenetic analysis of lymphocytes revealed his karyotype to be 46,XY,r(8)(p23q24.3)[27]/45,XY,-8[3]. A de novo 7.9 Mb terminal 8p23.3p23.1 deletion, a 27.1 Mb 8p23.1p11.22 duplication, and a 4.4 Mb intact segment with a normal copy number located between them, as well as a 154-kb maternal LINGO2 gene deletion (9p21.2) with unknown clinical significance were identified by aCGH + SNP array. These aberrations were confirmed by real-time PCR. According to FISH analysis, the 8p23.1-p11.22 duplication was inverted. The ring chromosome originated from maternal chromosome 8. Targeted massive parallel sequencing did not reveal the KCNK9 mutations associated with Birk-Barel syndrome. Our data allow to assume that autosomal monosomy with inactive allele of imprinted gene arising from the loss of a ring chromosome in some somatic cells may be an etiological mechanism of mosaic imprinting disorders, presumably with less severe phenotype.