A New Leu714Arg Variant in the Converter Domain of MYH7 is Associated with a Severe Form of Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy is the most frequent autosomal dominant disease, yet due to genetic heterogeneity, incomplete penetrance, and phenotype variability, the prognosis of the disease course in pathogenic variant carriers remains an issue. Identifying common patterns among the effects of different genetic variants is important. METHODS: We investigated the cause of familial hypertrophic cardiomyopathy (HCM) in a family with two patients suffering from a particularly severe disease. Searching for the genetic variants in HCM genes was performed using different sequencing methods. RESULTS: A new missense variant, p.Leu714Arg, was identified in exon 19 of the beta-myosin heavy chain gene (MYH7). The mutation was found in a region that encodes the 'converter domain' in the globular myosin head. This domain is essential for the conformational change of myosin during ATP cleavage and contraction cycle. Most reports on different mutations in this region describe severe phenotypic consequences. The two patients with the p.Leu714Arg mutation had heart failure early in life and died from HCM complications. CONCLUSIONS: This case presents a new likely pathogenic variant in MYH7 and supports the hypothesis that myosin converter mutations constitute a subclass of HCM mutations with a poor prognosis for the patient.

Pathogenetic Significance of Long Non-Coding RNAs in the Development of Thoracic and Abdominal Aortic Aneurysms.

Aortic aneurysm (AA) is a life-threatening condition with a high prevalence and risk of severe complications. The aim of this review was to summarize the data on the role of long non-coding RNAs (lncRNAs) in the development of AAs of various location. Within less than a decade of studies on the role of lncRNAs in AA, using experimental and bioinformatic approaches, scientists have obtained the data confirming the involvement of these molecules in metabolic pathways and pathogenetic mechanisms critical for the aneurysm development. Regardless of the location of pathological process (thoracic or abdominal aorta), AA was found to be associated with changes in the expression of various lncRNAs in the tissue of the affected vessels. The consistency of changes in the expression level of lncRNA, mRNA and microRNA in aortic tissues during AA development has been recordedand regulatory networks implicated in the AA pathogenesis in which lncRNAs act as competing endogenous RNAs (ceRNA networks) have been identified. It was found that the same lncRNA can be involved in different ceRNA networks and regulate different biochemical and cellular events; on the other hand, the same pathological process can be controlled by different lncRNAs. Despite some similarities in pathogenesis and overlapping of involved lncRNAs, the ceRNA networks described for abdominal and thoracic AA are different. Interactions between lncRNAs and other molecules, including those participating in epigenetic processes, have also been identified as potentially relevant to the AA pathogenesis. The expression levels of some lncRNAs were found to correlate with clinically significant aortic features and biochemical parameters. Identification of regulatory RNAs functionally significant in the aneurysm development is important for clarification of disease pathogenesis and will provide a basis for early diagnostics and development of new preventive and therapeutic drugs.

iPSC-Derived Endothelial Cells Reveal LDLR Dysfunction and Dysregulated Gene Expression Profiles in Familial Hypercholesterolemia.

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.

Apoptosis Genes as a Key to Identification of Inverse Comorbidity of Huntington's Disease and Cancer.

Cancer and neurodegenerative disorders present overwhelming challenges for healthcare worldwide. Epidemiological studies showed a decrease in cancer rates in patients with neurodegenerative disorders, including the Huntington disease (HD). Apoptosis is one of the most important processes for both cancer and neurodegeneration. We suggest that genes closely connected with apoptosis and associated with HD may affect carcinogenesis. We applied reconstruction and analysis of gene networks associated with HD and apoptosis and identified potentially important genes for inverse comorbidity of cancer and HD. The top 10 high-priority candidate genes included APOE, PSEN1, INS, IL6, SQSTM1, SP1, HTT, LEP, HSPA4, and BDNF. Functional analysis of these genes was carried out using gene ontology and KEGG pathways. By exploring genome-wide association study results, we identified genes associated with neurodegenerative and oncological disorders, as well as their endophenotypes and risk factors. We used publicly available datasets of HD and breast and prostate cancers to analyze the expression of the identified genes. Functional modules of these genes were characterized according to disease-specific tissues. This integrative approach revealed that these genes predominantly exert similar functions in different tissues. Apoptosis along with lipid metabolism dysregulation and cell homeostasis maintenance in the response to environmental stimulus and drugs are likely key processes in inverse comorbidity of cancer in patients with HD. Overall, the identified genes represent the promising targets for studying molecular relations of cancer and HD.

Calling and Phasing of Single-Nucleotide and Structural Variants of the LDLR Gene Using Oxford Nanopore MinION.

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.

Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy.

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.

Meier-Gorlin Syndrome: Clinical Misdiagnosis, Genetic Testing and Functional Analysis of ORC6 Mutations and the Development of a Prenatal Test.

Meier−Gorlin syndrome (MGS) is a rare genetic developmental disorder that causes primordial proportional dwarfism, microtia, the absence of or hypoplastic patellae and other skeletal anomalies. Skeletal symptoms overlapping with other syndromes make MGS difficult to diagnose clinically. We describe a 3-year-old boy with short stature, recurrent respiratory infections, short-rib dysplasia, tower head and facial dysmorphisms who was admitted to the Tomsk Genetic Clinic to verify a clinical diagnosis of Jeune syndrome. Clinical exome sequencing revealed two variants (compound heterozygosity) in the ORC6 gene: c.2T>C(p.Met1Thr) and c.449+5G>A. In silico analysis showed the pathogenicity of these two mutations and predicted a decrease in donor splicing site strength for c.449+5G>A. An in vitro minigene assay indicated that variant c.449+5G>A causes complete skipping of exon 4 in the ORC6 gene. The parents requested urgent prenatal testing for MGS for the next pregnancy, but it ended in a miscarriage. Our results may help prevent MGS misdiagnosis in the future. We also performed in silico and functional analyses of ORC6 mutations and developed a restriction fragment length polymorphism and haplotype-based short-tandem-repeat assay for prenatal genetic testing for MGS. These findings should elucidate MGS etiology and improve the quality of genetic counselling for affected families.

Induced pluripotent stem cell line ICGi038-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to compound heterozygous c.1246C > T/c.940 + 3_940 + 6del mutations in LDLR.

The development of cellular models for familial hypercholesterolemia (FH) is an important direction for creating new approaches to atherosclerosis treatment. Pathogenic mutations in the LDLR gene are the main FH source. We generated an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous c.1246C > T/c.940 + 3_940 + 6del LDLR mutation. The resulting iPSC line with confirmed patient-specific mutations maintains a normal karyotype and a typical undifferentiated state, including morphology, pluripotent gene expression, and in vitro differentiation potential. This iPSC line can be further differentiated toward relevant cells to better understand FH pathogenesis.

Induced pluripotent stem cell line ICGi037-A, obtained by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia due to heterozygous p.Trp443Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder increasing premature cardiovascular diseases risk due to atherosclerosis. Pathogenic mutations in the LDLR gene cause most FH cases. Available treatments are effective not for all LDLR mutations. Testing drugs on FH cell models help develop new efficient treatments. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with heterozygous p.Trp443Arg LDLR mutation. The iPSCs with confirmed patient-specific mutations express pluripotency markers, spontaneously differentiate into three germ layers and demonstrate normal karyotype. Patient-specific iPSCs-derived hepatocyte-like and endothelial cells are promising to develop new targeted therapies for FH.

Induced pluripotent stem cell line ICGi036-A generated by reprogramming peripheral blood mononuclear cells from a patient with familial hypercholesterolemia caused due to compound heterozygous p.Ser177Leu/p.Cys352Arg mutations in LDLR.

Familial hypercholesterolemia (FH) is a monogenic disease, leading to atherosclerosis due to a high level of low-density lipoprotein cholesterol. Most cases of the disease are based on pathological variants in the LDLR gene. Hepatocyte-like and endothelial cells derived from individual iPSCs are a good model for developing new approaches to therapy. We obtained an iPSC line from peripheral blood mononuclear cells of the patient with compound heterozygous p.Ser177Leu/p.Cys352Arg mutation in LDLR using non-integrating vectors. The iPSCs with a confirmed patient-specific mutation demonstrate pluripotency markers, normal karyotype, and the ability to differentiate into derivatives of three germ layers.

Human exome sequence data in support of somatic mosaicism in carotid atherosclerosis.

Understanding the mechanisms underlying the connection between somatic mosaicism and cardiovascular disease is likely essential for the future of personalized medicine. This article is aimed at providing data on somatic mosaicism in human carotid atherosclerosis. An advanced carotid atherosclerotic plaque and white blood cells were collected simultaneously from each patient (eight Slavic males, aged 67 ± 3.8 years [mean ± SD]) to assess the spectrum of germline and somatic genetic variants. Exome sequencing of DNA from the samples was performed with the SureSelect Clinical Research Exome Enrichment Kit (Agilent Technologies) and HiSeq 1500 (Illumina). The dataset contains germline and somatic single-nucleotide variants and small indels identified in the advanced carotid atherosclerotic plaque and white blood cells of each patient. This dataset does not include copy number variants owing to a lack of suitable tools for reliable calculation of copy numbers from exome sequencing data on cancer-unrelated samples. The dataset should help to understand somatic mosaicism in cardiovascular diseases and to identify copy number variants by means of more appropriate newer tools in the future.

ABCA1 and ABCG1 DNA methylation in epicardial adipose tissue of patients with coronary artery disease.

BACKGROUND: Recent studies have focused on the potential role of epicardial adipose tissue (EAT) in the development of coronary artery disease (CAD). ABCA1 and ABCG1 transporters regulate cell cholesterol content and reverse cholesterol transport. We aimed to determine whether DNA methylation and mRNA levels of the ABCA1 and ABCG1 genes in EAT and subcutaneous adipose tissue (SAT) were associated with CAD. METHODS: Paired EAT and SAT samples were collected from 82 patients undergoing elective cardiac surgery either for coronary artery bypass grafting (CAD group, N = 66) or valve surgery (NCAD group, N = 16). ABCA1 and ABCG1 mRNA levels in EAT and SAT samples were analyzed using real time polymerase chain reaction, ABCA1 protein levels in EAT samples were assessed by western blotting. ABCA1 and ABCG1 DNA methylation analysis was performed in 24 samples from the CAD group and 9 samples from the NCAD group via pyrosequencing. RESULTS: DNA methylation levels in the ABCA1 promoter and ABCG1 cg27243685 and cg06500161 CpG sites were higher in EAT samples from patients with CAD compared with NCAD (21.92% vs 10.81%, p = 0.003; 71.51% vs 68.42%, p = 0.024; 46.11% vs 37.79%, p = 0.016, respectively). In patients with CAD, ABCA1 and ABCG1 DNA methylation levels were higher in EAT than in SAT samples (p < 0.05). ABCA1 mRNA levels in EAT samples were reduced in the subgroup of patients with CAD and concomitant carotid artery disease or peripheral artery disease compared with the NCAD group (p = 0.024). ABCA1 protein levels in EAT samples tended to be lower in CAD patients than in the NCAD group (p = 0.053). DNA methylation levels at the ABCG1 cg27243685 site positively correlated with plasma triglyceride concentration (r = 0.510, p = 0.008), body mass index (r = 0.556, p = 0.013) and waist-to-hip ratio (r = 0.504, p = 0.012) in SAT samples. CONCLUSION: CAD is associated with ABCA1 and ABCG1 DNA hypermethylation in EAT. CAD with concomitant carotid artery disease or peripheral artery disease is accompanied by decreased ABCA1 gene expression in EAT. DNA methylation levels at the ABCG1 cg27243685 locus in SAT are associated with hypertriglyceridemia and obesity.

Genomic structural variations for cardiovascular and metabolic comorbidity.

The objective of this study was to identify genes targeted by both copy number and copy-neutral changes in the right coronary arteries in the area of advanced atherosclerotic plaques and intact internal mammary arteries derived from the same individuals with comorbid coronary artery disease and metabolic syndrome. The artery samples from 10 patients were screened for genomic imbalances using array comparative genomic hybridization. Ninety high-confidence, identical copy number variations (CNVs) were detected. We also identified eight copy-neutral changes (cn-LOHs) > 1.5 Mb in paired arterial samples in 4 of 10 individuals. The frequencies of the two gains located in the 10q24.31 (ERLIN1) and 12q24.11 (UNG, ACACB) genomic regions were evaluated in 33 paired arteries and blood samples. Two patients contained the gain in 10q24.31 (ERLIN1) and one patient contained the gain in 12q24.11 (UNG, ACACB) that affected only the blood DNA. An additional two patients harboured these CNVs in both the arteries and blood. In conclusion, we discovered and confirmed a gain of the 10q24.31 (ERLIN1) and 12q24.11 (UNG, ACACB) genomic regions in patients with coronary artery disease and metabolic comorbidity. Analysis of DNA extracted from blood indicated a possible somatic origin for these CNVs.

A comparison of genome-wide DNA methylation patterns between different vascular tissues from patients with coronary heart disease.

Epigenetic mechanisms of gene regulation in context of cardiovascular diseases are of considerable interest. So far, our current knowledge of the DNA methylation profiles for atherosclerosis affected and healthy human vascular tissues is still limited. Using the Illumina Infinium Human Methylation27 BeadChip, we performed a genome-wide analysis of DNA methylation in right coronary artery in the area of advanced atherosclerotic plaques, atherosclerotic-resistant internal mammary arteries, and great saphenous veins obtained from same patients with coronary heart disease. The resulting DNA methylation patterns were markedly different between all the vascular tissues. The genes hypomethylated in athero-prone arteries to compare with atherosclerotic-resistant arteries were predominately involved in regulation of inflammation and immune processes, as well as development. The great saphenous veins exhibited an increase of the DNA methylation age in comparison to the internal mammary arteries. Gene ontology analysis for genes harboring hypermethylated CpG-sites in veins revealed the enrichment for biological processes associated with the development. Four CpG-sites located within the MIR10B gene sequence and about 1 kb upstream of the HOXD4 gene were also confirmed as hypomethylated in the independent dataset of the right coronary arteries in the area of advanced atherosclerotic plaques in comparison with the other vascular tissues. The DNA methylation differences observed in vascular tissues of patients with coronary heart disease can provide new insights into the mechanisms underlying the development of pathology and explanation for the difference in graft patency after coronary artery bypass grafting surgery.