Novel GLCCI1-BRAF fusion drives kinase signaling in a case of pheochromocytomatosis.

Introduction: Recurrent and metastatic pheochromocytoma (PCC) are rare advanced endocrine neoplasms with limited treatment options. Insight into the pathogenic molecular alterations in patients with advanced PCC can provide therapeutic options for precisely targeting dysregulated pathways. Objective: We report the discovery and characterization of a novel BRAF-containing fusion transcript and its downstream molecular alterations in a patient with recurrent PCC with peritoneal seeding (pheochromocytomatosis). Methods: We reviewed the medical record of a patient with pheochromocytomatosis. A comprehensive pan-cancer molecular profiling using next-generation sequencing (NGS) as well as confirmatory real-time-quantitative PCR were performed on surgical specimens. BRAF rearrangement and downstream molecular changes were assayed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Western blot was used to assess the in vitro activation of the mitogen-activated protein kinase (MAPK) signaling pathway and the EMT markers in transfected HEK-293 cells. Results: The NGS analysis of a specimen from a 72-year-old female patient with pheochromocytomatosis showed an in-frame fusion of exon 3 of Glucocorticoid Induced 1 (GLCCI1) to exon 9 of BRAF. The upstream auto-inhibitory domain of BRAF was excluded from the GLCCI1-BRAF fusion; however, the downstream BRAF kinase domain was intact. A BRAF rearrangement was confirmed via a BRAF-specific break-apart FISH assay. Four separate tumor foci harbored GLCCI1-BRAF fusion. IHC demonstrated increased phosphorylated MEK. HEK-293 cells transfected with the GLCCI1-BRAF fusion demonstrated increased phosphorylated MEK as well as higher expression of EMT markers SNAI1 and ZEB1 in vitro. Conclusion: We demonstrate a novel pathogenic gene fusion of GLCCI1 with the oncogenic kinase domain of BRAF, resulting in an activation of the MAPK signaling pathway and EMT markers. Thus, this patient may benefit from clinically available MEK and/or BRAF inhibitors when systemic therapy is indicated. Summary Statement: This report is the first of GLCCI1 fused to BRAF in a human neoplasm and only the second BRAF-containing fusion transcript in PCC. Detailed molecular characterization of PCC can be a valuable tool in managing patients with recurrent PCC and pheochromocytomatosis that represents a significant clinical challenge.

Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification.

The 12q13-q14 chromosomal region is recurrently amplified in 25% of fusion-positive (FP) rhabdomyosarcoma (RMS) cases and is associated with a poor prognosis. To identify amplified oncogenes in FP RMS, we compared the size, gene composition, and expression of 12q13-q14 amplicons in FP RMS with those of other cancer categories (glioblastoma multiforme, lung adenocarcinoma, and liposarcoma) in which 12q13-q14 amplification frequently occurs. We uncovered a 0.2 Mb region that is commonly amplified across these cancers and includes CDK4 and 6 other genes that are overexpressed in amplicon-positive samples. Additionally, we identified a 0.5 Mb segment that is only recurrently amplified in FP RMS and includes 4 genes that are overexpressed in amplicon-positive RMS. Among these genes, only serine hydroxymethyltransferase 2 (SHMT2) was overexpressed at the protein level in an amplicon-positive RMS cell line. SHMT2 knockdown in amplicon-positive RMS cells suppressed growth, transformation, and tumorigenesis, whereas overexpression in amplicon-negative RMS cells promoted these phenotypes. High SHMT2 expression reduced sensitivity of FP RMS cells to SHIN1, a direct SHMT2 inhibitor, but sensitized cells to pemetrexed, an inhibitor of the folate cycle. In conclusion, our study demonstrates that SHMT2 contributes to tumorigenesis in FP RMS and that SHMT2 amplification predicts differential response to drugs targeting this metabolic pathway.

A Fusion Transcription Factor-Driven Cancer Progresses to a Fusion-Independent Relapse via Constitutive Activation of a Downstream Transcriptional Target.

Targeted monotherapies usually fail due to development of resistance by a subgroup of cells that evolve into recurrent tumors. Alveolar rhabdomyosarcoma is an aggressive myogenic soft-tissue cancer that is associated with a characteristic PAX3-FOXO1 gene fusion encoding a novel fusion transcription factor. In our myoblast model of PAX3-FOXO1-induced rhabdomyosarcoma, deinduction of PAX3-FOXO1 simulates a targeted therapy that antagonizes the fusion oncoprotein. This simulated therapy results initially in regression of the primary tumors, but PAX3-FOXO1-independent recurrent tumors eventually form after a delay. We report here that upregulation of FGF8, a direct transcriptional target of PAX3-FOXO1, is a mechanism responsible for PAX3-FOXO1-independent tumor recurrence. As a transcriptional target of PAX3-FOXO1, FGF8 promoted oncogenic activity in PAX3-FOXO1-expressing primary tumors that developed in the myoblast system. In the recurrent tumors forming after PAX3-FOXO1 deinduction, FGF8 expression was necessary and sufficient to induce PAX3-FOXO1-independent tumor growth through an autocrine mechanism. FGF8 was also expressed in human PAX3-FOXO1-expressing rhabdomyosarcoma cell lines and contributed to proliferation and transformation. In a human rhabdomyosarcoma cell line with reduced PAX3-FOXO1 expression, FGF8 upregulation rescued oncogenicity and simulated recurrence after PAX3-FOXO1-targeted therapy. We propose that deregulated expression of a PAX3-FOXO1 transcriptional target can generate resistance to therapy directed against this oncogenic transcription factor and postulate that this resistance mechanism may ultimately be countered by therapeutic approaches that antagonize the corresponding downstream pathways. SIGNIFICANCE: In a model of cancer initiated by a fusion transcription factor, constitutive activation of a downstream transcriptional target leads to fusion oncoprotein-independent recurrences, thereby highlighting a novel progression mechanism and therapeutic target.

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

The HDAC3-SMARCA4-miR-27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.

The expression and function of PAX3 in development and disease.

The PAX3 gene encodes a member of the PAX family of transcription factors that is characterized by a highly conserved paired box motif. The PAX3 protein is a transcription factor consisting of an N-terminal DNA binding domain (containing a paired box and homeodomain) and a C-terminal transcriptional activation domain. This protein is expressed during development of skeletal muscle, central nervous system and neural crest derivatives, and regulates expression of target genes that impact on proliferation, survival, differentiation and motility in these lineages. Germline mutations of the murine Pax3 and human PAX3 genes cause deficiencies in these developmental lineages and result in the Splotch phenotype and Waardenburg syndrome, respectively. Somatic genetic rearrangements that juxtapose the PAX3 DNA binding domain to the transcriptional activation domain of other transcription factors deregulate PAX3 function and contribute to the pathogenesis of the soft tissue cancers alveolar rhabdomyosarcoma and biphenotypic sinonasal sarcoma. The wild-type PAX3 protein is also expressed in other cancers related to developmental lineages that normally express this protein and exerts phenotypic effects related to its normal developmental role.

The complex genetics of hypoplastic left heart syndrome.

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.

PAX3-FOXO1 is essential for tumour initiation and maintenance but not recurrence in a human myoblast model of rhabdomyosarcoma.

The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalized human myoblasts. Although myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours, whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours showed transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated with a CRISPR/Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1-independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3-FOXO1 expression. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Phylogenetic relationship and time of divergence of Mus terricolor with reference to other Mus species.

Mitochondrial DNA control region of Mus terricolor, three aboriginal species M. spretus, M. macedonicus, M. spicilegus; the Asian lineage M. caroli, M. cervicolor, M. cookii; and the two house mice, M. musculus domesticus and M. m. castaneus were analysed to estimate the substitution rate, phylogenetic relationship and the probable time of divergence. Results showed that M. spretus, M. caroli and M. terricolor are highly diverged from each other (caroli/terricolor = 0.146, caroli/spretus = 0.147 and terricolor/spretus = 0.122), whereas M. spretus showed less divergence with two house mice species (0.070 and 0.071). Sequence divergence between M. terricolor and the Palearctic group were found to be ranging from 0.121 to 0.134. Phylogenetic analysis by minimum evolution, neighbour-joining, unweighed pair group method with arithmetic mean and maximum parsimony showed almost similar topology. Two major clusters were found, one included the Asian lineage, M. caroli, M. cookii and M. cervicolor and the other included the house mice M. m. domesticus, M. m. castaneus and the aboriginal mice M. macedonicus and M. spicilegus along with M. spretus, forming the Palearctic clade. M. terricolor was positioned between the Palearctic and Asian clades. Results showed that Palearctic-terricolor and the Asian lineages diverged 5.47 million years ago (Mya), while M. terricolor had split around 4.63 Mya from their ancestor. M. cervicolor, M. cookii and M. caroli diverged between 4.70 and 3.36 Mya, which indicates that M. terricolor and the Asian lineages evolved simultaneously. M. spretus is expected to have diverged nearly 2.9 Mya from their most recent common ancestor.

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.

Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

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.

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.

Malignant round cell tumor of bone with EWSR1-NFATC2 gene fusion.

Gene rearrangements involving the Ewing sarcoma breakpoint region 1 (EWSR1) gene are seen in a broad range of sarcomas and some nonmesenchymal neoplasms. Ewing sarcoma is molecularly defined by a fusion of the EWSR1 gene (or rarely the related FUS gene) to a member of the E26 transformation-specific (ETS) family of transcription factors, frequently the EWSR1-FLI1 fusion. More recently, EWSR1 gene fusion to non-ETS family members, including the nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) gene, has been reported in a histological variant of Ewing sarcoma. Here, we report a malignant round cell tumor of bone with an EWSR1-NFATC2 fusion gene. This report builds upon the unusual morphological and clinical presentation of bone neoplasms containing an EWSR1-NFATC2 fusion gene.

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.

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.

High prevalence of respiratory ciliary dysfunction in congenital heart disease patients with heterotaxy.

BACKGROUND: Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. METHODS AND RESULTS: We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. CONCLUSIONS: Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.

Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models.

BACKGROUND: GJA1 gene encodes a gap junction protein known as connexin 43 (Cx43). Cx43 is abundantly expressed in the ventricular myocardium and in cardiac neural crest cells. Cx43 is proposed to play an important role in human congenital heart disease, as GJA1 knock-out mice die neonatally from outflow tract obstruction. In addition, patients with visceroatrial heterotaxia or hypoplastic left heart syndrome were reported to have point mutations in GJA1 at residues that affect protein kinase phosphorylation and gating of the gap junction channel. However, as these clinical findings were not replicated in subsequent studies, the question remains about the contribution of GJA1 mutations in human congenital heart disease (CHD). MATERIALS AND METHODS: We analyzed the GJA1 coding sequence in 300 patients with CHD from two clinical centers, focusing on outflow tract anomalies. This included 152 with Tetralogy of Fallot from over 200 patients exhibiting outflow tract anomalies, as well as other structural heart defects including atrioventricular septal defects and other valvar anomalies. Our sequencing analysis revealed only two silent nucleotide substitutions in 8 patients. To further assess the possible role of Cx43 in CHD, we also generated two knock-in mouse models with point mutations at serine residues subject to protein kinase C or casein kinase phosphorylation, sites that are known to regulate gating and trafficking of Cx43, respectively. RESULTS: Both heterozygous and homozygous knock-in mice were long term viable and did not exhibit overt CHD. CONCLUSION: The combined clinical and knock-in mouse mutant studies indicate GJA1 mutation is not likely a major contributor to CHD, especially those involving outflow tract anomalies.

Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics.

Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs) to examine polarized cell morphology and directional cell movement. Studies using embryonic fibroblasts from Cx43 knockout (Cx43KO) mice showed Cx43 deficiency caused cell polarity defects as characterized by a failure of the Golgi apparatus and the microtubule organizing center to reorient with the direction of wound closure. Actin stress fibers at the wound edge also failed to appropriately align, and stabilized microtubule (Glu-tubulin) levels were markedly reduced. Forced expression of Cx43 with deletion of its tubulin-binding domain (Cx43dT) in both wildtype MEFs and neural crest cell explants recapitulated the cell migration defects seen in Cx43KO cells. However, forced expression of Cx43 with point mutation causing gap junction channel closure had no effect on cell motility. TIRF imaging revealed increased microtubule instability in Cx43KO cells, and microtubule targeting of membrane localized Cx43 was reduced with expression of Cx43dT construct in wildtype cells. Together, these findings suggest the essential role of Cx43 gap junctions in development is mediated by regulation of the tubulin cytoskeleton and cell polarity by Cx43 via a nonchannel function.

Disruption of Mks1 localization to the mother centriole causes cilia defects and developmental malformations in Meckel-Gruber syndrome.

Meckel-Gruber syndrome (MKS) is a recessive disorder resulting in multiple birth defects that are associated with mutations affecting ciliogenesis. We recovered a mouse mutant with a mutation in the Mks1 gene (Mks1(del64-323)) that caused a 260-amino-acid deletion spanning nine amino acids in the B9 domain, a protein motif with unknown function conserved in two other basal body proteins. We showed that, in wild-type cells, Mks1 was localized to the mother centriole from which the cilium was generated. However, in mutant Mks1(del64-323) cells, Mks1 was not localized to the centriole, even though it maintained a punctate distribution. Resembling MKS patients, Mks1 mutants had craniofacial defects, polydactyly, congenital heart defects, polycystic kidneys and randomized left-right patterning. These defects reflected disturbance of functions subserved by motile and non-motile cilia. In the kidney, glomerular and tubule cysts were observed along with short cilia, and cilia were reduced in number to a near-complete loss. Underlying the left-right patterning defects were fewer and shorter nodal cilia, and analysis with fluorescent beads showed no directional flow at the embryonic node. In the cochlea, the stereocilia were mal-patterned, with the kinocilia being abnormally positioned. Together, these defects suggested disruption of planar cell polarity, which is known to regulate node, kidney and cochlea development. In addition, we also showed that Shh signaling was disrupted. Thus, in the neural tube, the floor plate was not specified posteriorly even as expression of the Shh mediator Gli2 increased. By contrast, the Shh signaling domain was expanded in the anterior neural tube and anterior limb bud, consistent with reduced Gli3-repressor (Gli3R) function. The latter probably accounted for the preaxial digit duplication exhibited by the Mks1(del64-323) mutants. Overall, these findings indicate that centriole localization of Mks1 is required for ciliogenesis of motile and non-motile cilia, but not for centriole assembly. On the basis of these results, we hypothesize a role for the B9 domain in mother centriole targeting, a possibility that warrants further future investigations.