Functionalized Copper Nanoparticles with Gold Nanoclusters: Part I. Highly Selective Electrosynthesis of Hydrogen Peroxide.

Copper nanoparticles (CuNPs) and gold nanoclusters (AuNCs) show a high catalytic performance in generating hydrogen peroxide (H2O2), a property that can be exploited to kill disease-causing microbes and to carry carbon-free energy. Some combinations of NPs/NCs can generate synergistic effects to produce stronger antiseptics, such as H2O2 or other reactive oxygen species (ROS). Herein, we demonstrate a novel facile AuNC surface decoration method on the surfaces of CuNPs using galvanic displacement. The Cu-Au bimetallic NPs presented a high selective production of H2O2 via a two-electron (2e-) oxygen reduction reaction (ORR). Their physicochemical analyses were conducted by scanning electron microscopy (SEM), transmitting electron microscopy (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). With the optimized Cu-Au1.5NPs showing their particle sizes averaged in 53.8 nm, their electrochemical analysis indicated that the pristine AuNC structure exhibited the highest 2e- selectivity in ORR, the CuNPs presented the weakest 2e- selectivity, and the optimized Cu-Au1.5NPs exhibited a high 2e- selectivity of 95% for H2O2 production, along with excellent catalytic activity and durability. The optimized Cu-Au1.5NPs demonstrated a novel pathway to balance the cost and catalytic performance through the appropriate combination of metal NPs/NCs.

Known pathogenic gene variants and new candidates detected in Sudden Unexpected Infant Death using Whole Genome Sequencing.

Purpose: To gain insights into potential genetic factors contributing to the infant's vulnerability to Sudden Unexpected Infant Death (SUID). Methods: Whole Genome Sequencing (WGS) was performed on 145 infants that succumbed to SUID, and 576 healthy adults. Variants were filtered by gnomAD allele frequencies and predictions of functional consequences. Results: Variants of interest were identified in 86 genes, 63.4% of our cohort. Seventy-one of these have been previously associated with SIDS/SUID/SUDP. Forty-three can be characterized as cardiac genes and are related to cardiomyopathies, arrhythmias, and other conditions. Variants in 22 genes were associated with neurologic functions. Variants were also found in 13 genes reported to be pathogenic for various systemic disorders. Variants in eight genes are implicated in the response to hypoxia and the regulation of reactive oxygen species (ROS) and have not been previously described in SIDS/SUID/SUDP. Seventy-two infants met the triple risk hypothesis criteria (Figure 1). Conclusion: Our study confirms and further expands the list of genetic variants associated with SUID. The abundance of genes associated with heart disease and the discovery of variants associated with the redox metabolism have important mechanistic implications for the pathophysiology of SUID.

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome.

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine.

Correction: Genome Dynamics of Campylobacter jejuni in Response to Bacteriophage Predation.

[This corrects the article DOI: 10.1371/journal.ppat.0030119.].

Non-Toxigenic Clostridioides difficile Strain E4 (NTCD-E4) Prevents Establishment of Primary C. difficile Infection by Epidemic PCR Ribotype 027 in an In Vitro Human Gut Model.

Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI.

Transcriptomic Signatures of Single-Suture Craniosynostosis Phenotypes.

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.

Clinical, neuroimaging and molecular characteristics of PPP2R5D-related neurodevelopmental disorders: an expanded series with functional characterisation and genotype-phenotype analysis.

BACKGROUND: Variants in PPP2R5D, 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. METHODS: Individuals with PPP2R5D variants 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. RESULTS: We studied 76 individuals with PPP2R5D variants, 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. CONCLUSION: Our study delineates the most common features of PPP2R5D-related neurodevelopmental disorders, expands the clinical and molecular spectrum and identifies genotype-phenotype correlations.

Anti-tumor activity of a T-helper 1 multiantigen vaccine in a murine model of prostate cancer.

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.

The arginine methyltransferase Carm1 is necessary for heart development.

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.

Monoallelic and biallelic mutations in RELN underlie a graded series of neurodevelopmental disorders.

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.

Machine Learning Prediction of Non-Coding Variant Impact in Human Retinal cis-Regulatory Elements.

Purpose: Prior studies have demonstrated 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 utilized 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. Eighty percent 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 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 were scored. Variants with negative impact scores correlated with 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. Translational Relevance: This workflow and resulting dataset serve as a promising genomic tool to facilitate the clinical prioritization of functionally disruptive non-coding mutations in the retina.

Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.

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.

Cell-specific cis-regulatory elements and mechanisms of non-coding genetic disease in human retina and retinal organoids.

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.

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

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.

Spiny mice activate unique transcriptional programs after severe kidney injury regenerating organ function without fibrosis.

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 controlling temporal patterning, neurogenesis, and cell-fate specification in mammalian retina.

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.

The Autoimmune Risk R262W Variant of the Adaptor SH2B3 Improves Survival in Sepsis.

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 Ly6Chi 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.

Adaptations in Hippo-Yap signaling and myofibroblast fate underlie scar-free ear appendage wound healing in spiny mice.

Spiny mice (Acomys cahirinus) are terrestrial mammals that evolved unique scar-free regenerative wound-healing properties. Myofibroblasts (MFs) are the major scar-forming cell type in skin. We found that following traumatic injury to ear pinnae, MFs appeared rapidly in both Acomys and mouse yet persisted only in mouse. The timing of MF loss in Acomys correlated with wound closure, blastema differentiation, and nuclear localization of the Hippo pathway target protein Yap. Experiments in vitro revealed an accelerated PP2A-dependent dephosphorylation activity that maintained nuclear Yap in Acomys dermal fibroblasts (DFs) and was not detected in mouse or human DFs. Treatment of Acomys in vivo with the nuclear Yap-TEAD inhibitor verteporfin prolonged MF persistence and converted tissue regeneration to fibrosis. Forced Yap activity prevented and rescued TGF-ß1-induced human MF formation in vitro. These results suggest that Acomys evolved modifications of Yap activity and MF fate important for scar-free regenerative wound healing in vivo.

Haploinsufficiency of SF3B2 causes craniofacial microsomia.

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.

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome.

PURPOSE: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. METHODS: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. RESULTS: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. CONCLUSION: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.