Thrombosis is a leading cause of morbidity and mortality. We used a mouse forward genetic ENU screen to identify genomic variants that suppress F5 L/L Tfpi +/- lethal thrombosis. Surviving F5 L/L Tfpi +/- mice from our M odifier of F actor 5 L eiden 16 ( MF5L16 ) ENU line were subjected to whole-genome sequencing analysis. This revealed that instead of an ENU-induced mutation, four mutations introduced from our F5 L/L breeding stock were responsible for survival, which we named sMF5L1-4 for s pontaneous M odifier of F actor 5 L eiden. In our colony, F5 L/L female breeders carrying all four sMF5L mutations produced more litters and offspring than breeders with three or fewer mutations (p<0.006). Genotyping of 13 additional MF5L lines demonstrated that the four sMF5L mutations were present in all lines and were consistently associated with survival. Of these four mutations, a single G to A intergenic variant on Chromosome 18 (Chr18 A , sMF5L4 ), was most significantly associated with survival (p=0.003), with ∼15% penetrance for conferring the survival phenotype. Furthermore, platelet aggregation was significantly reduced in Chr18 A mice, suggesting an additional mechanism by which Chr18 A could suppress lethal thrombosis. Comparative transcriptomics analysis of livers from Chr18 A mice versus wildtype littermate controls revealed a small number of differentially expressed genes both known and unknown to affect thrombosis. In summary, we have identified four variants exerting a significant selective breeding advantage along with antithrombotic effects. Superimposing our mutagenesis screen on a selective background illustrates the interplay of natural strain background variants and de novo ENU mutations in suppressing F5 L/L Tfpi +/- lethal thrombosis.

The gamma-tubulin ring complex (γ-TuRC) plays a role in coordinating centrosome and spindle pole body formation during cell division. TUBG1 encodes a critical component of the γ-TuRC. Pathogenic TUBG1 variants can cause a range of alterations in cortical gyral patterning, microcephaly, and other neurological manifestations. We describe two missense variants in TUBG1 and their associated clinical phenotypes. One individual has microcephaly, epilepsy, and a simplified gyral pattern with a TUBG1 variant interpreted as pathogenic. The other individual has a likely pathogenic TUBG1 variant that explains the milder presentation of autism spectrum disorder, intellectual disability, later-onset well-controlled epilepsy, a normocephalic head size, and no detectable structural abnormalities on neuroimaging.

BackgroundVariants inPPP2R5D, affecting the regulatory B56δ subunit of protein phosphatase 2A (PP2A), have been identified in individuals with neurodevelopmental abnormalities. However, the molecular and clinical spectra remain incompletely understood.MethodsIndividuals withPPP2R5Dvariants were enrolled through Simons Variation in Individuals Project/Simons Searchlight. Data were collected from medical history interviews, medical record review, online validated instruments and neuroimaging review. Genetic variants were biochemically characterised.ResultsWe studied 76 individuals withPPP2R5Dvariants, including 68 with pathogenic de novo variants, four with a variant of uncertain significance (VUS) and four siblings with a novel dominantly inherited pathogenic variant. Among 13 pathogenic variants, eight were novel and two (p.Glu198Lys and p.Glu200Lys) were highly recurrent. Functional analysis revealed impaired PP2A A/C-subunit binding, decreased short linear interaction motif-dependent substrate binding or both—with the most severe phenotypes associated with variants that completely retained one of these binding characteristics and lost the other—further supporting a dominant-negative disease mechanism. p.Glu198Lys showed the highest C-binding defect and a more severe clinical phenotype. The inherited p.Glu197Gly variant had a mild substrate binding defect, and three of four VUS had no biochemical impact. Common clinical phenotypes were language, intellectual or learning disabilities (80.6%), hypotonia (75.0%), macrocephaly (66.7%), seizures (45.8%) and autism spectrum disorder (26.4%). The mean composite Vineland score was 59.8, and most participants were in the ‘moderate to low’ and ‘low’ adaptive levels in all domains.ConclusionOur study delineates the most common features ofPPP2R5D-related neurodevelopmental disorders, expands the clinical and molecular spectrum and identifies genotype–phenotype correlations.

Craniosynostosis is a birth defect where calvarial sutures close prematurely, as part of a genetic syndrome or independently, with unknown cause. This study aimed to identify differences in gene expression in primary calvarial cell lines derived from patients with four phenotypes of single-suture craniosynostosis, compared to controls. Calvarial bone samples (N = 388 cases/85 controls) were collected from clinical sites during reconstructive skull surgery. Primary cell lines were then derived from the tissue and used for RNA sequencing. Linear models were fit to estimate covariate adjusted associations between gene expression and four phenotypes of single-suture craniosynostosis (lambdoid, metopic, sagittal, and coronal), compared to controls. Sex-stratified analysis was also performed for each phenotype. Differentially expressed genes (DEGs) included 72 genes associated with coronal, 90 genes associated with sagittal, 103 genes associated with metopic, and 33 genes associated with lambdoid craniosynostosis. The sex-stratified analysis revealed more DEGs in males (98) than females (4). There were 16 DEGs that were homeobox (HOX) genes. Three TFs (SUZ12, EZH2, AR) significantly regulated expression of DEGs in one or more phenotypes. Pathway analysis identified four KEGG pathways associated with at least one phenotype of craniosynostosis. Together, this work suggests unique molecular mechanisms related to craniosynostosis phenotype and fetal sex.

To discover genes implicated in human congenital disorders, we performed ENU mutagenesis in the mouse and screened for mutations affecting embryonic development. In this work, we report defects of heart development in mice homozygous for a mutation of coactivator-associated arginine methyltransferase 1 (Carm1). While Carm1 has been extensively studied, it has never been previously associated with a role in heart development. Phenotype analysis combining histology and microcomputed tomography imaging shows a range of cardiac defects. Most notably, many affected midgestation embryos appear to have cardiac rupture and hemorrhaging in the thorax. Mice that survive to late gestation show a variety of cardiac defects, including ventricular septal defects, double outlet right ventricle, and persistent truncus arteriosus. Transcriptome analyses of the mutant embryos by mRNA-seq reveal the perturbation of several genes involved in cardiac morphogenesis and muscle development and function. In addition, we observe the mislocalization of cardiac neural crest cells at E12.5 in the outflow tract. The cardiac phenotype of Carm1 mutant embryos is similar to that of Pax3 null mutants, and PAX3 is a putative target of CARM1. However, our analysis does not support the hypothesis that developmental defects in Carm1 mutant embryos are primarily due to a functional defect of PAX3.

The Megalencephaly-capillary malformation (MCAP) syndrome is an overgrowth syndrome caused by mosaic gain-of-function (activating) variants in the PIK3CA gene. This multi-system disorder is characterized by megalencephaly (MEG) or hemimegalencaphly (HMEG), cutaneous vascular malformations, variable somatic overgrowth, digital anomalies, and connective tissue laxity. Epilepsy is commonly associated with MCAP, and a subset of affected individuals have cortical malformations which may require resective epilepsy surgery. Similar to other mosaic disorders, establishing a molecular diagnosis for individuals with MCAP is largely achieved by sequencing affected or lesional tissues (such as brain or skin), with a very low diagnostic yield from testing peripheral samples (such as peripheral blood). Therefore, in individuals where lesional tissues are scarce or unavailable, including individuals with MCAP who are not candidates for epilepsy surgery, establishing a molecular diagnosis can be challenging. CSF-derived cell free DNA (cfDNA) has recently emerged as novel tool for genomic profiling in brain tumors, as well more recently other types of PIK3CA-related somatic disorders including lymphatic and vascular malformations. Here we report on the utility of CSF-derived cfDNA for the molecular diagnosis of an individual with MCAP syndrome harboring a highly mosaic PIK3CA variant (c.3139C>T, p.His1047Tyr). The proband is a male who was identified shortly after birth to have megalencephaly, right hemihypertrophy as well as abnormal skin pigmentation with extensive deep purple-red vascular markings widely distributed over the body. Brain magnetic resonance imaging (MRI) revealed significant megalencephaly, with asymmetric cerebral hemispheres and cerebellar tonsillar herniation. On follow-up evaluations, he developed progressive hydrocephalus requiring ventriculoperitoneal shunt placement at age six months. He also underwent laser ablation treatment for the extensive capillary malformations on his upper lip and right cheek. Medical history is also notable for intestinal lymphangiectasia leading to episodes of diarrhea and nutritional deficiency during early childhood. Developmentally, he had severe delays in gross motor development, including inability to walk independently until after three years of age, moderate speech delays and significant neurobehavioral issues. Serial physical examinations confirmed his progressive macrocephaly as well as right hemihypertrophy involving the face and extremities with several measurements performed, as follows: at age three years, right ear was 6 cm (97th percentile) while the left was 4.7 cm (50th percentile); right hand measured 9.9 cm (3rd percentile), and left hand measured 9.3 cm (∼ 1st percentile). His occipito-frontal circumference (OFC) at three years of age was 58.5 cm (+5.8 SD) and later increased to 65.5 cm (+7 SD) at 19 years of age. He also had pinpoint elevated capillary malformations that range in size from 5 x 5 mm to approximately 1 x 1 cm, with a prominent venous pattern all over the scalp. Following laser ablation, he had residual vascular staining of his right cheek, on his right upper lip, and more extensive irregular vascular pattern over his left arm. During his late childhood, he had recurrent lymphedema, protein-losing enteropathy and pleural effusions. At age 19 years, he was admitted to the pediatric intensive care unit (PICU) due to recurrent pleural effusions. He was later found to have atypical lymphocytes in pleural fluid with increased fluorodeoxyglucose (FDG) uptake in bilateral cervical, mediastinal, abdominal, and pelvic lymph nodes on entire body Positron Emission Tomography (PET) imaging. Excisional biopsy of cervical lymph nodes showed sheets of atypical cells with large, vesicular nuclei with prominent nucleoli and scanty cytoplasm. Immunocytochemical staining and flow cytometry immunotyping confirmed the diagnosis of diffuse large B-cell lymphoma (DLBCL, stage III b). He underwent a lumbar puncture (LP) for tumor staging as well as during the course of chemotherapy, and additional CSF was collected for molecular diagnostics as well. CSF-derived cfDNA was collected in Cell-Free DNA BCT tubes and processed using established protocols. He completed chemotherapy without further remission. Brain MRI obtained during this period showed asymmetric megalencephaly with mildly abnormal cortical gyral pattern, asymmetric dysplastic ventricles and cerebellar tonsillar ectopia. He only had three brief seizures for a short period of time during his chemotherapy which responded well to levetiracetam. The proband first presented to our genetic team and underwent molecular diagnostic testing around the time when his lymphoma was diagnosed. We performed targeted sequencing using the Megaplex panel at the University of Washington. This panel offers ultra-deep sequencing of 63 megalencephaly and overgrowth genes. Testing was performed on several samples including CSF-derived cfDNA, peripheral blood and cultured fibroblasts from affected skin from the left arm. GRCh37/hg19 assembly was used. A mosaic variant in PIK3CA (NM_006218.2: c.3139 C>T, p.His1047Tyr) was identified in CSF cfDNA at variant allele fractions (VAF) of 3.08% (var/ref read: 14/440), as well as in peripheral blood (VAF 2%; var/ref read: 8/394), and skin fibroblasts (VAF 37.31%; var/ref read: 673/1131). These results molecularly confirmed his diagnosis of MCAP as well as provided insights on the range of mosaicism across these samples. Cell-free DNA-based diagnostic approaches have become robust and reliable molecular tools in prenatal genetics, as well as for cancer diagnosis, treatment, and monitoring. Plasma and cystic fluid cfDNA have also been recently successfully used to detect somatic variants associated with PIK3CA- associated lymphatic and vascular malformations. Altogether, these lines of evidence suggest that cfDNA from various body fluids in direct contact with pathological tissues can be utilized as a "proxy" for molecular diagnostics. In this study, we report on the molecular diagnostic yield of cfDNA from CSF in an individual with MCAP syndrome who had significant neurological involvement. Using CSF-derived cfDNA has several important clinical implications in mosaic brain disorders. cfDNA from CSF provides a more practical and less invasive option to establish a molecular diagnosis in individuals with these disorders. These approaches are particularly useful for individuals with isolated or very tissue-restricted mosaicism in the brain, or for individuals who are not candidates for epilepsy surgery. Further, establishing a molecular diagnosis by the identification of a PI3K-AKT-MTOR pathogenic variant prior to undergoing epilepsy surgery could shift the existing paradigm for testing and treatment strategies, especially as repurposed PI3K-AKT-MTOR pathway-inhibitors are becoming more widely available, and may become earlier lines of therapy for epilepsy in affected individuals.

Patients undergoing cardiac surgery face significant inflammatory induced by exposure to cardiopulmonary bypass (CPB), contributing to heightened morbidity and mortality. The molecular and cellular mechanisms that underpin this inflammatory process remain unknown. To address this knowledge gap, we performed snRNA/ATAC-Sequencing on leukocytes from neonatal CPB patients. Classical monocytes become more prevalent and have dysregulation of inflammatory genes after CPB, indicating their role in CPB-associated inflammation. A genome-wide CRISPR screen and in vitro experiments in non-adherent monocytic cells identified two novel genes, SPTAN1 and RAF1, as effectors of hemodynamic stress. SPTAN1 and RAF1 activate store-operated calcium entry that results inflammation and cell death. snATAC-Seq revealed dynamically changing patterns of chromatin accessibility and AP-1 transcription factor binding after CPB exposure. These findings provide novel insights into the pathogenesis of CPB-associated inflammation, with broad implications for understanding the early stages of sterile inflammation and how non-adherent cells sense shear stress.### Competing Interest StatementThe authors have declared no competing interest.

Purpose Prior studies demonstrate the significance of specific cis -regulatory variants in retinal disease, however determining the functional impact of regulatory variants remains a major challenge. In this study, we utilize a machine learning approach, trained on epigenomic data from the adult human retina, to systematically quantify the predicted impact of cis -regulatory variants. Methods We used human retinal DNA accessibility data (ATAC-seq) to determine a set of 18.9k high-confidence putative cis -regulatory elements. 80% of these elements were used to train a machine learning model utilizing a gapped k-mer support vector machine-based approach. In silico saturation mutagenesis and variant scoring was applied to predict the functional impact of all potential single nucleotide variants within cis -regulatory elements. Impact scores were tested in a 20% hold-out dataset and compared to allele population frequency, phylogenetic conservation, transcription factor (TF) binding motifs, and existing massively parallel reporter assay (MPRA) data. Results We generated a model that distinguishes between human retinal regulatory elements and negative test sequences with 95% accuracy. Among a hold-out test set of 3.7k human retinal CREs, all possible single nucleotide variants (SNVs) were scored. Variants with negative impact scores correlated with reduced population allele frequency, higher phylogenetic conservation of the reference allele, disruption of predicted TF binding motifs, and massively-parallel reporter expression. Conclusions We demonstrated the utility of human retinal epigenomic data to train a machine learning model for the purpose of predicting the impact of non-coding regulatory sequence variants. Our model accurately scored sequences and predicted putative transcription factor binding motifs. This approach has the potential to expedite the characterization of pathogenic non-coding sequence variants in the context of unexplained retinal disease.

Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Prostate cancer is one of the few malignancies that includes vaccination as a treatment modality. Elements of an effective cancer vaccine should include the ability to elicit a Type I T-cell response and target multiple antigenic proteins expressed early in the disease. Using existing gene datasets encompassing normal prostate tissue and tumors with Gleason Score ≤ 6 and ≥ 8, 10 genes were identified that were upregulated and conserved in prostate cancer regardless of the aggressiveness of disease. These genes encoded proteins also expressed in prostatic intraepithelial neoplasia. Putative Class II epitopes derived from these proteins were predicted by a combination of algorithms and, using human peripheral blood, epitopes which selectively elicited IFN-γ or IL-10 dominant antigen specific cytokine secretion were determined. Th1 selective epitopes were identified for eight antigens. Epitopes from three antigens elicited Th1 dominant immunity in mice; PSMA, HPN, and AMACR. Each single antigen vaccine demonstrated significant anti-tumor activity inhibiting growth of implanted Myc-Cap cells after immunization as compared to control. Immunization with the combination of antigens, however, was superior to each alone in controlling tumor growth. When vaccination occurred simultaneously to tumor implant, multiantigen immunized mice had significantly smaller tumors than controls (p = 0.002) and a significantly improved overall survival (p = 0.0006). This multiantigen vaccine shows anti-tumor activity in a murine model of prostate cancer.

Patients undergoing cardiac surgery face significant inflammatory induced by exposure to cardiopulmonary bypass (CPB), contributing to heightened morbidity and mortality. The molecular and cellular mechanisms that underpin this inflammatory process remain unknown. To address this knowledge gap, we performed snRNA/ATAC-Sequencing on leukocytes from neonatal CPB patients. Classical monocytes become more prevalent and have dysregulation of inflammatory genes after CPB, indicating their role in CPB-associated inflammation. A genome-wide CRISPR screen and in vitro experiments in non-adherent monocytic cells identified two novel genes, SPTAN1 and RAF1, as effectors of hemodynamic stress. SPTAN1 and RAF1 activate store-operated calcium entry that results inflammation and cell death. snATAC-Seq revealed dynamically changing patterns of chromatin accessibility and AP-1 transcription factor binding after CPB exposure. These findings provide novel insights into the pathogenesis of CPB-associated inflammation, with broad implications for understanding the early stages of sterile inflammation and how non-adherent cells sense shear stress.

Reelin, a large extracellular protein, plays several critical roles in brain development and function. It is encoded by RELN, first identified as the gene disrupted in the reeler mouse, a classic neurological mutant exhibiting ataxia, tremors and a 'reeling' gait. In humans, biallelic variants in RELN have been associated with a recessive lissencephaly variant with cerebellar hypoplasia, which matches well with the homozygous mouse mutant that has abnormal cortical structure, small hippocampi and severe cerebellar hypoplasia. Despite the large size of the gene, only 11 individuals with RELN-related lissencephaly with cerebellar hypoplasia from six families have previously been reported. Heterozygous carriers in these families were briefly reported as unaffected, although putative loss-of-function variants are practically absent in the population (probability of loss of function intolerance = 1). Here we present data on seven individuals from four families with biallelic and 13 individuals from seven families with monoallelic (heterozygous) variants of RELN and frontotemporal or temporal-predominant lissencephaly variant. Some individuals with monoallelic variants have moderate frontotemporal lissencephaly, but with normal cerebellar structure and intellectual disability with severe behavioural dysfunction. However, one adult had abnormal MRI with normal intelligence and neurological profile. Thorough literature analysis supports a causal role for monoallelic RELN variants in four seemingly distinct phenotypes including frontotemporal lissencephaly, epilepsy, autism and probably schizophrenia. Notably, we observed a significantly higher proportion of loss-of-function variants in the biallelic compared to the monoallelic cohort, where the variant spectrum included missense and splice-site variants. We assessed the impact of two canonical splice-site variants observed as biallelic or monoallelic variants in individuals with moderately affected or normal cerebellum and demonstrated exon skipping causing in-frame loss of 46 or 52 amino acids in the central RELN domain. Previously reported functional studies demonstrated severe reduction in overall RELN secretion caused by heterozygous missense variants p.Cys539Arg and p.Arg3207Cys associated with lissencephaly suggesting a dominant-negative effect. We conclude that biallelic variants resulting in complete absence of RELN expression are associated with a consistent and severe phenotype that includes cerebellar hypoplasia. However, reduced expression of RELN remains sufficient to maintain nearly normal cerebellar structure. Monoallelic variants are associated with incomplete penetrance and variable expressivity even within the same family and may have dominant-negative effects. Reduced RELN secretion in heterozygous individuals affects only cortical structure whereas the cerebellum remains intact. Our data expand the spectrum of RELN-related neurodevelopmental disorders ranging from lethal brain malformations to adult phenotypes with normal brain imaging.

Cis-regulatory elements (CREs) play a critical role in the development and disease-states of all human cell types. In the retina, CREs have been implicated in several inherited disorders. To better characterize human retinal CREs, we performed single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) on the developing and adult human retina and on induced pluripotent stem cell (iPSC)-derived retinal organoids. These analyses identified developmentally dynamic, cell-class-specific CREs, enriched transcription-factor-binding motifs, and putative target genes. CREs in the retina and organoids are highly correlated at the single-cell level, and this supports the use of organoids as a model for studying disease-associated CREs. As a proof of concept, we disrupted a disease-associated CRE at 5q14.3, confirming its principal target gene as the miR-9-2 primary transcript and demonstrating its role in neurogenesis and gene regulation in mature glia. This study provides a resource for characterizing human retinal CREs and showcases organoids as a model to study the function of CREs that influence development and disease.

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10 −10 ), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2 . Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases.

The single-nucleotide polymorphism (SNP) rs3184504 is broadly associated with increased risk for multiple autoimmune and cardiovascular diseases. Although the allele is uniquely enriched in European descent, the mechanism for the widespread selective sweep is not clear. In this study, we find the rs3184504*T allele had a strong association with reduced mortality in a human sepsis cohort. The rs3184504*T allele associates with a loss-of-function amino acid change (p.R262W) in the adaptor protein SH2B3, a likely causal variant. To better understand the role of SH2B3 in sepsis, we used mouse modeling and challenged SH2B3-deficient mice with a polymicrobial cecal-ligation puncture (CLP) procedure. We found SH2B3 deficiency improved survival and morbidity with less organ damage and earlier bacterial clearance compared with control mice. The peritoneal infiltrating cells exhibited augmented phagocytosis in Sh2b3(-/-) mice with enriched recruitment of Ly6C(hi) inflammatory monocytes despite equivalent or reduced chemokine expression. Rapid cycling of monocytes and progenitors occurred uniquely in the Sh2b3(-/-) mice following CLP, suggesting augmented myelopoiesis. To model the hypomorphic autoimmune risk allele, we created a novel knockin mouse harboring a similar point mutation in the murine pleckstrin homology domain of SH2B3. At baseline, phenotypic changes suggested a hypomorphic allele. In the CLP model, homozygous knockin mice displayed improved mortality and morbidity compared with wild-type or heterozygous mice. Collectively, these data suggest that hypomorphic SH2B3 improves the sepsis response and that balancing selection likely contributed to the relative frequency of the autoimmune risk variant.

The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease. SPLiT-seq single-nucleus RNA sequencing of the developing human cerebellum reveals cell-type complexities and prolonged maturation compared to mouse with important disease implications.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of B-ALL often associated with genetic variants that alter cytokine receptor signaling, including mutations in the interleukin-7 receptor ( IL7R ). To investigate whether IL7R variants are leukemia-initiating, we built mouse models expressing activated Il7r (aIL7R). B-cell intrinsic aIL7R mice developed spontaneous B-ALL, demonstrating sufficiency of Il7r activating mutations in leukemogenesis. Concomitant introduction of a knock-out allele in the associated adapter protein Lnk (encoded by Sh2b3 ) or a dominant-negative variant of the transcription factor Ikaros ( Ikzf1 ) increased disease penetrance. The resulting murine leukemias displayed monoclonality and recurrent somatic Kras mutations and efficiently engrafted into immunocompetent mice. Phosphoproteomic analyses of aIL7R leukemic cells revealed constitutive Stat5 signaling and B cell receptor (BCR)-like signaling despite the absence of surface pre-BCR. Finally, in vitro treatment of aIL7R leukemic B-cells with Jak, mTOR, or Syk inhibitors blocked growth, confirming that each pathway is active in this mouse model of IL7R -driven B-ALL.

Summary Fibrosis-driven solid organ failure is an enormous burden on global health. Spiny mice (Acomys) are terrestrial mammals that can regenerate severe skin wounds without scars to avoid predation. Whether spiny mice also regenerate internal organ injuries is unknown. Here, we show that despite equivalent acute obstructive or ischemic kidney injury, spiny mice fully regenerate nephron structure and organ function without fibrosis, whereas C57Bl/6 or CD1 mice progress to complete organ failure with extensive renal fibrosis. Two mechanisms for vertebrate regeneration have been proposed that emphasize either extrinsic (pro-regenerative macrophages) or intrinsic (surviving cells of the organ itself) controls. Comparative transcriptome analysis revealed that the Acomys genome appears poised at the time of injury to initiate regeneration by surviving kidney cells, whereas macrophage accumulation was not detected until about day 7. Thus, we provide evidence for rapid activation of a gene expression signature for regenerative wound healing in the spiny mouse kidney.

Gene regulatory networks (GRNs), consisting of transcription factors and their target sites, control neurogenesis and cell-fate specification in the developing central nervous system. In this study, we use integrated single-cell RNA and single-cell ATAC sequencing (scATAC-seq) analysis in developing mouse and human retina to identify multiple interconnected, evolutionarily conserved GRNs composed of cell-type-specific transcription factors that both activate genes within their own network and inhibit genes in other networks. These GRNs control temporal patterning in primary progenitors, regulate transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirm that NFI transcription factors selectively activate expression of genes promoting late-stage temporal identity in primary retinal progenitors and identify other transcription factors that regulate rod photoreceptor specification in postnatal retina. This study inventories cis- and trans-acting factors that control retinal development and can guide cell-based therapies aimed at replacing retinal neurons lost to disease.