Antiapoptotic PON2 expression and its clinical implications in locally advanced oral squamous cell carcinoma.

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.

A Mechanistic Review of Methotrexate and Celecoxib as a Potential Metronomic Chemotherapy for Oral Squamous Cell Carcinoma.

The combination of low-dose methotrexate and celecoxib as metronomic chemotherapy (MCT) is a novel therapy, believed to act by modulating the immune response, inhibiting angiogenesis and its cytotoxic action, though the exact mechanism of action is unclear. Clinically, MCT was found to be very effective in delaying tumor progression in patients with head and neck squamous cell carcinoma in both curative and palliative settings. This review was aimed to give a brief insight into the mechanism of action and potential molecular alterations of MCT in the treatment of oral cancers taking into consideration the various in vivo and in vitro studies.

Circulating cell-free DNA as a diagnostic and prognostic marker for cervical cancer.

Circulating cell-free DNA (cfDNA) is a promising tool for liquid biopsy-based tests. cfDNA has been reported to help in the diagnosis, quantification of minimal residual disease, prognosis, and identification of mutations conferring resistance in various types of cancers. Cervical cancer is the fourth most common cancer among women worldwide. High-risk human papillomavirus (hr-HPV) infections have been associated with almost all cervical cancers. Lack of HPV vaccines in national vaccination programs and irregular screening strategies in nations with low or moderate levels of human development index have led to cervical cancer becoming the second leading cause of cancer mortality in women. As HPV integration and overexpression of E6/E7 oncoprotein are crucial steps in the development of cancer, HPV cfDNA could potentially be used as a specific biomarker for the detection of cervical cancer. Many studies have used HPV cfDNA and other gene mutations or mRNA expression profiles for diagnosis and disease surveillance in patients with cervical cancer at various stages of disease progression. In this review we present an overview of different studies discussing the utility of cfDNA in cervical cancer and summarize the evidence supporting its potential use in diagnosis and treatment monitoring.

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Human papillomavirus 16 promotes microhomology-mediated end-joining.

Squamous cell carcinomas (SCCs) arising from aerodigestive or anogenital epithelium that are associated with the human papillomavirus (HPV) are far more readily cured with radiation therapy than HPV-negative SCCs. The mechanism behind this increased radiosensitivity has been proposed to be secondary to defects in DNA repair, although the specific repair pathways that are disrupted have not been elucidated. To gain insight into this important biomarker of radiosensitivity, we first examined genomic patterns reflective of defects in DNA double-strand break repair, comparing HPV-associated and HPV-negative head and neck cancers (HNSCC). Compared to HPV-negative HNSCC genomes, HPV+ cases demonstrated a marked increase in the proportion of deletions with flanking microhomology, a signature associated with a backup, error-prone double-strand break repair pathway known as microhomology-mediated end-joining (MMEJ). Then, using 3 different methodologies to comprehensively profile double-strand break repair pathways in isogenic paired cell lines, we demonstrate that the HPV16 E7 oncoprotein suppresses canonical nonhomologous end-joining (NHEJ) and promotes error-prone MMEJ, providing a mechanistic rationale for the clinical radiosensitivity of these cancers.

Global genetic analysis in mice unveils central role for cilia in congenital heart disease.

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.

Liquid biopsy approaches for pleural effusion in lung cancer patients.

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.

DNAH6 and Its Interactions with PCD Genes in Heterotaxy and Primary Ciliary Dyskinesia.

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.

Novel Jbts17 mutant mouse model of Joubert syndrome with cilia transition zone defects and cerebellar and other ciliopathy related anomalies.

Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome.

ARMC4 mutations cause primary ciliary dyskinesia with randomization of left/right body asymmetry.

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.

Role of cilia in structural birth defects: insights from ciliopathy mutant mouse models.

Structural birth defect (SBD) is a major cause of morbidity and mortality in the newborn period. Although the etiology of SBD is diverse, a wide spectrum of SBD associated with ciliopathies points to the cilium as having a central role in the pathogenesis of SBDs. Ciliopathies are human diseases arising from disruption of cilia structure and/or function. They are associated with developmental anomalies in one or more organ systems and can involve defects in motile cilia, such as those in the airway epithelia or from defects in nonmotile (primary cilia) that have sensory and cell signaling function. Availability of low cost next generation sequencing has allowed for explosion of new knowledge in genetic etiology of ciliopathies. This has led to the appreciation that many genes are shared in common between otherwise clinically distinct ciliopathies. Further insights into the relevance of the cilium in SBD has come from recovery of pathogenic mutations in cilia-related genes from many large-scale mouse forward genetic screens with differing developmental phenotyping focus. Our mouse mutagenesis screen for congenital heart disease (CHD) using noninvasive fetal echocardiography has yielded a marked enrichment for pathogenic mutations in genes required for motile or primary cilia function. These novel mutant mouse models will be invaluable for modeling human ciliopathies and further interrogating the role of the cilium in the pathogenesis of SBD and CHD. Overall, these findings suggest a central role for the cilium in the pathogenesis of a wide spectrum of developmental anomalies associated with CHD and SBDs.

Ion Torrent sequencing for conducting genome-wide scans for mutation mapping analysis.

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1(c.204+1G>A), an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.

Expression of PTGS2 along with genes regulating VEGF signalling pathway and association with high-risk factors in locally advanced oral squamous cell carcinoma.

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.

Prostaglandin-endoperoxide synthase 2 (PTGS2) gene expression and its association with genes regulating the VEGF signaling pathway in head and neck squamous cell carcinoma.

Introduction: The PTGS2 gene codes for the cyclooxygenase-2 (COX-2) enzyme that catalyzes the committed step in prostaglandin (PG) synthesis. Various in-vivo and in-vitro data suggest that prostaglandin E2 mediates as a signaling molecule for activating the VEGF signaling pathway (VSP), forming an association between COX-2 and VSP. Several chemotherapy regimens increasingly rely on preventing the synthesis of PGs. The targeted and metronomic chemotherapy agents, which suppress the COX-2 enzymes, have a major role in suppressing the oral cancer cascade. Hence, this study was designed to understand the pattern of PTGS2 expression and genes regulating VSP in head and neck cancers. Methods: PTGS2 expression was analyzed in the TCGA database computationally with the help of the UALCAN web-server. The expression of VEGF signaling pathway genes was mined, and their expression pattern was determined. Co-expression analysis was done to elucidate the association between VEGF signaling genes and PTGS2. The ShineyGo web server was used for gene set enrichment. Results: Significantly high PTGS2 expression was observed in tumor samples. Further genes regulating VEGF signaling were significantly overexpressed in tumor samples. Co-expression analysis results showed a significant positive correlation between PTGS2 and angiogenesis-regulating genes. The majority of the genes were enriched for angiogenesis pathways. Conclusion: PTGS2 was significantly expressed in head and neck cancer, and its expression was associated with genes regulating angiogenesis.

Increased Gene Expression of C1orf74 Is Associated with Poor Prognosis in Cervical Cancer.

C1orf74, 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 C1orf74 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 C1orf74 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 C1orf74 in HPV-positive cervical cancer cell lines and most cervical cancer patients. The promoter methylation levels of the C1orf74 gene in cervical cancer tissues were lower compared to normal cervical tissues (p < 0.05). Collectively, our study indicates that higher expression of the C1orf74 gene caused by hypomethylation of its promoter is associated with poor overall survival in cervical cancer patients. Thus, C1orf74 is a novel prognostic marker in cervical cancer.

Precision Radiotherapy: Reduction in Radiation for Oropharyngeal Cancer in the 30 ROC Trial.

BACKGROUND: Patients with human papillomavirus-related oropharyngeal cancers have excellent outcomes but experience clinically significant toxicities when treated with standard chemoradiotherapy (70 Gy). We hypothesized that functional imaging could identify patients who could be safely deescalated to 30 Gy of radiotherapy. METHODS: In 19 patients, pre- and intratreatment dynamic fluorine-18-labeled fluoromisonidazole positron emission tomography (PET) was used to assess tumor hypoxia. Patients without hypoxia at baseline or intratreatment received 30 Gy; patients with persistent hypoxia received 70 Gy. Neck dissection was performed at 4 months in deescalated patients to assess pathologic response. Magnetic resonance imaging (weekly), circulating plasma cell-free DNA, RNA-sequencing, and whole-genome sequencing (WGS) were performed to identify potential molecular determinants of response. Samples from an independent prospective study were obtained to reproduce molecular findings. All statistical tests were 2-sided. RESULTS: Fifteen of 19 patients had no hypoxia on baseline PET or resolution on intratreatment PET and were deescalated to 30 Gy. Of these 15 patients, 11 had a pathologic complete response. Two-year locoregional control and overall survival were 94.4% (95% confidence interval = 84.4% to 100%) and 94.7% (95% confidence interval = 85.2% to 100%), respectively. No acute grade 3 radiation-related toxicities were observed. Microenvironmental features on serial imaging correlated better with pathologic response than tumor burden metrics or circulating plasma cell-free DNA. A WGS-based DNA repair defect was associated with response (P = .02) and was reproduced in an independent cohort (P = .03). CONCLUSIONS: Deescalation of radiotherapy to 30 Gy on the basis of intratreatment hypoxia imaging was feasible, safe, and associated with minimal toxicity. A DNA repair defect identified by WGS was predictive of response. Intratherapy personalization of chemoradiotherapy may facilitate marked deescalation of radiotherapy.

Detection of Early Human Papillomavirus-Associated Cancers by Liquid Biopsy.

PURPOSE: A circulating tumor DNA (ctDNA) test to detect plasma Epstein-Barr viral DNA can be used to screen for early nasopharyngeal cancers; however, the reported sensitivity of viral ctDNA tests to detect human papillomavirus (HPV)-associated cancers is modest. We assessed the utility of droplet digital polymerase chain reaction (ddPCR) to detect early-stage HPV-associated cancers using sequential HPV16 and HPV33 assays that account for HPV subtype distribution and subtype sequence variants. PATIENTS AND METHODS: We collected plasma specimens from 97 HPV-positive patients with oropharyngeal squamous cell carcinoma and eight patients with HPV-positive anal squamous cell carcinoma, each with locoregionally confined disease. Negative controls included samples from seven patients with HPV-negative head and neck cancers and 20 individuals without cancer. RESULTS: Of 97 patients with nonmetastatic, locoregionally confined oropharyngeal squamous cell carcinoma, 90 patients had detectable HPV16 ctDNA and three patients had HPV33 ctDNA, indicating an overall sensitivity of 95.6%. Seven of eight patients with early anal cancer were HPV16 ctDNA positive. No HPV ctDNA was detected in 27 negative controls, indicating 100% specificity. HPV16 ctDNA was detected in 19 of 19 patients with low-volume disease, defined as patients with a single, asymptomatic positive lymph node (N1) or an isolated T1-2 asymptomatic primary tumor. HPV16 ctDNA levels directly corresponded to tumor responses to chemoradiation and surgery. CONCLUSION: With an updated understanding of HPV subtypes and sequence variation, HPV ctDNA by ddPCR is highly sensitive and specific, identifying HPV16 and HPV33 subtypes in a similar distribution as reported in major genomic profiling studies. The detection of small tumors indicates that HPV16 and HPV33 ctDNA ddPCR could be readily used in early detection screening trials and in disease response monitoring, analogous to Epstein-Barr virus DNA.

Coincident myelomeningocele and gastroschisis: report of 2 cases.

Myelomeningocele and gastroschisis, on their own, are both relatively common entities encountered in pediatric surgical care. Coexistence of these pathologies, however, is exceedingly rare. The authors report on 2 patients who presented with myelomeningocele and gastroschisis at birth. They obtained blood for whole-exome analysis for one of the patients and identified 3 mutations that could be related to the underlying anomalies: homozygous mutations in FAM171B and ABCA1 and a hemizygous (X-linked) mutation in COL4A5. Of these, FAM171B and ABCA1 both have function that may be related to the underlying disease.

Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.

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.

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences.

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.