TPM1 Research Articles

The Differential Expression of Circular RNAs and the Role of circAFF1 in Lens Epithelial Cells of High-Myopic Cataract.

High-myopic cataract (HMC) is a complex cataract with earlier onset and more rapid progress than age-related cataract (ARC). Circular RNAs (circRNAs) have been implicated in many diseases. However, their involvement in HMC remain largely unexplored. To investigate the role of dysregulated circRNAs in HMC, lens epithelium samples from 24 HMC and 24 ARC patients were used for whole transcriptome sequencing. Compared with ARC, HMC had 3687 uniquely expressed circRNAs and 1163 significantly differentially expressed circRNAs (DEcRs) (|log2FC| > 1, p < 0.05). A putative circRNA-miRNA-mRNA network was constructed based on correlation analysis. We validated the differential expression of 3 DEcRs by quantitative polymerase chain reaction (qPCR) using different sets of samples. We further investigated the role of circAFF1 in cultured lens epithelial cells (LECs) and found that the overexpression of circAFF1 promoted cell proliferation, migration and inhibited apoptosis. We also showed that circAFF1 upregulated Tropomyosin 1 (TPM1) expression by sponging miR-760, which was consistent with the network prediction. Collectively, our study suggested the involvement of circRNAs in the pathogenesis of HMC and provide a resource for further study on this topic.

Nucleotide sequence variant of the TPM1 gene in a family with different phenotypes of left ventricular non-compaction

Left ventricular non-compaction (LVNC) is a rare, genetically and phenotypically heterogeneous disease, which is often accompanied by diagnostic difficulties.Aim. To demonstrate several generations of a family with LVNC with various clinical and phenotypic manifestations of the disease (dilated and isolated types of LVNC) with an identified rs397516387 variant of the TPM1 gene.Material and methods. Based on the multicenter registry "Myocardial Non-compaction", a family with a familial form of LVNC was selected. Next generation sequencing (NGS) was performed on an Ion S5 system (Thermo Fisher Scientific, USA) using Ampliseq technology. Variant was verified using Sanger sequencing on an Applied Biosystem 3500 Genetic Analyzer (Thermo Fisher Scientific, USA). For clinical interpretation, variants in the genes associated with LVNC with a minor allele frequency &lt;0,1% were selected in the gnomAD database (v2.1.1). Results. Variant rs397516387 was found in 5 family members, including the proband. Further examination revealed LVNC in 2 additional family members. The proband and the proband’s uncle had a dilated type of LVNC, and the proband’s mother had an isolated type.Conclusion. The paper presents several generations of a family with different phenotypic manifestations of LVNC and rs397516387 variant in the TPM1 gene. The beginning of genetic screening from a proband, a thorough collection of a family history and further detailed genetic screening of relatives led to the identification of rs397516387 variant in 4 more family members, which in turn made it possible to conduct an additional examination to confirm the diagnosis and prescribe timely drug therapy.

Effects of tropomyosin 3 on pyroptosis of cardiomyocytes and fibroblast activation induced by hypoxia/reoxygenation in rats

To explore the role of tropomyosin 3 (TPM3) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte pyroptosis and fibroblast activation. Rat cardiomyocytes (H9c2 cells) were treated with H/R method to simulate myocardial ischemia/reperfusion (I/R) injury, and cell proliferation activity was evaluated with cell counting kit-8 (CCK8). The expression of TPM3 mRNA and protein was detected by quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting. H9c2 cells with stable TPM3-short hairpin RNA (shRNA) expression were constructed and treated with H/R (hypoxia for 3 hours, and reoxygenation for 4 hours). The expression of TPM3 was measured by RT-qPCR. The expressions of TPM3, pyroptosis-related proteins including caspase-1, NOD-like receptor protein 3 (NLRP3) and Gasdermin family proteins-N (GSDMD-N) were measured by Western blotting. The expression of caspase-1 was also observed by immunofluorescence assay. The levels of human interleukins (IL-1β, IL-18) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA) to elucidate the effect of sh-TPM3 on pyroptosis of cardiomyocytes. Rat myocardial fibroblasts were incubated with the above cell supernatant, and the expressions of human collagen I, collagen III, matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase inhibitor 2 (TIMP2) were detected by Western blotting to determine the effect of TPM3-interfered cardiomyocytes on the activation of fibroblasts under H/R conditions. Compared with the control group, H/R treatment for 4 hours significantly decreased the survival rate of H9c2 cells [(25.81±1.90)% vs. (99.40±5.54)%, P < 0.01], promoted the expression of TPM3 mRNA and protein [TPM3/GAPDH (2-ΔΔCt): 3.87±0.50 vs. 1, TPM3/β-Tubulin: 0.45±0.05 vs. 0.14±0.01, both P < 0.01], and promoted the expressions of caspase-1, NLRP3, GSDMD-N, and the enhanced release of cytokines IL-1β and IL-18 [cleaved caspase-1/caspase-1: 0.89±0.04 vs. 0.42±0.03, NLRP3/β-Tubulin: 0.39±0.03 vs. 0.13±0.02, GSDMD-N/β-Tubulin: 0.69±0.05 vs. 0.21±0.02, IL-1β (μg/L): 13.84±1.89 vs. 4.31±0.33, IL-18 (μg/L): 17.56±1.94 vs. 5.36±0.63, all P < 0.01]. However, compared with the H/R group, sh-TPM3 significantly weakened the promoting effects of H/R on these proteins and cytokines [cleaved caspase-1/caspase-1: 0.57±0.05 vs. 0.89±0.04, NLRP3/β-Tubulin: 0.25±0.04 vs. 0.39±0.03, GSDMD-N/β-Tubulin: 0.27±0.03 vs. 0.69±0.05, IL-1β (μg/L): 8.56±1.22 vs. 13.84±1.89, IL-18 (μg/L): 9.34±1.04 vs. 17.56±1.94, all P < 0.01]. In addition, the expressions of collagen I, collagen III, TIMP2, and MMP-2 in myocardial fibroblasts were significantly increased by the cultured supernatants from the H/R group (collagen I/β-Tubulin: 0.62±0.05 vs. 0.09±0.01, collagen III/β-tubulin: 0.44±0.03 vs. 0.08±0.00, TIMP2/β-tubulin: 0.73±0.04 vs. 0.20±0.03, TIMP2/β-Tubulin: 0.74±0.04 vs. 0.17±0.01, all P < 0.01). However, these boosting effects were weakened by sh-TPM3 (collagen I/β-Tubulin: 0.18±0.01 vs. 0.62±0.05, collagen III/β-Tubulin: 0.21±0.03 vs. 0.44±0.03, TIMP2/β-Tubulin: 0.37±0.03 vs. 0.73±0.04, TIMP2/β-Tubulin: 0.45±0.03 vs. 0.74±0.04, all P < 0.01). Interference with TPM3 can alleviate H/R-induced cardiomyocyte pyroptosis and fibroblast activation, suggesting that TPM3 may be a potential target of myocardial I/R injury.

Sarcomeric gene variants among Indians with hypertrophic cardiomyopathy: A scoping review.

Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease that frequently causes sudden cardiac death (SCD) among young adults. Several pathogenic mutations in genes encoding the cardiac sarcomere have been identified as diagnostic factors for HCM and proposed as prognostic markers for SCD. The objective of this review was to determine the scope of available literature on the variants encoding sarcomere proteins associated with SCD reported among Indian patients with HCM. The eligibility criteria for the scoping review included full text articles that reported the results of genetic screening for sarcomeric gene mutations in HCM patients of Indian south Asian ancestry. We systematically reviewed studies from the databases of Medline, Scopus, Web of Science core collection and Google Scholar. The electronic search strategy included a combination of generic terms related to genetics, disease and population. The protocol of the study was registered with Open Science Framework (https://osf.io/53gde/). A total of 19 articles were identified that reported pathogenic or likely pathogenic (P/LP) variants within MYH7, MYBPC3, TNNT2, TNNI3 and TPM1 genes, that included 16 singletons, one de novo and one digenic mutation (MYH7/ TPM1) associated with SCD among Indian patients. Evidence from functional studies and familial segregation implied a plausible mechanistic role of these P/LP variants in HCM pathology. This scoping review has compiled all the P/LP variants reported to-date among Indian patients and summarized their association with SCD. Single homozygous, de novo and digenic mutations were observed to be associated with severe phenotypes compared to single heterozygous mutations. The abstracted genetic information was updated with reference sequence ID (rsIDs) and compiled into freely accessible HCMvar database, available at https://hcmvar.heartfailure.org.in/. This can be used as a population specific genetic database for reference by clinicians and researchers involved in the identification of diagnostic and prognostic markers for HCM.

De Novo Asp219Val Mutation in Cardiac Tropomyosin Associated with Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), caused by mutations in thin filament proteins, manifests as moderate cardiac hypertrophy and is associated with sudden cardiac death (SCD). We identified a new de novo variant, c.656A>T (p.D219V), in the TPM1 gene encoding cardiac tropomyosin 1.1 (Tpm) in a young SCD victim with post-mortem-diagnosed HCM. We produced recombinant D219V Tpm1.1 and studied its structural and functional properties using various biochemical and biophysical methods. The D219V mutation did not affect the Tpm affinity for F-actin but increased the thermal stability of the Tpm molecule and Tpm-F-actin complex. The D219V mutation significantly increased the Ca2+ sensitivity of the sliding velocity of thin filaments over cardiac myosin in an in vitro motility assay and impaired the inhibition of the filament sliding at low Ca2+ concentration. The molecular dynamics (MD) simulation provided insight into a possible molecular mechanism of the effect of the mutation that is most likely a cause of the weakening of the Tpm interaction with actin in the "closed" state and so makes it an easier transition to the “open” state. The changes in the Ca2+ regulation of the actin-myosin interaction characteristic of genetic HCM suggest that the mutation is likely pathogenic.

A novel mutation in the tropomyosin 1 gene in a Chinese patient with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is an inherited heart disease characterized by left ventricular hypertrophy. Although sarcomeric gene mutations can explain many HCM cases, the genetic basis of approximately half of HCM cases remains elusive. Here, we report the case of a 30-year-old woman with HCM after imaging and pathological examinations. We identified a novel mutation in a pathogenic gene from the HCM patient through whole-exome sequencing. A heterozygous missense mutation in exon 1 of the α-tropomyosin (TPM1) gene (NM_000366.5:exon1:c.71A > G) was found based on patient sequencing data. The mutation resulted in glutamine replacing arginine (p.Gln24Arg). This mutation was expected to cause significant and deleterious changes in the structure of the TPM1 protein. Therefore, according to pathogenicity assessment guidelines, the mutation was considered likely pathogenic. We found that most of these mutations are related to cardiomyopathy and majority of them are in exon 1 and are missense mutations. TPM1 mutations are thus a common cause of HCM and other congenital heart defects. Thus, a novel missense variant, p.Gln24Arg, in the TPM1 gene is associated with the autosomal dominant disease HCM. These results expand the knowledge spectrum of TPM1 gene function.

Silencing of Tropomyosin 1 suppresses the proliferation, invasion and metastasis of oral squamous cell carcinoma in vitro

We focused on the expression of Tropomyosin 1 (TPM1) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. TPM1 is an actin binding and microfilament-associated protein, which is thought to affect the tumor behavior of various malignancies. In this study, we examined the role of TPM1 in proliferation, invasion and metastasis of OSCC cells in vitro as we tried to understand its usefulness as a potential biomarker for OSCC. Human OSCC cell lines were transfected by TPM1-siRNA. MTT assay, cell migration assay and wound healing assay were carried out to understand the characteristics of the TPM1-siRNA-transfected cells. Our data showed that suppression of TPM1 increased the proliferative ability, wound healing ability, and migratory ability of OSCC cells. These findings suggest that low expression of TPM1 could contribute to cancer cell invasion and metastasis in OSCC patients, and that TPM1 may have potential as a useful biomarker for OSCC. Availability of data and materialsAll data generated or analyzed during the present study are included in this published article.

LncRNA SFTA1P promotes cervical cancer progression by interaction with PTBP1 to facilitate TPM4 mRNA degradation

Long non-coding RNAs (lncRNAs) play key roles in cancer development and progression. However, the biological function and clinical significance of most lncRNAs in cervical cancer remain elusive. In this study, we explore the function and mechanism of lncRNA surfactant associated 1 (SFTA1P) in cervical cancer. We firstly identified SFTA1P by analyzing the RNA sequencing data of cervical cancer from our previous study and from The Cancer Genome Atlas (TCGA). We then verified SFTA1P expression by qRT-PCR. The cell proliferation and migration capacity of SFTA1P was assessed by using CCK-8, colony formation, transwell and wound healing assays. RNA pull-down, RNA immunoprecipitation (RIP), RNA stability and western blot assays were used to reveal potential mechanisms. Athymic nude mice were used to evaluate tumorigenicity and metastasis in vivo. SFTA1P is upregulated in cervical tumor tissues and its high expression is associated with poor prognosis. Biologically, knockdown of SFTA1P inhibited the proliferation, migration, and invasion of cervical cancer cells in vitro, as well as tumorigenesis and metastasis in vivo. Mechanistically, SFTA1P was shown to interact with polypyrimidine tract binding protein 1 (PTBP1) to regulate the stability of tropomyosin 4 (TPM4) mRNA, thereby resulting in malignant cell phenotypes. TPM4 knockdown could attenuate the suppression of cell progression induced by either SFTA1P or PTBP1 knockdown. Our findings demonstrate that SFTA1P can promote tumor progression by mediating the degradation of TPM4 mRNA through its interaction with PTBP1 protein. This provides a potential therapeutic strategy to target the SFTA1P-PTBP1-TPM4 axis in cervical cancer.

TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway

BackgroundMicroglia, the innate immune cells in the central nervous system, play an essential role in brain homeostasis, neuroinflammation and brain infections. Dysregulated microglia, on the other hand, are associated with neurodegenerative diseases, yet the mechanisms underlying pro-inflammatory gene expression in microglia are incompletely understood.MethodsWe investigated the role of the actin-associated protein tropomyosin 1 (TPM1) in regulating pro-inflammatory phenotype of microglia in the retina by using a combination of cell culture, immunocytochemistry, Western blot, qPCR, TUNEL, RNA sequencing and electroretinogram analysis. TREM2−/− mice were used to investigate whether TPM1 regulated pro-inflammatory responses downstream of TREM2. To conditionally deplete microglia, we backcrossed CX3CR1CreER mice with Rosa26iDTR mice to generate CX3CR1CreER:Rosa26iDTR mice.ResultsWe revealed a vital role for TPM1 in regulating pro-inflammatory phenotype of microglia. We found that TPM1 drove LPS-induced inflammation and neuronal death in the retina via the PKA/CREB pathway. TPM1 knockdown ameliorated LPS-induced inflammation in WT retinas yet exaggerated the inflammation in TREM2−/− retinas. RNA sequencing revealed that genes associated with M1 microglia and A1 astrocytes were significantly downregulated in LPS-treated WT retinas but upregulated in LPS-treated TREM2−/− retinas after TPM1 knockdown. Mechanistically, we demonstrated that CREB activated by TPM1 knockdown mediated anti-inflammatory genes in LPS-treated WT retinas but pro-inflammatory genes in LPS-treated TREM2−/− retinas, suggesting a novel role for TREM2 as a brake on TPM1-mediated inflammation. Furthermore, we identified that TPM1 regulated inflammation downstream of TREM2 and in a microglia-dependent manner.ConclusionsWe demonstrate that TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway. Our findings suggest that TPM1 could be a potential target for therapeutic intervention in brain diseases.

Combining Gene Mutation with Expression of Candidate Genes to Improve Diagnosis of Escobar Syndrome.

Escobar syndrome is a rare, autosomal recessive disorder that affects the musculoskeletal system and the skin. Mutations in the CHRNG and TPM2 genes are associated with this pathology. In this study, we conducted a clinical and genetic investigation of five patients and also explored via in silico and gene expression analysis their phenotypic variability. In detail, we identified a patient with a novel composite heterozygous variant of the CHRNG gene and two recurrent mutations in both CHRNG and TPM2 in the rest of the patients. As for the clinical particularities, we reported a list of modifier genes in a patient suffering from myopathy. Moreover, we identified decreased expression of IGF-1, which could be related to the short stature of Escobar patients, and increased expression of POLG1 specific to patients with TPM2 mutation. Through this study, we identified the genetic spectrum of Escobar syndrome in the Tunisian population, which will allow setting up genetic counseling and prenatal diagnosis for families at risk. In addition, we highlighted relevant biomarkers that could differentiate between patients with different genetic defects.

Investigate the stemness of adult adipose-derived stromal cells based on single-cell RNA-sequencing.

The cellular heterogeneity and genetic features of stemness of adipose-derived stromal cells (ADSCs) remain unclear. Using single-cell RNA sequencing (scRNA-seq), we investigated the genomic features of the stemness gene in ADSCs with genetic variability. We cultured the ADSCs isolated from the fat waste of a healthy adult volunteers undergoing cosmetic plastic surgery to the third generation, used the BD Rhapsody platform to perform scRNA-seq, then used Monocle2 to analyze the growth and development trajectory of ADSCs, Cellular Trajectory Reconstruction Analysis Using Gene Counts and Expression (CytoTRACE) to evaluate the stemness gene characteristics in ADSCs clusters, and Beam to analyze the expression change characteristics of the main stemness related genes of ADSCs. According to the scRNA-seq data of 5325 ADSCs, they could be classified into nine cell clusters. According to CytoTRACE analysis, Cluster 3 of ADSCs had the highest stemness, whereas Cluster 8 had the lowest stemness. Pseudotime analysis revealed that Cluster 3 of ADSCs was primarily dispersed in the middle part of the growth and development trajectory, whereas Cluster 8 was primarily distributed at the end. We summarized the stemness of Cluster 3 in ADSCs with high expression of TPM1 and CCND1 genes in the metaphase of growth and development is the strongest, whereas the stemness of Cluster 8 with high expression of FICD, CREBRF, SDF2L1, HERPUD1, and HYOU1 genes in the telophase of growth and development is the weakest, providing a theoretical basis for screening and improving the therapeutic effect of ADSCs in cell transplantation research.

An Immune-Related Prognostic Risk Model in Colon Cancer by Bioinformatics Analysis.

Colon cancer is one of the leading malignancies with poor prognosis worldwide. Immune cell infiltration has a potential prognostic value for colon cancer. This study aimed to establish an immune-related prognostic risk model for colon cancer by bioinformatics analysis. A total of 1670 differentially expressed genes (DEGs), including 177 immune-related genes, were identified from The Cancer Genome Atlas (TCGA) dataset. A prognostic risk model was constructed based on six critical immune-related genes (C-X-C motif chemokine ligand 1 (CXCL1), epiregulin (EREG), C-C motif chemokine ligand 24 (CCL24), fatty acid binding protein 4 (FABP4), tropomyosin 2 (TPM2), and semaphorin 3G (SEMA3G)). This model was validated using the microarray dataset GSE35982. In addition, Cox regression analysis showed that age and clinical stage were correlated with prognostic risk scores. Kaplan–Meier survival analysis showed that high risk scores correlated with low survival probabilities in patients with colon cancer. Downregulated TPM2, FABP4, and SEMA3G levels were positively associated with the activated mast cells, monocytes, and macrophages M2. Upregulated CXCL1 and EREG were positively correlated with macrophages M1 and activated T cells CD4 memory, respectively. Based on these results, we can conclude that the proposed prognostic risk model presents promising novel signatures for the diagnosis and prognosis prediction of colon cancer. This model may provide therapeutic benefits for the development of immunotherapy for colon cancer.

Clinical Characterization and Prognostic Value of TPM4 and Its Correlation with Epithelial-Mesenchymal Transition in Glioma.

Tropomyosin 4 (TPM4) has been reported as an oncogenic gene across different malignancies. However, the role of TPM4 in glioma remains unclear. This study aimed to determine the clinical characterization and prognostic value of TPM4 in gliomas. Transcriptome expression and clinical information were collected from the CGGA and TCGA datasets, which included 998 glioma patients. ScRNA-seq data were obtained from CGGA. R software was utilized for statistical analyses. There was a positive correlation between TPM4 and WHO grades. IDH-wildtype and mesenchymal subtype gliomas were accompanied by TPM4 upregulation. GO and GSEA analysis suggested that TPM4 was profoundly associated with epithelial-to-mesenchymal transition (EMT). Subsequent GSVA revealed a robust correlation between TPM4 and three signaling pathways of EMT (hypoxia, TGF-β, PI3K/AKT). Furthermore, TPM4 showed a synergistic effect with mesenchymal biomarkers, particularly with N-cadherin, Slug, Snail, TWIST1, and vimentin. ScRNA-seq analysis suggested that higher TPM4 was mainly attributed to tumor cells and macrophages and associated with tumor cell progression and macrophage polarization. Finally, high TPM4 was significantly associated with unfavorable outcomes. In conclusion, our findings indicate that TPM4 is significantly correlated with more malignant characteristics of gliomas, potentially through involvement in EMT. TPM4 could predict worse survival for patients with glioma.

MiR-107 inhibits the malignant biological behavior of esophageal squamous cell carcinoma by targeting TPM3.

BackgroundEsophageal squamous cell carcinoma (ESCC) is a highly lethal and aggressive tumor. Our previous study revealed that tropomyosin 3 (TPM3) is up-regulated in the late stage of ESCC and promotes epithelial-mesenchymal transition (EMT) via MMP2/MMP9. However, we have not yet explored the upstream regulator of TPM3. In this study, miR-107, a microRNA molecule, was predicted as an inhibitor targeting TPM3, and in vivo and in vitro experiments confirmed this hypothesis.MethodsWestern blot and fluorescence quantitative polymerase chain reaction (qPCR) were applied to analyze the expression of miR-107 and TPM3. Flow cytometry, cell counting kit-8 (CCK8) assay, wound-healing assay, colony formation assay, transwell assay, and a BALB/c nude mouse (male, 8 weeks old, 20±2 g) model were used to detect the function of miR-107 and TPM3 in ESCC. Dual-luciferase assay was used to analyze the suppressed TPM3 expression induced by miR-107. Rescue experiments were also conducted in our research.ResultsThe cell and nude mouse models verified that TPM3 promotes proliferation, invasion and metastasis, and inhibits apoptosis, which is the opposite effect of miR-107 in ESCC. Meanwhile, the expression of TPM3 was up-regulated in the ESCC sample and cell lines, and miR-107 was down-regulated correspondingly. Dual-luciferase detection confirmed that miR-107 decreased the expression of TPM3 by targeting the 3’-untranslated region (3’-UTR) at the end of the TPM3 transcript. Further experiments verified that TPM3 could rescue the tumor suppression effect derived from miR-107.ConclusionsMiR-107 negatively regulates TPM3 expression and plays a tumor suppression role in ESCC.

MYC-Induced Upregulation of Lncrna ELFN1-AS1 Contributes to Tumor Growth in Colorectal Cancer via Epigenetically Silencing TPM1.

This study revealed a novel molecular pathway in colorectal cancer progression, which may provide new method for early diagnosis and treatment of colorectal cancer.

Endometriosis and inflammatory immune responses: Indian experience.

Endometriosis is a public health disorder affecting ∼ 247 million women globally and ∼ 42 million women in India. Women with endometriosis suffer from dysmenorrhea, chronic pelvic pain, dyspareunia, dyschezia, fatigue, depression, and infertility leading to significant socioeconomic impact and morbidity. The etiology of endometriosis is not understood well even after 100 years of research. Currently, there is no permanent cure for endometriosis. The inflammatory immune response is one of the important features of etiopathogenesis of endometriosis and therefore understanding the inflammatory immune response would lead to a better understanding of this enigmatic disorder and may also lead to biomarker discovery for diagnosis of endometriosis. We investigated the autoimmune etiology of endometriosis in the Indian population. Using the proteomics approach, anti-endometrial antibodies (AEAs) were detected in Indian women with endometriosis [anti-endometrial antibodies - tropomyosin 3 (TPM3), stomatin-like protein2 (SLP-2), and tropomodulin 3 (TMOD3)]. The studies on AEAs provided a better understanding of autoimmune mechanisms in endometriosis. All three subtypes of endometriosis; superficial peritoneal, ovarian endometrioma, and deep infiltrating endometriosis were reported in Indian women. In this review, we discuss our experiences of the inflammatory immune response, autoimmunity, comorbidities, and clinical phenotypes in women with endometriosis in India.

Proteomic Analysis of Serum Proteins from Patients with Severe Coronary Artery Calcification.

Proteomic studies investigating novel molecular markers of coronary artery calcification (CAC) are scarce.This study compared the protein expression in the serum of patients with severe CAC and non-CAC. The serum from 30 patients with severe CAC and 30 matched-controls were screened by data-independent acquisition(DIA)-based proteomic technology. Bioinformatics analysis tools were used to analyze the underlying molecular mechanisms of the differentially expressed proteins. Candidate proteins were further validated by an enzyme-linked immunosorbent assay (ELISA) in an independent cohort. A receiver operating characteristic (ROC) curve was used to estimate the diagnostic power of the candidate proteins. Among the 110 identified proteins, the expression of 81 was significantly upregulated, whereas 29 proteins were downregulated (fold change 1.5; p 0.05) between patients with and without CAC. Bioinformatics analysis indicated that the differential proteins are involved in complement and coagulation cascades, platelet activation, regulation of actin cytoskeleton, or glycolysis/gluconeogenesis pathways. Further verification showed that serum levels of complement C5 (C5), fibrinogen gamma (FGG), pyruvate kinase isoform M2 (PKM2), and tropomyosin 4 (TPM4) were consistent with the proteomic findings, which could allow discrimination between CAC and non-CAC patients. This study revealed that high serum levels of serum C5, FGG, PKM2, and TPM4 proteins were linked to severe CAC. These proteins may be developed as biomarkers to predict coronary calcification.

A pathogenic mechanism associated with myopathies and structural birth defects involves TPM2-directed myogenesis.

Nemaline myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle–specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as cap myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found 4 variants significantly affected muscle development and muscle function. Transient overexpression of the 4 variants also disrupted the morphogenesis of mouse myotubes in vitro and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize 2 potentially novel TPM2 variants and 1 recurring variant that we identified in patients with DA (V129A, E139K, A155T, respectively) and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our patients with DA, suggesting disrupted myogenesis is a potentially novel pathomechanism of TPM2 disorders and that our myogenic assays can predict the clinical severity of TPM2 variants.

Abstract 986: RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness

Abstract NGS studies implicate dysregulation of the splicing machinery in the development of diverse cancers. The X-chromosome RBM10 gene encodes an RNA-binding protein that modulates transcriptome-wide cassette exon splicing. Truncation and missense RBM10 mutations are enriched (11%) in non-anaplastic thyroid cancers of patients who died of metastatic disease. MSK-MET, an integrated pan-cancer cohort of tumor genomic and clinical outcome data showed RBM10 alterations associate with metastatic burden in thyroid cancer. Within the MSK-IMPACT thyroid cancer cohort, 44% of RBM10 alterations co-occur with RAS mutations. We developed a murine Rbm10 floxed allele, which results in a non-functional transcript. Thyroid-specific Rbm10 inactivation through Tpo-Cre did not induce a phenotype. When crossed with FR-HrasG12V mice, which upon recombination generate endogenous expression of HrasG12V, Hras/Rbm10 mice developed thyroid cancers, ~ 20% of which were metastatic to lung. Cell lines derived from these tumors showed high penetrance of lung metastases after tail vein or orthotopic implantation into the thyroid. We identified splicing targets of RBM10 by high depth RNAseq of 5 isogenic human thyroid cancer cell lines (2 RBM10-null with dox-induced RBM10; 3 RBM10 WT with RBM10 shRNA). The common abnormalities associated with RBM10 loss were exon inclusion events. Ingenuity Pathway Analysis of global transcriptomes in RBM10 isogenic human thyroid cancer cell lines showed enrichment in pro-migratory, aberrant integrin and RHO/RAC signaling expression signatures. Enriched GO analysis confined to genes subject to aberrant splicing by RBM10 loss extended these findings, with the top terms being cell adhesion, cytoskeleton, cadherin and integrin binding. RBM10 loss was associated with increased cell migration velocity in vitro as measured by time lapse imaging. Key cytoskeletal and extracellular matrix genes subject to exon inclusion events included vinculin (VCL), tenascin C (TNC), CD44, fibronectin (FN1) and tropomyosin 1 (TPM1). Isoform-specific knockdown of the VCL splice inclusion cassette exon that leads to illegitimate expression of metavinculin (mVCL) reduced cell migration, whereas isoform-specific knockdown of TNC and CD44 exon inclusion events reduced cell invasiveness in vitro. Rho GTPases transduce signals from the ECM to integrin receptors to regulate cell adhesion, migration, and invasiveness. Consistent with this, RBM10 overexpression in RBM10 null cells reduced RAC1-GTP levels. Finally, RBM10 re-expression in RBM10 null cells reversed metastatic competency in vivo. In conclusion, RBM10 loss alters the ratio of cassette exon inclusion events in a subset of transcripts that regulate interactions between the ECM and the cytoskeleton, leading to RHO/RAC activation and governing a process favoring increased cell movement and metastatic competence. Citation Format: Gnana P. Krishnamoorthy, Anthony Glover, Brian Untch, Mahesh Saqcena, Dina Vukel, Katherine Berman, Omar Abdel-Wahab, Robert K. Bradley, Jeffrey A. Knauf, James A. Fagin. RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 986.

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling

BackgroundRare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. ObjectivesTo characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. Patients/MethodsIndex case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. ResultsFive family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. ConclusionsThis study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.