Systematic analysis of variants escaping nonsense-mediated decay uncovers candidate Mendelian diseases.

Protein-truncating variants (PTVs) near the 3' end of genes may escape nonsense-mediated decay (NMD). PTVs in the NMD-escape region (PTVescs) can cause Mendelian disease but are difficult to interpret given their varying impact on protein function. Previously, PTVesc burden was assessed in an epilepsy cohort, but no large-scale analysis has systematically evaluated these variants in rare disease. We performed a retrospective analysis of 29,031 neurodevelopmental disorder (NDD) parent-offspring trios referred for clinical exome sequencing to identify PTVesc de novo mutations (DNMs). We identified 1,376 PTVesc DNMs and 133 genes that were significantly enriched (binomial p < 0.001). The PTVesc-enriched genes included those with PTVescs previously described to cause dominant Mendelian disease (e.g., SEMA6B, PPM1D, and DAGLA). We annotated ClinVar variants for PTVescs and identified 948 genes with at least one high-confidence pathogenic variant. Twenty-two known Mendelian PTVesc-enriched genes had no prior evidence of PTVesc-associated disease. We found 22 additional PTVesc-enriched genes that are not well established to be associated with Mendelian disease, several of which showed phenotypic similarity between individuals harboring PTVesc variants in the same gene. Four individuals with PTVesc mutations in RAB1A had similar phenotypes including NDD and spasticity. PTVesc mutations in IRF2BP1 were found in two individuals who each had severe immunodeficiency manifesting in NDD. Three individuals with PTVesc mutations in LDB1 all had NDD and multiple congenital anomalies. Using a large-scale, systematic analysis of DNMs, we extend the mutation spectrum for known Mendelian disease-associated genes and identify potentially novel disease-associated genes.

A novel SMARCC1 BAFopathy implicates neural progenitor epigenetic dysregulation in human hydrocephalus.

Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery in children. Recent studies have implicated SMARCC1, a component of the BRG1-associated factor (BAF) chromatin remodelling complex, as a candidate congenital hydrocephalus gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, congenital hydrocephalus-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo. Here, we aimed to assess the prevalence of SMARCC1 variants in an expanded patient cohort, describe associated clinical and radiographic phenotypes, and assess the impact of Smarcc1 depletion in a novel Xenopus tropicalis model of congenital hydrocephalus. To do this, we performed a genetic association study using whole-exome sequencing from a cohort consisting of 2697 total ventriculomegalic trios, including patients with neurosurgically-treated congenital hydrocephalus, that total 8091 exomes collected over 7 years (2016-23). A comparison control cohort consisted of 1798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents were sourced from the Simons Simplex Collection. Enrichment and impact on protein structure were assessed in identified variants. Effects on the human fetal brain transcriptome were examined with RNA-sequencing and Smarcc1 knockdowns were generated in Xenopus and studied using optical coherence tomography imaging, in situ hybridization and immunofluorescence. SMARCC1 surpassed genome-wide significance thresholds, yielding six rare, protein-altering de novo variants localized to highly conserved residues in key functional domains. Patients exhibited hydrocephalus with aqueductal stenosis; corpus callosum abnormalities, developmental delay, and cardiac defects were also common. Xenopus knockdowns recapitulated both aqueductal stenosis and cardiac defects and were rescued by wild-type but not patient-specific variant SMARCC1. Hydrocephalic SMARCC1-variant human fetal brain and Smarcc1-variant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2. These results suggest de novo variants in SMARCC1 cause a novel human BAFopathy we term 'SMARCC1-associated developmental dysgenesis syndrome', characterized by variable presence of cerebral ventriculomegaly, aqueductal stenosis, developmental delay and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodelling complex for human brain morphogenesis and provide evidence for a 'neural stem cell' paradigm of congenital hydrocephalus pathogenesis. These results highlight utility of trio-based whole-exome sequencing for identifying pathogenic variants in sporadic congenital structural brain disorders and suggest whole-exome sequencing may be a valuable adjunct in clinical management of congenital hydrocephalus patients.

Human genetics and molecular genomics of Chiari malformation type 1.

Chiari malformation type 1 (CM1) is the most common structural brain disorder involving the craniocervical junction, characterized by caudal displacement of the cerebellar tonsils below the foramen magnum into the spinal canal. Despite the heterogeneity of CM1, its poorly understood patho-etiology has led to a 'one-size-fits-all' surgical approach, with predictably high rates of morbidity and treatment failure. In this review we present multiplex CM1 families, associated Mendelian syndromes, and candidate genes from recent whole exome sequencing (WES) and other genetic studies that suggest a significant genetic contribution from inherited and de novo germline variants impacting transcription regulation, craniovertebral osteogenesis, and embryonic developmental signaling. We suggest that more extensive WES may identify clinically relevant, genetically defined CM1 subtypes distinguished by unique neuroradiographic and neurophysiological endophenotypes.

De novo variants implicate chromatin modification, transcriptional regulation, and retinoic acid signaling in syndromic craniosynostosis.

Craniosynostosis (CS) is the most common congenital cranial anomaly. Several Mendelian forms of syndromic CS are well described, but a genetic etiology remains elusive in a substantial fraction of probands. Analysis of exome sequence data from 526 proband-parent trios with syndromic CS identified a marked excess (observed 98, expected 33, p = 4.83 × 10-20) of damaging de novo variants (DNVs) in genes highly intolerant to loss-of-function variation (probability of LoF intolerance > 0.9). 30 probands harbored damaging DNVs in 21 genes that were not previously implicated in CS but are involved in chromatin modification and remodeling (4.7-fold enrichment, p = 1.1 × 10-11). 17 genes had multiple damaging DNVs, and 13 genes (CDK13, NFIX, ADNP, KMT5B, SON, ARID1B, CASK, CHD7, MED13L, PSMD12, POLR2A, CHD3, and SETBP1) surpassed thresholds for genome-wide significance. A recurrent gain-of-function DNV in the retinoic acid receptor alpha (RARA; c.865G>A [p.Gly289Arg]) was identified in two probands with similar CS phenotypes. CS risk genes overlap with those identified for autism and other neurodevelopmental disorders, are highly expressed in cranial neural crest cells, and converge in networks that regulate chromatin modification, gene transcription, and osteoblast differentiation. Our results identify several CS loci and have major implications for genetic testing and counseling.

Multiomic analyses implicate a neurodevelopmental program in the pathogenesis of cerebral arachnoid cysts.

Cerebral arachnoid cysts (ACs) are one of the most common and poorly understood types of developmental brain lesion. To begin to elucidate AC pathogenesis, we performed an integrated analysis of 617 patient-parent (trio) exomes, 152,898 human brain and mouse meningeal single-cell RNA sequencing transcriptomes and natural language processing data of patient medical records. We found that damaging de novo variants (DNVs) were highly enriched in patients with ACs compared with healthy individuals (P = 1.57 × 10-33). Seven genes harbored an exome-wide significant DNV burden. AC-associated genes were enriched for chromatin modifiers and converged in midgestational transcription networks essential for neural and meningeal development. Unsupervised clustering of patient phenotypes identified four AC subtypes and clinical severity correlated with the presence of a damaging DNV. These data provide insights into the coordinated regulation of brain and meningeal development and implicate epigenomic dysregulation due to DNVs in AC pathogenesis. Our results provide a preliminary indication that, in the appropriate clinical context, ACs may be considered radiographic harbingers of neurodevelopmental pathology warranting genetic testing and neurobehavioral follow-up. These data highlight the utility of a systems-level, multiomics approach to elucidate sporadic structural brain disease.

A novel SMARCC1 -mutant BAFopathy implicates epigenetic dysregulation of neural progenitors in hydrocephalus.

Importance: Hydrocephalus, characterized by cerebral ventriculomegaly, is the most common disorder requiring brain surgery. A few familial forms of congenital hydrocephalus (CH) have been identified, but the cause of most sporadic cases of CH remains elusive. Recent studies have implicated SMARCC1 , a component of the B RG1- a ssociated factor (BAF) chromatin remodeling complex, as a candidate CH gene. However, SMARCC1 variants have not been systematically examined in a large patient cohort or conclusively linked with a human syndrome. Moreover, CH-associated SMARCC1 variants have not been functionally validated or mechanistically studied in vivo . Objectives: The aims of this study are to (i) assess the extent to which rare, damaging de novo mutations (DNMs) in SMARCC1 are associated with cerebral ventriculomegaly; (ii) describe the clinical and radiographic phenotypes of SMARCC1 -mutated patients; and (iii) assess the pathogenicity and mechanisms of CH-associated SMARCC1 mutations in vivo . Design setting and participants: A genetic association study was conducted using whole-exome sequencing from a cohort consisting of 2,697 ventriculomegalic trios, including patients with neurosurgically-treated CH, totaling 8,091 exomes collected over 5 years (2016-2021). Data were analyzed in 2023. A comparison control cohort consisted of 1,798 exomes from unaffected siblings of patients with autism spectrum disorder and their unaffected parents sourced from the Simons simplex consortium. Main outcomes and measures: Gene variants were identified and filtered using stringent, validated criteria. Enrichment tests assessed gene-level variant burden. In silico biophysical modeling estimated the likelihood and extent of the variant impact on protein structure. The effect of a CH-associated SMARCC1 mutation on the human fetal brain transcriptome was assessed by analyzing RNA-sequencing data. Smarcc1 knockdowns and a patient-specific Smarcc1 variant were tested in Xenopus and studied using optical coherence tomography imaging, in situ hybridization, and immunofluorescence microscopy. Results: SMARCC1 surpassed genome-wide significance thresholds in DNM enrichment tests. Six rare protein-altering DNMs, including four loss-of-function mutations and one recurrent canonical splice site mutation (c.1571+1G>A) were detected in unrelated patients. DNMs localized to the highly conserved DNA-interacting SWIRM, Myb-DNA binding, Glu-rich, and Chromo domains of SMARCC1 . Patients exhibited developmental delay (DD), aqueductal stenosis, and other structural brain and heart defects. G0 and G1 Smarcc1 Xenopus mutants exhibited aqueductal stenosis and cardiac defects and were rescued by human wild-type SMARCC1 but not a patient-specific SMARCC1 mutant. Hydrocephalic SMARCC1 -mutant human fetal brain and Smarcc1 -mutant Xenopus brain exhibited a similarly altered expression of key genes linked to midgestational neurogenesis, including the transcription factors NEUROD2 and MAB21L2 . Conclusions: SMARCC1 is a bona fide CH risk gene. DNMs in SMARCC1 cause a novel human BAFopathy we term " S MARCC1- a ssociated D evelopmental D ysgenesis S yndrome (SaDDS)", characterized by cerebral ventriculomegaly, aqueductal stenosis, DD, and a variety of structural brain or cardiac defects. These data underscore the importance of SMARCC1 and the BAF chromatin remodeling complex for human brain morphogenesis and provide evidence for a "neural stem cell" paradigm of human CH pathogenesis. These results highlight the utility of trio-based WES for identifying risk genes for congenital structural brain disorders and suggest WES may be a valuable adjunct in the clinical management of CH patients. KEY POINTS: Question: What is the role of SMARCC1 , a core component of the B RG1- a ssociated factor (BAF) chromatin remodeling complex, in brain morphogenesis and congenital hydrocephalus (CH)? Findings: SMARCC1 harbored an exome-wide significant burden of rare, protein-damaging de novo mutations (DNMs) (p = 5.83 × 10 -9 ) in the largest ascertained cohort to date of patients with cerebral ventriculomegaly, including treated CH (2,697 parent-proband trios). SMARCC1 contained four loss-of-function DNMs and two identical canonical splice site DNMs in a total of six unrelated patients. Patients exhibited developmental delay, aqueductal stenosis, and other structural brain and cardiac defects. Xenopus Smarcc1 mutants recapitulated core human phenotypes and were rescued by the expression of human wild-type but not patient-mutant SMARCC1 . Hydrocephalic SMARCC1 -mutant human brain and Smarcc1 -mutant Xenopus brain exhibited similar alterationsin the expression of key transcription factors that regulate neural progenitor cell proliferation. Meaning: SMARCC1 is essential for human brain morphogenesis and is a bona fide CH risk gene. SMARCC1 mutations cause a novel human BAFopathy we term " S MARCC1- a ssociated D evelopmental D ysgenesis S yndrome (SaDDS)". These data implicate epigenetic dysregulation of fetal neural progenitors in the pathogenesis of hydrocephalus, with diagnostic and prognostic implications for patients and caregivers.

Multiethnic Prediction of Nicotine Biomarkers and Association With Nicotine Dependence.

INTRODUCTION: The nicotine metabolite ratio and nicotine equivalents are measures of metabolism rate and intake. Genome-wide prediction of these nicotine biomarkers in multiethnic samples will enable tobacco-related biomarker, behavioral, and exposure research in studies without measured biomarkers. AIMS AND METHODS: We screened genetic variants genome-wide using marginal scans and applied statistical learning algorithms on top-ranked genetic variants, age, ethnicity and sex, and, in additional modeling, cigarettes per day (CPD), (in additional modeling) to build prediction models for the urinary nicotine metabolite ratio (uNMR) and creatinine-standardized total nicotine equivalents (TNE) in 2239 current cigarette smokers in five ethnic groups. We predicted these nicotine biomarkers using model ensembles and evaluated external validity using dependence measures in 1864 treatment-seeking smokers in two ethnic groups. RESULTS: The genomic regions with the most selected and included variants for measured biomarkers were chr19q13.2 (uNMR, without and with CPD) and chr15q25.1 and chr10q25.3 (TNE, without and with CPD). We observed ensemble correlations between measured and predicted biomarker values for the uNMR and TNE without (with CPD) of 0.67 (0.68) and 0.65 (0.72) in the training sample. We observed inconsistency in penalized regression models of TNE (with CPD) with fewer variants at chr15q25.1 selected and included. In treatment-seeking smokers, predicted uNMR (without CPD) was significantly associated with CPD and predicted TNE (without CPD) with CPD, time-to-first-cigarette, and Fagerström total score. CONCLUSIONS: Nicotine metabolites, genome-wide data, and statistical learning approaches developed novel robust predictive models for urinary nicotine biomarkers in multiple ethnic groups. Predicted biomarker associations helped define genetically influenced components of nicotine dependence. IMPLICATIONS: We demonstrate development of robust models and multiethnic prediction of the uNMR and TNE using statistical and machine learning approaches. Variants included in trained models for nicotine biomarkers include top-ranked variants in multiethnic genome-wide studies of smoking behavior, nicotine metabolites, and related disease. Association of the two predicted nicotine biomarkers with Fagerström Test for Nicotine Dependence items supports models of nicotine biomarkers as predictors of physical dependence and nicotine exposure. Predicted nicotine biomarkers may facilitate tobacco-related disease and treatment research in samples with genomic data and limited nicotine metabolite or tobacco exposure data.

Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis.

HUWE1 mutations in Juberg-Marsidi and Brooks syndromes: the results of an X-chromosome exome sequencing study.

BACKGROUND: X linked intellectual disability (XLID) syndromes account for a substantial number of males with ID. Much progress has been made in identifying the genetic cause in many of the syndromes described 20-40 years ago. Next generation sequencing (NGS) has contributed to the rapid discovery of XLID genes and identifying novel mutations in known XLID genes for many of these syndromes. METHODS: 2 NGS approaches were employed to identify mutations in X linked genes in families with XLID disorders. 1 involved exome sequencing of genes on the X chromosome using the Agilent SureSelect Human X Chromosome Kit. The second approach was to conduct targeted NGS sequencing of 90 known XLID genes. RESULTS: We identified the same mutation, a c.12928 G>C transversion in the HUWE1 gene, which gives rise to a p.G4310R missense mutation in 2 XLID disorders: Juberg-Marsidi syndrome (JMS) and Brooks syndrome. Although the original families with these disorders were considered separate entities, they indeed overlap clinically. A third family was also found to have a novel HUWE1 mutation. CONCLUSIONS: As we identified a HUWE1 mutation in an affected male from the original family reported by Juberg and Marsidi, it is evident the syndrome does not result from a mutation in ATRX as reported in the literature. Additionally, our data indicate that JMS and Brooks syndromes are allelic having the same HUWE1 mutation.

Truncating mutations in LRP4 lead to a prenatal lethal form of Cenani-Lenz syndrome.

Cenani-Lenz syndrome (CLS) is an autosomal recessive skeletal dysplasia that results in malformations of the distal limb, renal anomalies, and characteristic facies. In 2010, this condition was found to be caused by mutations in LRP4, a member of the low-density lipoprotein family of receptors. LRP4 has been shown to antagonize LRP5/LRP6 activation of WNT and ß-catenin signaling. Loss of LRP4 function leads to excessive Wnt and ß-catenin signaling in the limb bud, which causes abnormal limb development. The large majority of patients with CLS reported in the literature have splicing and missense mutations, which result in syndactyly, oligodactyly, and minor renal malformations. More recently, a patient with CLS has been identified with a homozygous nonsense mutation and a more severe presentation of findings typically associated with this condition. Here we present two sibling fetuses with a prenatal lethal presentation of mesomelic limb reductions, oligosyndactyly, genitourinary malformation and compound heterozygosity for two novel truncating mutations in LRP4. These findings lend further support to the CLS genotype-phenotype correlation presented in recent publications.

The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization.

BACKGROUND: Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length - in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. RESULTS: Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. CONCLUSIONS: Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative.