An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Atlas of Metastatic Gastric Cancer Links Ferroptosis to Disease Progression and Immunotherapy Response.

BACKGROUND & AIMS: Metastases from gastric adenocarcinoma (GAC) lead to high morbidity and mortality. Developing innovative and effective therapies requires a comprehensive understanding of the tumor and immune biology of advanced GAC. Yet, collecting matched specimens from advanced, treatment-naïve patients with GAC poses a significant challenge, limiting the scope of current research, which has focused predominantly on localized tumors. This gap hinders deeper insight into the metastatic dynamics of GAC. METHODS: We performed in-depth single-cell transcriptome and immune profiling on 68 paired, treatment-naïve, primary metastatic tumors to delineate alterations in cancer cells and their tumor microenvironment during metastatic progression. To validate our observations, we conducted comprehensive functional studies both in vitro and in vivo, using cell lines and multiple patient-derived xenograft and novel mouse models of GAC. RESULTS: Liver and peritoneal metastases exhibited distinct properties in cancer cells and dynamics of tumor microenvironment phenotypes, supporting the notion that cancer cells and their local tumor microenvironments co-evolve at metastatic sites. Our study also revealed differential activation of cancer meta-programs across metastases. We observed evasion of cancer cell ferroptosis via GPX4 up-regulation during GAC progression. Conditional depletion of Gpx4 or pharmacologic inhibition of ferroptosis resistance significantly attenuated tumor growth and metastatic progression. In addition, ferroptosis-resensitizing treatments augmented the efficacy of chimeric antigen receptor T-cell therapy. CONCLUSIONS: This study represents the largest single-cell dataset of metastatic GACs to date. High-resolution mapping of the molecular and cellular dynamics of GAC metastasis has revealed a rationale for targeting ferroptosis defense in combination with chimeric antigen receptor T-cell therapy as a novel therapeutic strategy with potential immense clinical implications.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

CIC missense variants contribute to susceptibility for spina bifida.

Neural tube defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to the cerebral folate deficiency syndrome by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole-genome sequencing (WGS) data of 140 isolated spina bifida cases and identified eight missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased the FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants downregulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.

FKBP8 variants are risk factors for spina bifida.

Neural tube defects (NTDs) are a group of severe congenital malformations caused by a failure of neural tube closure during early embryonic development. Although extensively investigated, the genetic etiology of NTDs remains poorly understood. FKBP8 is critical for proper mammalian neural tube closure. Fkbp8-/- mouse embryos showed posterior NTDs consistent with a diagnosis of spina bifida (SB). To date, no publication has reported any association between FKBP8 and human NTDs. Using Sanger sequencing on genomic DNA samples from 472 SB and 565 control samples, we identified five rare (MAF ≤ 0.001) deleterious variants in SB patients, while no rare deleterious variant was identified in the controls (P = 0.0191). p.Glu140* affected FKBP8 localization to the mitochondria and created a truncated form of the FKBP8 protein, thus impairing its interaction with BCL2 and ultimately leading to an increase in cellular apoptosis. p.Ser3Leu, p.Lys315Asn and p.Ala292Ser variants decreased FKBP8 protein level. p.Lys315Asn further increased the cellular apoptosis. RNA sequencing on anterior and posterior tissues isolated from Fkbp8-/- and wildtype mice at E9.5 and E10.5 showed that Fkbp8-/- embryos have an abnormal expression profile within tissues harvested at posterior sites, thus leading to a posterior NTD. Moreover, we found that Fkbp8 knockout mouse embryos have abnormal expression of Wnt3a and Nkx2.9 during the early stage of neural tube development, perhaps also contributing to caudal specific NTDs. These findings provide evidence that functional variants of FKBP8 are risk factors for SB, which may involve a novel mechanism by which Fkbp8 mutations specifically cause SB in mice.

CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression.

Background Cerebral folate deficiency (CFD) syndrome is characterised by a low concentration of 5-methyltetrahydrofolate in cerebrospinal fluid, while folate levels in plasma and red blood cells are in the low normal range. Mutations in several folate pathway genes, including FOLR1 (folate receptor alpha, FRα), DHFR (dihydrofolate reductase) and PCFT (proton coupled folate transporter) have been previously identified in patients with CFD. Methods In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis on eight CFD trios and identified eight de novo mutations in seven trios. Results Notably, we found a de novo stop gain mutation in the capicua (CIC) gene. Using 48 sporadic CFD samples as a validation cohort, we identified three additional rare variants in CIC that are putatively deleterious mutations. Functional analysis indicates that CIC binds to an octameric sequence in the promoter regions of folate transport genes: FOLR1, PCFT and reduced folate carrier (Slc19A1; RFC1). The CIC nonsense variant (p.R353X) downregulated FOLR1 expression in HeLa cells as well as in the induced pluripotent stem cell (iPSCs) derived from the original CFD proband. Folate binding assay demonstrated that the p.R353X variant decreased cellular binding of folic acid in cells. Conclusion This study indicates that CIC loss of function variants can contribute to the genetic aetiology of CFD through regulating FOLR1 expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD.

Loss of RAD9B impairs early neural development and contributes to the risk for human spina bifida.

DNA damage response (DDR) genes orchestrating the network of DNA repair, cell cycle control, are essential for the rapid proliferation of neural progenitor cells. To date, the potential association between specific DDR genes and the risk of human neural tube defects (NTDs) has not been investigated. Using whole-genome sequencing and targeted sequencing, we identified significant enrichment of rare deleterious RAD9B variants in spina bifida cases compared to controls (8/409 vs. 0/298; p = .0241). Among the eight identified variants, the two frameshift mutants and p.Gln146Glu affected RAD9B nuclear localization. The two frameshift mutants also decreased the protein level of RAD9B. p.Ser354Gly, as well as the two frameshifts, affected the cell proliferation rate. Finally, p.Ser354Gly, p.Ser10Gly, p.Ile112Met, p.Gln146Glu, and the two frameshift variants showed a decreased ability for activating JNK phosphorylation. RAD9B knockdowns in human embryonic stem cells profoundly affected early differentiation through impairing PAX6 and OCT4 expression. RAD9B deficiency impeded in vitro formation of neural organoids, a 3D cell culture model for human neural development. Furthermore, the RNA-seq data revealed that loss of RAD9B dysregulates cell adhesion genes during organoid formation. These results represent the first demonstration of a DDR gene as an NTD risk factor in humans.

Approaches to studying the genomic architecture of complex birth defects.

Every year nearly 6 percent of children worldwide are born with a serious congenital malformation, resulting in death or lifelong disability. In the United States, birth defects remain one of the leading causes of infant mortality. Among the common structural congenital defects are conditions known as neural tube defects (NTDs). These are a class of malformation of the brain and spinal cord where the neural tube fails to close during the neurulation. Although NTDs remain among the most pervasive and debilitating of all human developmental anomalies, there is insufficient understanding of their etiology. Previous studies have proposed that complex birth defects like NTDs are likely omnigenic, involving interconnected gene regulatory networks with associated signals throughout the genome. Advances in technologies have allowed researchers to more critically investigate regulatory gene networks in ever increasing detail, informing our understanding of the genetic basis of NTDs. Employing a systematic analysis of these complex birth defects using massively parallel DNA sequencing with stringent bioinformatic algorithms, it is possible to approach a greater level of understanding of the genomic architecture underlying NTDs. Herein, we present a brief overview of different approaches undertaken in our laboratory to dissect out the genetics of susceptibility to NTDs. This involves the use of mouse models to identify candidate genes, as well as large scale whole genome/whole exome (WGS/WES) studies to interrogate the genomic landscape of NTDs. The goal of this research is to elucidate the gene-environment interactions contributing to NTDs, thus encouraging global research efforts in their prevention.

Acetylation promotes TyrRS nuclear translocation to prevent oxidative damage.

Tyrosyl-tRNA synthetase (TyrRS) is well known for its essential aminoacylation function in protein synthesis. Recently, TyrRS has been shown to translocate to the nucleus and protect against DNA damage due to oxidative stress. However, the mechanism of TyrRS nuclear localization has not yet been determined. Herein, we report that TyrRS becomes highly acetylated in response to oxidative stress, which promotes nuclear translocation. Moreover, p300/CBP-associated factor (PCAF), an acetyltransferase, and sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, regulate the nuclear localization of TyrRS in an acetylation-dependent manner. Oxidative stress increases the level of PCAF and decreases the level of SIRT1 and deacetylase activity, all of which promote the nuclear translocation of hyperacetylated TyrRS. Furthermore, TyrRS is primarily acetylated on the K244 residue near the nuclear localization signal (NLS), and acetylation inhibits the aminoacylation activity of TyrRS. Molecular dynamics simulations have shown that the in silico acetylation of K244 induces conformational changes in TyrRS near the NLS, which may promote the nuclear translocation of acetylated TyrRS. Herein, we show that the acetylated K244 residue of TyrRS protects against DNA damage in mammalian cells and zebrafish by activating DNA repair genes downstream of transcription factor E2F1. Our study reveals a previously unknown mechanism by which acetylation regulates an aminoacyl-tRNA synthetase, thus affecting the repair pathways for damaged DNA.

Genomic Staging of Multifocal Lung Squamous Cell Carcinomas Is Independent of the Comprehensive Morphologic Assessment.

INTRODUCTION: Morphologic and molecular data for staging of multifocal lung squamous cell carcinomas (LSCCs) are limited. In this study, whole exome sequencing (WES) was used as the gold standard to determine whether multifocal LSCC represented separate primary lung cancers (SPLCs) or intrapulmonary metastases (IPMs). Genomic profiles were compared with the comprehensive morphologic assessment. METHODS: WES was performed on 20 tumor pairs of multifocal LSCC and matched normal lymph nodes using the Illumina NovaSeq6000 S4-Xp (Illumina, San Diego, CA). WES clonal and subclonal analysis data were compared with histologic assessment by 16 thoracic pathologists. In addition, the immune gene profiling of the study cases was characterized by the HTG EdgeSeq Precision Immuno-Oncology Panel. RESULTS: By WES data, 11 cases were classified as SPLC and seven cases as IPM. Two cases were technically suboptimal. Analysis revealed marked genomic and immunogenic heterogeneity, but immune gene expression profiles highly correlated with mutation profiles. Tumors classified as IPM have a large number of shared mutations (ranging from 33.5% to 80.7%). The agreement between individual morphologic assessments for each case and WES was 58.3%. One case was unanimously interpreted morphologically as IPM and was in agreement with WES. In a further 17 cases, the number of pathologists whose morphologic interpretation was in agreement with WES ranged from two (one case) to 15 pathologists (one case) per case. Pathologists showed a fair interobserver agreement in the morphologic staging of multiple LSCCs, with an overall kappa of 0.232. CONCLUSIONS: Staging of multifocal LSCC based on morphologic assessment is unreliable. Comprehensive genomic analyses should be adopted for the staging of multifocal LSCC.

KDM6B Variants May Contribute to the Pathophysiology of Human Cerebral Folate Deficiency.

(1) Background: The genetic etiology of most patients with cerebral folate deficiency (CFD) remains poorly understood. KDM6B variants were reported to cause neurodevelopmental diseases; however, the association between KDM6B and CFD is unknown; (2) Methods: Exome sequencing (ES) was performed on 48 isolated CFD cases. The effect of KDM6B variants on KDM6B protein expression, Histone H3 lysine 27 epigenetic modification and FOLR1 expression were examined in vitro. For each patient, serum FOLR1 autoantibodies were measured; (3) Results: Six KDM6B variants were identified in five CFD patients, which accounts for 10% of our CFD cohort cases. Functional experiments indicated that these KDM6B variants decreased the amount of KDM6B protein, which resulted in elevated H3K27me2, lower H3K27Ac and decreased FOLR1 protein concentrations. In addition, FOLR1 autoantibodies have been identified in serum; (4) Conclusion: Our study raises the possibility that KDM6B may be a novel CFD candidate gene in humans. Variants in KDM6B could downregulate FOLR1 gene expression, and might also predispose carriers to the development of FOLR1 autoantibodies.

Hypermethylation of PI3K-AKT signalling pathway genes is associated with human neural tube defects.

Neural tube defects (NTDs) are a group of common and severe congenital malformations. The PI3K-AKT signalling pathway plays a crucial role in the neural tube development. There is limited evidence concerning any possible association between aberrant methylation in PI3K-AKT signalling pathway genes and NTDs. Therefore, we aimed to investigate potential associations between aberrant methylation of PI3K-AKT pathway genes and NTDs. Methylation studies of PI3K-AKT pathway genes utilizing microarray genome-methylation data derived from neural tissues of ten NTD cases and eight non-malformed controls were performed. Targeted DNA methylation analysis was subsequently performed in an independent cohort of 73 NTD cases and 32 controls to validate the methylation levels of identified genes. siRNAs were used to pull-down the target genes in human embryonic stem cells (hESCs) to examine the effects of the aberrant expression of target genes on neural cells. As a result, 321 differentially hypermethylated CpG sites in the promoter regions of 30 PI3K-AKT pathway genes were identified in the microarray data. In target methylation analysis, CHRM1, FGF19, and ITGA7 were confirmed to be significantly hypermethylated in NTD cases and were associated with increased risk for NTDs. The down-regulation of FGF19, CHRM1, and ITGA7 impaired the formation of rosette-like cell aggregates. The down-regulation of those three genes affected the expression of PAX6, SOX2 and MAP2, implying their influence on the differentiation of neural cells. This study for the first time reported that hypermethylation of PI3K-AKT pathway genes such as CHRM1, FGF19, and ITGA7 is associated with human NTDs.

The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma.

Tumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD. SIGNIFICANCE: While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy. This article is highlighted in the In This Issue feature, p. 2483.

Gene Environment Interactions in the Etiology of Neural Tube Defects.

Human structural congenital malformations are the leading cause of infant mortality in the United States. Estimates from the United States Center for Disease Control and Prevention (CDC) determine that close to 3% of all United States newborns present with birth defects; the worldwide estimate approaches 6% of infants presenting with congenital anomalies. The scientific community has recognized for decades that the majority of birth defects have undetermined etiologies, although we propose that environmental agents interacting with inherited susceptibility genes are the major contributing factors. Neural tube defects (NTDs) are among the most prevalent human birth defects and as such, these malformations will be the primary focus of this review. NTDs result from failures in embryonic central nervous system development and are classified by their anatomical locations. Defects in the posterior portion of the neural tube are referred to as meningomyeloceles (spina bifida), while the more anterior defects are differentiated as anencephaly, encephalocele, or iniencephaly. Craniorachischisis involves a failure of the neural folds to elevate and thus disrupt the entire length of the neural tube. Worldwide NTDs have a prevalence of approximately 18.6 per 10,000 live births. It is widely believed that genetic factors are responsible for some 70% of NTDs, while the intrauterine environment tips the balance toward neurulation failure in at risk individuals. Despite aggressive educational campaigns to inform the public about folic acid supplementation and the benefits of providing mandatory folic acid food fortification in the United States, NTDs still affect up to 2,300 United States births annually and some 166,000 spina bifida patients currently live in the United States, more than half of whom are now adults. Within the context of this review, we will consider the role of maternal nutritional status (deficiency states involving B vitamins and one carbon analytes) and the potential modifiers of NTD risk beyond folic acid. There are several well-established human teratogens that contribute to the population burden of NTDs, including: industrial waste and pollutants [e.g., arsenic, pesticides, and polycyclic aromatic hydrocarbons (PAHs)], pharmaceuticals (e.g., anti-epileptic medications), and maternal hyperthermia during the first trimester. Animal models for these teratogens are described with attention focused on valproic acid (VPA; Depakote). Genetic interrogation of model systems involving VPA will be used as a model approach to discerning susceptibility factors that define the gene-environment interactions contributing to the etiology of NTDs.

Wnt1 Lineage Specific Deletion of Gpr161 Results in Embryonic Midbrain Malformation and Failure of Craniofacial Skeletal Development.

Sonic hedgehog (Shh) signaling regulates multiple morphogenetic processes during embryonic neurogenesis and craniofacial skeletal development. Gpr161 is a known negative regulator of Shh signaling. Nullizygous Gpr161 mice are embryonic lethal, presenting with structural defects involving the neural tube and the craniofacies. However, the lineage specific role of Gpr161 in later embryonic development has not been thoroughly investigated. We studied the Wnt1-Cre lineage specific role of Gpr161 during mouse embryonic development. We observed three major gross morphological phenotypes in Gpr161 cKO (Gpr161 f/f; Wnt1-Cre) fetuses; protrusive tectum defect, encephalocele, and craniofacial skeletal defect. The overall midbrain tissues were expanded and cell proliferation in ventricular zones of midbrain was increased in Gpr161 cKO fetuses, suggesting that protrusive tectal defects in Gpr161 cKO are secondary to the increased proliferation of midbrain neural progenitor cells. Shh signaling activity as well as upstream Wnt signaling activity were increased in midbrain tissues of Gpr161 cKO fetuses. RNA sequencing further suggested that genes in the Shh, Wnt, Fgf and Notch signaling pathways were differentially regulated in the midbrain of Gpr161 cKO fetuses. Finally, we determined that cranial neural crest derived craniofacial bone formation was significantly inhibited in Gpr161 cKO fetuses, which partly explains the development of encephalocele. Our results suggest that Gpr161 plays a distinct role in midbrain development and in the formation of the craniofacial skeleton during mouse embryogenesis.

Somatic and de novo Germline Variants of MEDs in Human Neural Tube Defects.

BACKGROUND: Neural tube defects (NTDs) are among the most common and severe congenital defects in humans. Their genetic etiology is complex and remains poorly understood. The Mediator complex (MED) plays a vital role in neural tube development in animal models. However, no studies have yet examined the role of its human homolog in the etiology of NTDs. METHODS: In this study, 48 pairs of neural lesion site and umbilical cord tissues from NTD and 21 case-parent trios were involved in screening for NTD-related somatic and germline de novo variants. A series of functional cell assays were performed. We generated a Med12 p.Arg1784Cys knock-in mouse using CRISPR/Cas9 technology to validate the human findings. RESULTS: One somatic variant, MED12 p.Arg1782Cys, was identified in the lesion site tissue from an NTD fetus. This variant was absent in any other normal tissue from different germ layers of the same case. In 21 case-parent trios, one de novo stop-gain variant, MED13L p.Arg1760∗, was identified. Cellular functional studies showed that MED12 p.Arg1782Cys decreased MED12 protein level and affected the regulation of MED12 on the canonical-WNT signaling pathway. The Med12 p.Arg1784Cys knock-in mouse exhibited exencephaly and spina bifida. CONCLUSION: These findings provide strong evidence that functional variants of MED genes are associated with the etiology of some NTDs. We demonstrated a potentially important role for somatic variants in the occurrence of NTDs. Our study is the first study in which an NTD-related variant identified in humans was validated in mice using CRISPR/Cas9 technology.

Actuation enhances patterning in human neural tube organoids.

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

Variants identified in PTK7 associated with neural tube defects.

BACKGROUND: Variants in planar cell polarity (PCP) pathway genes have been repeatedly implicated in the pathogenesis of NTDs in both mouse models and in human cohorts. Mouse models indicate that the homogenous disruption of the Ptk7 gene, a PCP regulator, results in craniorachischisis; while embryos that are doubly heterozygous for Ptk7XST87 and Vangl2Lp mutations present with spina bifida. METHODS: In this study, we initially sequenced exons of the human PTK7 gene in 192 spina bifida patients and 190 controls from a California population. A phase II validation study was performed in 343 Chinese NTD cohort. Functional assays including immunoblotting and immunoprecipitation were used to study identified variants effect on PTK7 function. RESULTS: We identified three rare (MAF <0.001) missense heterozygous PTK7 variants (NM_001270398.1:c.581C>T, p.Arg630Ser and p.Tyr725Phe) in the spina bifida patients. In our functional analyses, p.Arg630Ser affected PTK7 mutant protein stability and increased interaction with Dvl2, while the p.Thr186Met variant decreased PTK7 interactions with Dvl2. No novel predicted-to-be-damaging variant or function-disrupted PTK7 variant was identified among the control subjects. We subsequently re-sequenced the PTK7 CDS region in 343 NTDs from China to validate the association between PTK7 and NTDs. The frequency of PTK7 rare missense variants in the Chinese NTD samples is significantly higher than in gnomAD controls. CONCLUSION: Our study suggests that rare missense variants in PTK7 contribute to the genetic risk of NTDs.

Whole-Exome Sequencing Identifies Damaging de novo Variants in Anencephalic Cases.

BACKGROUND: Anencephaly is a lethal neural tube defect (NTD). Although variants in several genes have been implicated in the development of anencephaly, a more complete picture of variants in the genome, especially de novo variants (DNVs), remains unresolved. We aim to identify DNVs that play an important role in the development of anencephaly by performing whole-exome DNA sequencing (WES) of proband-parent trios. RESULTS: A total of 13 DNVs were identified in 8 anencephaly trios by WES, including two loss of function (LoF) variants detected in pLI > 0.9 genes (SPHKAP, c.2629_2633del, and NCOR1, p.Y1907X). Damaging DNVs were identified in 61.5% (8/13) of the anencephalic cases. Independent validation was conducted in an additional 502 NTD cases. Gene inactivation using targeted morpholino antisense oligomers and rescue assays were conducted in zebrafish, and transfection expression in HEK293T cells. Four DNVs in four cases were identified and predicted to alter protein function, including p.R328Q in WD repeat domain phosphoinositide-interacting 1 (WIPI1). Three variants, p.G313R, p.T418M, and p.L406P, in the WIPI1 gene were identified from the independent replication cohort consisting of 502 cases. Functional analysis suggested that the wipi1 p.L406P and p.R328Q variants most likely displayed loss-of-function effects during embryonic development. CONCLUSION: De novo damaging variants are the main culprit for majority of anencephalic cases. Missense variants in WIPI1 may play a role in the genetic etiology of anencephaly, and LoF variants in SPHKAP and NCOR1 may also contribute to anencephaly. These findings add to our existing understanding of the genetic mechanisms of NTD formation.