TPM1 Research Articles

A novel homozygous TPM1 mutation in familial pediatric hypertrophic cardiomyopathy and in silico screening of potential targeting drugs.

Familial hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. While sarcomeric gene mutations explain many HCM cases, the genetic basis of about half of HCM cases remains elusive. Here we aimed to identify the gene causing HCM in a non-consanguineous Saudi Arabian family with affected family members and a history of sudden death. The impact of the identified mutation on protein structure and potential drug targets were evaluated in silico. Triplets (two HCM subjects and one patent ductus arteriosus (PDA) case) and unaffected parents were screened by targeted next-generation sequencing (NGS) for 181 candidate cardiomyopathy genes. In silico structural and functional analyses, including protein modeling, structure prediction, drug screening, drug binding, and dynamic simulations were performed to explore the potential pathogenicity of the variant and to identify candidate drugs. A homozygous missense mutation in exon 1 of TMP1 (assembly GRCh37-chr15: 63340781; G>A) was identified in the triplets [two HCM and one patent ductus arteriosus (PDA)] that substituted glycine for arginine at codon 3 (p.Gly3Arg). The parents were heterozygous for the variant. The mutation was predicted to cause a significant and deleterious change in the TPM1 protein structure that slightly affected drug binding, stability, and conformation. In addition, we identified several putative TPM1-targeting drugs through structure-based in silico screening. TPM1 mutations are a common cause of HCM and other congenital heart defects. To date, TPM1 has not been associated with isolated PDA; to our knowledge, this is the first report of the homozygous missense variation p.Gly3Arg in TPM1 associated with familial autosomal recessive pediatric HCM and PDA. The identified candidate TPM1 inhibitors warrant further prospective investigation.

Inhibition of miR-96 enhances the sensitivity of colorectal cancer cells to oxaliplatin by targeting TPM1.

Colorectal cancer (CRC) is one of the major threats to human health worldwide. In the treatment of CRC, chemoresistance affects the efficacy of platinum-based therapies. Oxaliplatin is one of the most commonly used first-line medications for the treatment of CRC; however, chemoresistance is common among patients receiving oxaliplatin treatment, which significantly decreases its therapeutic efficacy. The present study focused on the roles of microRNA (miR)-96 in the oxaliplatin resistance of CRC cells and the underlying mechanisms. First, the expression of miR-96 was compared between CRC and adjacent tissues. Furthermore, target genes of miR-96 were predicted, and a dual-luciferase reporter assay was employed to confirm whether the candidate tropomyosin 1 (TPM1) is a direct target of miR-96. In addition, CRC cells were transfected with miR-96 inhibitor, miR-negative control, small interfering RNA (siRNA) targeting TPM1 or siRNA NC, and then treated with oxaliplatin. CCK-8 assay and flow cytometry were performed to examine the proliferation and apoptosis of the CRC cell line SW480. Next, reverse transcription-quantitative PCR and western blot analysis were performed to determine the mRNA and/or protein levels of miR-96, Bcl-2, BAX and TPM1. The results indicated that miR-96 was upregulated in CRC compared with normal adjacent tissues, while TPM1 was downregulated. The luciferase activity was reduced following transfection with miR-96 mimics and luciferase reporter plasmid containing the wild-type sequence of the 3'-untranslated region of TPM1. Furthermore, knockdown of miR-96 combined with oxaliplatin reduced the viability and induced apoptosis of CRC cells, which was further verified by decreased expression of Bcl-2 and the increased expression of TPM1 and BAX. Taken together, the downregulation of miR-96 enhanced the sensitivity of CRC cells to oxaliplatin.

Suppression of long non-coding RNA MALAT1 inhibits survival and metastasis of esophagus cancer cells by sponging miR-1-3p/CORO1C/TPM3 axis.

Esophageal cancer (EC) is a malignancy causing lots of mortality worldwide. Long non-coding RNAs (lncRNAs) are involved in the progression of multiple cancer types. The present study aimed to explore the function and associated mechanisms of lncRNA metastasis-associated lung adenocarcinoma transcript1 (MALAT1) in EC development by focusing on its interaction with miR-1-3p. The levels of MALAT1 and miR-1-3p were investigated in clinical EC specimens. Then, the expression of MALAT1 was knocked down in EC cell lines, and the effects of MALAT1 inhibition on the viability, migration, and invasion, and miR-1-3p/Coronin-1C (CORO1C)/Tropomyosin3 (TPM3) axis in EC cells were detected. The interaction between MALAT1 and miR-1-3p in the progression of EC was further determined by suppressing the expression of miR-1-3p in MALAT1 inhibition cells. The results were further verified with EC xenograft mice model. MALAT1 level was downregulated, while miR-1-3p level was upregulated in EC specimens. The inhibition of MALAT1 suppressed the viability, migration, and invasion in EC cell lines. The changes in phenotypes of EC cells were associated with the upregulation of miR-1-3p level and inhibition of CORO1C/TPM3 activity. Furthermore, the results of dual-luciferase assay showed the direct binding of MALAT1 to the seed sequence of miR-1-3p. The suppressed level of miR-1-3p not only induced the activity of CORO1C/TPM3 signaling, but also upregulated MALAT1 expression, indicating the reciprocal regulation between the two factors. The inhibition of MALAT1 also inhibited tumor growth and epithelial-mesenchymal transition (EMT) in mice model, which was reversed by miR-1-3p inhibition. Collectively, MALAT1 was important to the survival and metastasis of EC cells by sponging miR-1-3p.

Tropomyosin 1 genetically constrains in vitro hematopoiesis

BackgroundIdentifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.ResultsOur model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.ConclusionsOur findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.

Demonstration of beta-tropomyosin (Tpm2) and duplication of the alpha-slow tropomyosin gene (TPM3) in Atlantic salmon Salmo salar.

Beta tropomyosin (Tpm2) is demonstrated for the first time at the protein level in a fish species, using a combination of electrophoresis, mass spectrometric peptide mapping and end-group analysis. Tpm2 accounts for 50% of the total tropomyosin in slow trunk muscle of the adult Atlantic salmon as determined by quantitative carboxypeptidase digestion and is also present in the head and pectoral fin. It is absent in the fast skeletal (lighter-toned) trunk muscle, the most abundant muscle, which is composed solely of an alpha-fast (Tpm1) isoform. In contrast to the mammalian homologues, salmon Tpm2 migrates faster than salmon Tpm1 in the presence of anionic detergent. Other distinguishing characteristics are a reduced content of cysteine (one per chain) and tyrosine (five per chain) and a unique carboxyl-terminal region (residues 276-284). Two isoforms (paralogs) of alpha-slow tropomyosin (Tpm3) having different contents of methionine and histidine exist in slow trunk muscle indicating duplication of the TPM3 gene. Minor skeletal muscles, surveyed for the first time, contain a mix of at least two tropomyosins - Tpm2 (~ 50% of total) in pectoral fin, jaw and tongue and another isoform, either Tpm1 (pectoral fin) or alpha-1-like Tpm (jaw and tongue). Cheek muscle contains Tpm1 and alpha 1-like Tpm in varying proportion depending upon the section (light or dark). Of the two tropomyosins in tongue, Tpm2 displays comparatively weaker affinity for troponin-Sepharose. A feature of the major sarcomeric tropomyosins in Atlantic salmon is a pair of neighbouring glycines situated between residues 20-90.

Can Genetic Profile of Patients Undergoing Heart Transplantation Alter Clinical Decisions?

Although a significant proportion of cardiomyopathies are determined by genetic factors, the genetic testing of these patients has not been introduced routinely into clinical practice. The aim of this study is the genotyping of patients with advanced cardiomyopathy who have been bridged for heart transplantation or have been already transplanted, in order to evaluate the usefulness of genetic testing in the early diagnosis of the disease, and in the clinical management of these patients and their relatives. Fifteen heart transplanted patients and 5 patients bridged for heart transplantation due to familial dilated, hypertrophic or restrictive cardiomyopathy were genotyped with next generation sequencing, using Illumina's Trusight Cardio sequencing panel, covering 174 clinically relevant genes to cardiac diseases. Detected variants were characterized according to the recommendations of the American College of Medical Genetics and Genomics. Fifteen patients (75%) were found positive for rare single mutations after genotyping. Nine of these mutations (45% of patients) in PLN, LAMP2, RBM20, TNNI3, LMNA and TTN genes were characterized as pathogenic or likely pathogenic and 7 of them were previously described in the literature. A new likely pathogenic mutation in LMNA gene (p.Arg50Cys) and a new frameshift mutation in TTN gene characterized as pathogenic (p.Ile29015Thr*15), were identified in two families with DCM. The PLN gene mutation (p.Leu39*) was detected in 3 of the 20 patients (15%) indicating that it could be a founder mutation in the Greek population. Six patients (30%) carried variants of unknown significance (vus), while 5 patients (25%) didn't harbor any mutation. One of the vus in the TPM1 gene is of great interest since it was detected in three heart transplanted members of a DCM family. A significant proportion of rare variants in genes associated with cardiomyopathies has been identified in a cohort of patients undergoing heart transplantation. Detection of these variants may be critical in early diagnosis and management of the patient and his family and might prevent sudden cardiac death or alter heart transplantation or VAD implantation clinical decisions.

Pharmacokinetic-Pharmacodynamic-Efficacy Modeling of ONO-7579, a Novel Pan-Tropomyosin Receptor Kinase Inhibitor, in a Murine Xenograft Tumor Model.

The orally available and novel small molecule ONO-7579 (N-{2-[4-(2-amino-5-chloropyridin-3-yl)phenoxy]pyrimidin-5-yl}-N'-[2-(methanesulfonyl)-5-(trifluoromethyl)phenyl]urea) is a highly potent and selective pan-tropomyosin receptor kinase (TRK) inhibitor. The objective of the present study was to characterize the pharmacokinetic (PK), pharmacodynamic (PD), and antitumor efficacy relationships of ONO-7579 in mice xenografted with a human colorectal cancer cell line, KM12 (harboring the tropomyosin 3 (TPM3) -neurotrophic tyrosine receptor kinase 1 fusion gene), via a PK/PD modeling approach. Plasma and tumor concentrations of ONO-7579, tumor levels of phosphorylated TPM3-TRKA (pTRKA), and tumor volumes in the murine model were measured with a single or multiple dose of ONO-7579 (0.06-0.60 mg/kg) administered once daily. The PK/PD/efficacy models were developed in a sequential manner. Changes in plasma concentrations of ONO-7579 were described with an oral one-compartment model. Tumor concentrations of ONO-7579 were higher than plasma concentrations, and changes in ONO-7579 tumor concentrations were described with an additional tumor compartment that had no influence on plasma concentrations. pTRKA in tumors was described with a direct Emax model, and the tumor ONO-7579 concentration causing 50% of the maximum effect was estimated to be 17.6 ng/g. In addition, a pTRKA-driven tumor growth inhibition model indicated that ONO-7579 started to sharply increase the antitumor effect at pTRKA inhibition rates >60% and required >91.5% to reduce tumors. In conclusion, the developed PK/PD/efficacy models revealed a "switch-like" relationship between pTRKA inhibition rate and antitumor effect in a murine KM12 xenograft model, demonstrating that pTRKA in tumors could serve as an effective biomarker for scheduling the dose regimen in early-stage clinical studies. SIGNIFICANCE STATEMENT: In recent years, clinical development of TRK inhibitors in patients with neurotrophic tyrosine receptor kinase fusion-positive solid tumors has been accelerated. This research found that phosphorylated TRKA was a useful biomarker for explaining the antitumor efficacy of TRK inhibitors using a pharmacokinetic/pharmacodynamic modeling approach in xenograft mice. This finding suggests a rational dosing regimen in early-stage clinical studies for ONO-7579 (N-{2-[4-(2-amino-5-chloropyridin-3-yl)phenoxy]pyrimidin-5-yl}-N'-[2-(methanesulfonyl)-5-(trifluoromethyl)phenyl]urea), a novel pan-TRK inhibitor.

Tropomyosin isoforms regulate cofilin 1 activity by modulating actin filament conformation

Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F–Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.

The R168G heterozygous mutation of tropomyosin 3 (TPM3) was identified in three family members and has manifestations ranging from asymptotic to serve scoliosis and respiratory complications

According to existing reports, mutations in the slow tropomyosin gene (TPM3) may lead to congenital fiber-type disproportion (CFTD), nemaline myopathy (NM) and cap myopathy (CD). They are all congenital myopathies and are associated with clinical, pathological and genetic heterogeneity. A ten-year-old girl with scoliosis was unable to wean from mechanical ventilation after total intravenous anesthesia. The girl has scoliosis, respiratory insufficiency, motion delay and muscle weakness; her younger brother has a similar physiology but does not have scoliosis or respiratory insufficiency, and her parents are healthy. We conducted genetic testing and found a c.502C > G (p.R168G) heterozygous mutation in the family. This mutation originated from the father and was autosomal dominant. Muscle biopsy results indicated that no special structures were present, and the type I fiber ratio was not notably high compared to previous reports. Although the family members have the same mutations, their clinical manifestations are quite different.

Arecoline suppresses epithelial cell viability by upregulating tropomyosin-1 through the transforming growth factor-β/Smad pathway

Context Oral submucous fibrosis (OSF) is a chronic and progressive disease. Arecoline, present in betel nuts, has been proposed as a vital aetiological factor. However, the underlying mechanism remains unclear. Objectives This research elucidates the expression of tropomyosin-1 (TPM1) and its regulation mechanism in HaCaT cells treated with arecoline. Materials and methods HaCaT cells were assigned into three groups: (1) Control; (2) Treated with arecoline (0.16 mM) for 48 h (3) Treated with arecoline (0.16 mM) and transfected with small interfering RNA (siRNA) for TPM1 (50 nM) for 48 h. CCK8, cell cycle, and apoptosis phenotypic analyses were performed. PCR and western blot analyses were performed to detect the expression level of TPM1 and examine the related signalling pathway. Results The IC50 of arecoline was approximately 50 μg/mL (0.21 mM). The arecoline dose (0.16 mM) and time (48 h) markedly increased TPM1 expression at the mRNA and protein levels in HaCaT cells. Arecoline suppressed the cell growth, caused cell cycle arrest at the G1 phase, and induced cell apoptosis in HaCaT cells. siRNA-mediated knockdown of TPM1 attenuated the effect of arecoline on cell proliferation, apoptosis, and cell cycle arrest at the G1 phase. Furthermore, blocking of the transforming growth factor (TGF)-β receptor using SB431542 significantly suppressed TPM1 expression in the cells treated with arecoline. Discussion and conclusions Arecoline suppresses HaCaT cell viability by upregulating TPM1 through the TGF-β/Smad signalling pathway. This research provides a scientific basis for further study of arecoline and TPM1 in OSF and can be generalised to broader pharmacological studies. TPM1 may be a promising molecular target for treating OSF.

Delivery of platelet TPM3 mRNA into breast cancer cells via microvesicles enhances metastasis.

Platelets are implicated in the pathophysiology of breast and other cancers through their role in exchanging biomolecules with tumor cells in the tumor microenvironment. Such exchange results in tumor‐educated platelets with altered RNA expression profiles. Multiple lines of evidence indicate that platelet RNA profiles may be suitable as diagnostic biomarkers for cancer‐related biological processes. In this study, we characterized the gene expression signatures of platelets in breast cancer (BC) by high‐throughput sequencing and quantitative real‐time RT‐PCR. Our results indicate that the expression of TPM3 (tropomyosin 3) mRNA is significantly elevated in platelets from patients with BC compared with age‐matched healthy control subjects. Furthermore, up‐regulation of TPM3 mRNA in platelets was found to be significantly correlated with metastasis in patients with BC. Finally, we report that platelet TPM3 mRNA is delivered into BC cells through microvesicles and leads to enhanced migrative phenotype of BC cells. In summary, our findings suggest that the transfer of platelet TPM3 mRNA into cancer cells via microvesicles promotes cancer cell migration, and thus platelet‐derived TPM3 mRNA may be a suitable biomarker for early diagnosis of metastatic BC.

Tropomyosin 1 Genetically Constrains in Vitro Megakaryopoiesis

Platelet size and count are heritable, quantifiable outcomes of hematopoiesis. A better understanding of genetic mechanisms that regulate hematopoiesis could yield insights into blood disorders and augment clinical translational efforts to generate platelets in vitro. For example, induced pluripotent stem cell-derived blood cells could someday ameliorate adverse effects and increase transfusable platelet supplies. However, these methods remain inefficient. Genome wide association studies (GWAS) have linked hundreds of DNA loci with altered human platelet traits, but little is known about how these GWAS loci mechanistically impact hematopoiesis, megakaryocyte or platelet biology. Using publicly available genome-wide association and epigenetic data sets, we applied a machine-learning framework to identify epigenetic features enriched at established platelet trait association sites. From these results, we derived a quantitative prediction model that identified hematopoiesis-, megakaryocyte-, and platelet-relevant genomic loci and related genes more accurately than any prior computational method. In addition to specifying exact genetic variants known to regulate platelet traits and function, our model highlighted several novel variants in established platelet trait variation GWAS loci that were predicted to disrupt key transcription factor binding sites. Among loci nominated by our statistical model was a variant (rs11071720) that alters Tropomyosin 1 (TPM1) gene expression. TPM1 regulates cytoskeletal biology in many cell types, and cytoskeletal functions critically impact hematopoiesis and terminal blood cell biology. We used CRISPR/Cas9-mediated genome editing to create TPM1-knockout human induced pluripotent stem cells. TPM1KO cells were healthy and showed normal developmental progression through primitive streak, mesoderm, and early primitive hematopoietic differentiation programs. Unexpectedly, TPM1KO cells demonstrated enhanced formation of hemogenic endothelium and hematopoietic progenitor cells, increasing total megakaryocyte yield by more than 2-fold. TPM1KO megakaryocytes demonstrated normal morphology, gene expression patterns and functional responses to platelet agonists. Our findings help explain human genetics associations and identify a novel strategy to enhance in vitro hematopoiesis. More broadly, our now-validated machine learning-based approach might specify additional functional loci underlying hematopoiesis, megakaryopoiesis, or thrombopoiesis. Disclosures No relevant conflicts of interest to declare.

Association of single nucleotide polymorphisms in the 3'UTR region of TPM1 gene with dilated cardiomyopathy: A case-control study.

Tropomyosin 1 (TPM1) is a protein that constitutes the sarcomere filaments and is encoded by the TPM1 gene. The aim of the present study is to investigate the correlation between the 3′ untranslated region (3′UTR) single nucleotide polymorphisms (SNPs) of the TPM1 gene and dilated cardiomyopathy (DCM).A total of 245 patients with DCM and 245 healthy controls were recruited with 5 ml of venous blood. Genomic DNA was extracted to analyze the TPM1 gene rs12148828, rs11558748, rs707602, rs6738, rs7178040 loci genotypes, and the plasma miR-21 level was analyzed by reverse transcription-PCR (RT-PCR).The risk of DCM development in the rs6738 locus G allele carriers were 1.69 times more than A allele carriers (95% CI: 1.22-2.33, P = .001). Age and gender had no effect on the association of TPM1 gene SNPs with DCM risk (P > .05). The plasma miR-21 level of TPM1 gene rs6738 locus AA carriers was significantly higher than that of the AG and GG genotypes (P < .001).The SNPs of TPM1 gene rs6738 locus is associated with the risk of DCM, which may be related to the abnormal increase of miR-21 level in DCM patients, but further research is needed to prove the causal relationship between miR-21 level and DCM risk.

Tanshinone IIA attenuates high glucose induced human VSMC proliferation and migration through miR-21-5p-mediated tropomyosin 1 downregulation

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the development and progression of diabetes-related vascular complications. Recently, microRNAs (miRNAs) have been suggested to be involved in the pathogenesis of vascular diseases. This study was designed to investigate the influences of tanshinone IIA, an active compound extracted from Chinese herb Salvia miltiorrhiza, on the proliferation and migration of human aortic VSMCs (HASMCs).cultured in a high glucose medium and the underlying mechanisms related miRNAs. Using a miRNA microarray method, we profiled the miRNA expression signature in human aortic VSMCs (HASMCs) exposed to normal glucose, high glucose with and without Tanshinone IIA. Cell proliferation was measured with 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. Cell migration was evaluated using transwell migration assay and wound scratch assay. Western blot was used to examine the expression of tropomyosin 1 (TPM1) and miRNA level was quantified by real-time PCR. The results showed that several miRNAs that were highly expressed in the high glucose group were significantly decreased in the high glucose with Tanshinone IIA group compared with the normal glucose group (P < 0.05). Among these miRNAs, miR-21–5p was significantly upregulated in the high glucose group and downregulated after Tanshinone IIA treatment (P < 0.05). The depletion of miR-21–5p in HASMCs resulted in decreased cell proliferation and migration (P < 0.05). Moreover, we found that Tanshinone IIA inhibited proliferation and migration partly through miR-21-5p-mediated TPM1 downregulation (P < 0.05). In conclusion, the present study demonstrates that Tanshinone IIA is able to protect HASMCs from high glucose-induced proliferation and migration through regulating expression of miRNAs.

Cardiomyopathy-associated mutations in tropomyosin differently affect actin-myosin interaction at single-molecule and ensemble levels.

In the heart, mutations in the TPM1 gene encoding the α-isoform of tropomyosin lead, in particular, to the development of hypertrophic and dilated cardiomyopathies. We compared the effects of hypertrophic, D175N and E180G, and dilated, E40K and E54K, cardiomyopathy mutations in TPM1 gene on the properties of single actin-myosin interactions and the characteristics of the calcium regulation in an ensemble of myosin molecules immobilised on a glass surface and interacting with regulated thin filaments. Previously, we showed that at saturating Ca2+ concentration the presence of Tpm on the actin filament increases the duration of the interaction. Here, we found that the studied Tpm mutations differently affected the duration: the D175N mutation reduced it compared to WT Tpm, while the E180G mutation increased it. Both dilated mutations made the duration of the interaction even shorter than with F-actin. The duration of the attached state of myosin to the thin filament in the optical trap did not correlate to the sliding velocity of thin filaments and its calcium sensitivity in the in vitro motility assay. We suppose that at the level of the molecular ensemble, the cooperative mechanisms prevail in the manifestation of the effects of cardiomyopathy-associated mutations in Tpm.

MiRNA‑183‑5p.1 promotes the migration and invasion of gastric cancer AGS cells by targeting TPM1.

MicroRNA-183 (miR-183) is a small, non-coding RNA that is involved in post-transcriptional processes, is upregulated in gastric cancer and acts as an oncogene in cancer migration. Although fragmentary reports have demonstrated the importance of miR-183 in gastric cancer, its biofunctions and regulatory effects are still unknown. In the present study, the gene and protein expression levels were determined by reverse transcription-quantitative PCR and western blot analysis. The connection between miR-183-5p.1 and tropomyosin 1 (TPM1) was tested through luciferase reporter experiments. Cell viability, apoptosis and related proteins were detected by MTT assay, flow cytometry, immunofluorescence and western blotting, respectively. The migration and invasion of AGS cells modulated by miR-183-5p.1 were analyzed by Transwell assay. TPM1 expression was found to be decreased in gastric cancer tissues and cell lines when compared with normal and adjacent tissues and gastric epithelial cells, and was regulated by miR-183-5p.1 targeting TPM1. miR-183-5p.1 overexpression facilitated the growth and suppressed the death of AGS cells through Bcl-2 and P53 proteins. In addition, miR-183-5p.1 restricted TPM1, TPM2 and TPM3 protein expression in AGS cells. The excessive levels of miR-183-5p.1 promoted the migration and invasion of AGS cells, and inhibited the apoptosis of AGS cells. However, the knockdown of miR-183-5p.1 induced the opposite in AGS cells. In conclusion, miR-183-5p.1 promotes cell proliferation, migration and invasion by downregulating TPM1 and deactivating the Bcl-2/P53 signaling pathways in gastric cancer, indicating that miR-183-5p.1 and TPM1 may be potential targets for the diagnosis or therapy of gastric cancer in the future.

YIELD OF GENETIC TESTING FOR HYPERTROPHIC CARDIOMYOPATHY ACCORDING TO CONTEMPORARY VARIANT INTERPRETATION GUIDELINES: RESULTS FROM A LARGE CANADIAN REFERRAL CENTRE

The yield of genetic testing for hypertrophic cardiomyopathy (HCM) has been reported to be between 30% and 60%. Differences in populations studied are likely to be one explanation for this wide variation. Changes in the way we interpret genetic variants is likely to be a contributing factor. We aimed to analyze the genetic results from a large Canadian referral centre interpreted according to contemporary guidelines in order to examine the yield of genetic testing in the Canadian population. HCM index cases who underwent genetic testing between 2005 and January 2018 were included. All variants were re-classified according to the guidelines of the American College of Medical Genetics (ACMG) and subsequent gene-specific adaptations. Of 1,730 cases tested 393 (23%) were tested for 5 sarcomeric genes and 1,337 (77%) were tested for 8. In 325 patients (19%) a pathogenic (P) or likely pathogenic (LP) variant was identified and variants of unknown significance in 247 (14%). The majority (57%) of P/LP variants were found in the MYBPC3 gene, followed by the MYH7 gene (31%). The yield of TNNI3, TPM1 and TNNT2 genes was 4%, 3% and 2%, respectively. The 3 remaining genes combined (MYL2, MYL3 and ACTC1) were accountable for 3% of P/LP variants. Of the MYBPC3 variants 89% were loss-of-function (nonsense, frameshift or splice-site), one was an in-frame deletion and the rest (11%) missense. The yield of genetic testing in our centre was lower than previously reported. Possible explanations include lack of a founder mutation in the heterogenous Canadian population and higher referral rates of non-familial cases which are less likely to be gene-positive. Finally, using current and stricter guidelines for variant interpretation may have led to less variants being classified as P/LP, and may also explain the higher percentage of MYBPC3 variants among gene-positive cases. Further studies are required in order to determine whether this reflects the true genetic landscape of HCM or the limitations of current variant interpretation methods.

Establishment of a human induced pluripotent stem cell line (SDQLCHi004-A) from a patient with nemaline myopathy-4 disease carrying heterozygous mutation in TPM2 gene.

Nemaline myopathy-4 (NEM4) is a very rare inherited muscle disorder caused by a heterozygous mutation in tropomyosin-2 (TPM2) gene. We established an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a 3-month-old girl with NEM4 carrying a heterozygous mutation (c.397C>T (p.R133W)) in TPM2 gene. This iPSC line showed a normal karyotype, expressed pluripotency markers, showed differentiation potential and harbored the original mutation of c.397C>T in the TPM2 gene.

TPM2 as a potential predictive biomarker for atherosclerosis.

Cardiac-cerebral vascular disease (CCVD), is primarily induced by atherosclerosis, and is a leading cause of mortality. Numerous studies have investigated and attempted to clarify the molecular mechanisms of atherosclerosis; however, its pathogenesis has yet to be completely elucidated. Two expression profiling datasets, GSE43292 and GSE57691, were obtained from the Gene Expression Omnibus (GEO) database. The present study then identified the differentially expressed genes (DEGs), and functional annotation of the DEGs was performed. Finally, an atherosclerosis animal model and neural network prediction model was constructed to verify the relationship between hub gene and atherosclerosis. The results identified a total of 234 DEGs between the normal and atherosclerosis samples. The DEGs were mainly enriched in actin filament, actin binding, smooth muscle cells, and cytokine-cytokine receptor interactions. A total of 13 genes were identified as hub genes. Following verification of animal model, the common DEG, Tropomyosin 2 (TPM2), was found, which were displayed at lower levels in the atherosclerosis models and samples. In summary, DEGs identified in the present study may assist clinicians in understanding the pathogenesis governing the occurrence and development of atherosclerosis, and TPM2 exhibits potential as a promising diagnostic and therapeutic biomarker for atherosclerosis.

Syndrome of Primary Myocardial Hypertrophy: Clinical and Morphological, Genetic Diagnostics and Comparison of Sarcomerial Variants of Cardiomyopathy and its Phenocopy

Aim. To study the nosological spectrum in the syndrome of primary left ventricle hypertrophy (PLVH) using morphological and genetic diagnostics and to compare the clinical course of true hypertrophic cardiomyopathy (HCM) and its phenocopy.Material and methods. Fifty five adult patients (29 men, 48.2±17.0 years) with PLVH (12 mm and more) were included. The exclusion criteria were athletic heart, hypertensive heart disease, severe valvular disease and other causes of secondary left ventricle (LV) hypertrophy. We performed 11 endomyocardial biopsy, 8 intraoperative biopsy, 1 study of explanted heart, 1 autopsy with virus investigation (real-time polymerase chain reaction) of the blood and myocardium. Mutational screening had included simultaneous sequencing of the MYBPC3, TAZ, TPM1, LDB3, MYL2, ACTC1, MYL3, MYH7, TNNI3 and TNNT2 genes based on NGS technology (Ion Torrent PGMTM) with following Sanger resequencing of potentially significant genetic variants. For patients with a phenotype of particular genetic syndrome the Sanger sequencing of target gene(s) for performed first. Clinical examination had included electrocardiography, Holter monitoring, echocardiography, coronary angiography, computer tomography/magnetic resonance imaging (by indication). The mean follow-up was 8 [3;32] month.Results. Isolated HCM was found in 28 patients, and 10 have a combination of HCM and noncompaction myocardium (NCM). Mutations in the MYH7 and MYBPC3 genes were detected in six cases. In 17 cases (30.9%) the non-sarcomeric causes of LVHS were detected. Three patients had Fabry disease, 2 ‒ had Danon disease, in 10patients we found amyloidoses, in 1 – Friedreich ataxia, and 1 patient was diagnosed with LEOPARD syndrome (all cases were confirmed by DNA diagnostics). Genotype-positive diagnosis was established in 23.6% of patients. In patients with HCM were significantly more frequent asymmetric septal hypertrophy with obstruction and muscle bridges, in other forms of primary hypertrophy – right ventricular hypertrophy, low QRS voltage, QS complexes and increasing of ejection fraction (EF) (55.7±12.5% vs 62.5±10.1% in HCM, p=0.08). The morphologic signs of myocarditis were in 46.7% of patients with HCM detected: in 3 patients with NCM and in 4 patients with isolated HCM. The viral genome in the myocardium was in 11 patients with HCM (73.3%) detected, previously human herpes virus type 6 (it was correlation with myocarditis) and parvovirus B19. Eleven patients died due to a stroke/heart failure without no significant differences between patients with HCM and phenocopy.Conclusion. The spectrum of causes of the primary left ventricular hypertrophy is very wide. The frequency of myocarditis associated with sarcomeric HCM was 46.7%. When lower EF and heart failure in patients with HCM can be result of myocarditis, in patients with storage disease they are the result of disease itself.