Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is ...strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling.
Locally advanced oral squamous cell carcinoma poses a significant challenge in oncology due to its rising incidence and mortality rates. Despite therapeutic progress, understanding molecular ...intricacies is essential. This study explored the role of PON2, a multifunctional enzyme implicated in antiapoptotic mechanisms. Aberrant PON2 expression in oral cancers raises questions regarding its involvement in evading programmed cell death and treatment resistance. Patients with locally advanced disease were enrolled, and molecular analyses were undertaken on the collected tumor and normal tissues. Utilizing computational datasets, this study used in silico gene expression analysis, differential gene expression analysis in our patient cohort, survival analysis, and gene set enrichment analysis to unravel role of PON2 in disease prognosis. The results showed elevated PON2 levels in advanced tumor stages, correlating with factors such as tobacco exposure, higher tumor grade, and nodal metastasis. Survival analysis revealed prognostic relevance of PON2, with lower expression linked to extended survival rates. Gene set enrichment analysis identified pathways aiding in cancer metastasis influenced by PON2. This study underscores the significance of PON2 expression as a prognostic marker for oral malignancies, with increased expression associated with advanced disease stages. Understanding the molecular profile of the PON2 gene suggests its potential as a valuable biomarker for the management of cancer.
Role of PON2 in regulating diverse molecular pathways influenced by PON2 during cancer progression and metastasis.
Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate ...diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and other PCD genes potentially can cause heterotaxy. DNAH6 was initially identified as a candidate heterotaxy/PCD gene by filtering exome-sequencing data from 25 heterotaxy patients stratified by whether they have airway motile cilia defects. dnah6 morpholino knockdown in zebrafish disrupted motile cilia in Kupffer's vesicle required for left-right patterning and caused heterotaxy with abnormal cardiac/gut looping. Similarly DNAH6 shRNA knockdown disrupted motile cilia in human and mouse respiratory epithelia. Notably a heterotaxy patient harboring heterozygous DNAH6 mutation was identified to also carry a rare heterozygous PCD-causing DNAI1 mutation, suggesting a DNAH6/DNAI1 trans-heterozygous interaction. Furthermore, sequencing of 149 additional heterotaxy patients showed 5 of 6 patients with heterozygous DNAH6 mutations also had heterozygous mutations in DNAH5 or other PCD genes. We functionally assayed for DNAH6/DNAH5 and DNAH6/DNAI1 trans-heterozygous interactions using subthreshold double-morpholino knockdown in zebrafish and showed this caused heterotaxy. Similarly, subthreshold siRNA knockdown of Dnah6 in heterozygous Dnah5 or Dnai1 mutant mouse respiratory epithelia disrupted motile cilia function. Together, these findings support an oligogenic disease model with broad relevance for further interrogating the genetic etiology of human ciliopathies.
Genomic profiling of tumors has become the mainstay for diagnosis, treatment monitoring and a guide to precision medicine. However, in clinical practice, the detection of driver mutations in tumors ...has several procedural limitations owing to progressive disease and tumor heterogeneity. The current era of liquid biopsy promises a better solution. This diagnostic utility of liquid biopsy has been demonstrated by numerous studies for the detection of cell-free DNA (cfDNA) in plasma for disease diagnosis, prognosis, and prediction. However, cfDNAs are limited in blood circulation and still hurdles to achieve promising precision medicine. Malignant pleural effusion (MPE) is usually detected in advanced lung malignancy, which is rich in tumor cells. Extracellular vesicles and cfDNAs are the two major targets currently explored using MPE. Therefore, MPE can be used as a source of biomarkers in liquid biopsy for investigating tumor mutations. This review focuses on the liquid biopsy approaches for pleural effusion which may be explored as an alternative source for liquid biopsy in lung cancer patients to diagnose early disease progression.
Background
PTGS2 encodes cyclooxygenase‐2 (COX‐2), which catalyses the committed step in prostaglandin synthesis. Various in vivo and in vitro data suggest that COX‐2 mediates the VEGF signalling ...pathway. In silico analysis performed in TCGA, PanCancer Atlas for head and neck cancers, demonstrated significant expression and co‐expression of PTGS2 and genes that regulate VEGF signalling. This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma (OSCC).
Methodology
Tumour and normal tissue samples were collected from patients with locally advanced OSCC. RNA was isolated from tissue samples, followed by cDNA synthesis. The cDNA was used for gene expression analysis (RT‐PCR) using target‐specific primers. The results obtained were compared with the in silico gene expression of the target genes in the TCGA datasets. Co‐expression analysis was performed to establish an association between PTGS2 and VEGF signalling genes.
Results
Tumour and normal tissue samples were collected from 24 OSCC patients. Significant upregulation of PTGS2 expression was observed. Furthermore, VEGFA, KDR, CXCR1 and CXCR2 were significantly upregulated in tumour samples compared with paired normal samples, except for VEGFB, whose expression was not statistically significant. A similar expression pattern was observed in silico, except for CXCR2 which was highly expressed in the normal samples. Co‐expression analysis showed a significant positive correlation between PTGS2 and VEGF signalling genes, except for VEGFB which showed a negative correlation.
Conclusion
PTGS2 and VEGF signalling genes are upregulated in OSCC, which has a profound impact on clinical outcomes.
This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma. Significant upregulation of PTGS2, VEGFA, KDR, CXCR1, and CXCR2 expression were observed in tumour samples compared with paired normal samples. This study underscores the clinical relevance of these genes, pointing to potential links between disease aggressiveness and adverse prognostic factors.
Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in ...humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with TTAGGG
6
sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.
, also known as URCL4, has been reported to have higher expression and be associated with poor prognosis in lung adenocarcinoma patients, and its role in regulation of the EGFR/AKT/mTORC1 pathway has ...been recently elucidated. In the current study, we used publicly available data and experimental validation of
gene expression and its association with prognosis in cervical cancer patients. qRT-PCR was performed using RNA from cervical cancer cell lines and twenty-five cervical cancer patients. Data from TNMplot revealed that mRNA expression of the
gene in primary tumor tissues, as well as metastatic tissues from cervical cancer patients, was significantly higher compared to normal cervical tissues. HPV-positive tumors had higher expression of this gene compared to HPV-negative tumors. qPCR analysis also demonstrated higher expression of
in HPV-positive cervical cancer cell lines and most cervical cancer patients. The promoter methylation levels of the
gene in cervical cancer tissues were lower compared to normal cervical tissues (
< 0.05). Collectively, our study indicates that higher expression of the
gene caused by hypomethylation of its promoter is associated with poor overall survival in cervical cancer patients. Thus,
is a novel prognostic marker in cervical cancer.
Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) ...in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development.
Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere ...with DNA synthesis
in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human TTAGGG
10 repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates TTGGGG
4 and TTTTGGGG
4 are thermally more stable compared to human TTAGGG
4. Consistent with this, replication of vectors with ciliate TTGGGG
10 repeats yielded a 3-fold higher mutant rate compared to the human TTAGGG
10 vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.
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